R. M. Cassidy

Karolinska Institutet, Solna, Stockholm, Sweden

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Publications (29)124.8 Total impact

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    ABSTRACT: Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.
    Nature Neuroscience 04/2009; 12(3):259-67. · 15.25 Impact Factor
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    ABSTRACT: New neurons are generated from stem cells in a few regions of the adult mammalian brain. Here we provide evidence for the generation of dopaminergic projection neurons of the type that are lost in Parkinson's disease from stem cells in the adult rodent brain and show that the rate of neurogenesis is increased after a lesion. The number of new neurons generated under physiological conditions in substantia nigra pars compacta was found to be several orders of magnitude smaller than in the granular cell layer of the dentate gyrus of the hippocampus. However, if the rate of neuronal turnover is constant, the entire population of dopaminergic neurons in substantia nigra could be replaced during the lifespan of a mouse. These data indicate that neurogenesis in the adult brain is more widespread than previously thought and may have implications for our understanding of the pathogenesis and treatment of neurodegenerative disorders such as Parkinson's disease.
    Proceedings of the National Academy of Sciences 07/2003; 100(13):7925-30. · 9.81 Impact Factor
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    Robert Cassidy, Jonas Frisén
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    ABSTRACT: Embryonic stem cells readily generate neurons in vitro, but steering their differentiation into specific neuronal subtypes is a major challenge. It has now been shown that mechanisms that regulate neuronal specification during development can be applied to embryonic stem cells in vitro; this may lead to new ways of generating cells for therapy.
    Current Biology 11/2002; 12(20):R705-6. · 9.49 Impact Factor
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    ABSTRACT: Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.
    Experimental Cell Research 10/2002; 279(1):34-9. · 3.56 Impact Factor
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    ABSTRACT: Over the past decade, it has become clear that neural stem cells in the adult mammalian brain continuously generate new neurons, predominantly in the hippocampus and olfactory bulb. However, the central issue of whether these new neurons participate in functional synaptic circuitry has yet to be resolved. Here, we use virus-based transsynaptic neuronal tracing and c-Fos mapping of odor-induced neuronal activity to demonstrate that neurons generated in the adult functionally integrate into the synaptic circuitry of the brain.
    Current Biology 05/2002; 12(7):606-8. · 9.49 Impact Factor
  • R Cassidy, J Frisén
    Nature 09/2001; 412(6848):690-1. · 38.60 Impact Factor
  • Robert Cassidy, Jonas Frisén
    Nature 01/2001; 412:690-691. · 38.60 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8alpha(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs. CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses. RBP-J/Su(H)/Lag1, the main transcriptional mediator of Notch signaling, binds DNA with the consensus sequence YRTGDGAD. Notch target genes can be controlled by two opposing activities of RBP-J. The interaction of the Notch intracellular domain with RBP-J induces a weak transcriptional activation and requires an additional tissue-specific transcriptional activator such as bHLH proteins or GATA to mediate strong target gene expression. For example, during Drosophila sensory organ precursor (SOP) cell development, proneural bHLH interacts with Da, a Drosophila orthologue of E2A, to form a tissue-specific activator of Su(H), the Drosophila orthologue of RBP-J. This complex and Su(H) act synergistically to promote the epidermal cell fate. In contrast, a complex of Su(H) with Hairless, a Drosophila functional homologue of MINT, has transcriptional repression activity that promotes SOP differentiation to neurons. Recent conditional loss-of-function studies demonstrated that transcriptional networks involving RBP-J, MINT, and E2A are conserved in mammalian cell differentiation, including multiple steps of lymphocyte development, and probably also in neuronal maturation in adult neurogenesis. During neurogenesis, Notch-RBP-J signaling was thought historically to be involved mainly in the maintenance of undifferentiated neural progenitors. However, the identification of a tissue-specific transcriptional activator of RBP-J-Notch has revealed new roles of RBP-J in the promotion of neuronal maturation. Finally, the Notch-independent function of RBP-J was recently discovered and will be reviewed here. Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2(+) oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2(+) cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. Deletion or duplication of the human chromosome 22q11.2 is associated with many behavioral traits and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia. However, why phenotypes vary widely among individuals with identical deletions or duplications of 22q11.2 and which specific 22q11.2 genes contribute to these phenotypes are still poorly understood. Previous studies have identified a approximately 200 kb 22q11.2 region that contributes to behavioral phenotypes in mice. We tested the role of Septin 5 (Sept5), a gene encoded in the approximately 200 kb region, in affective behaviors, cognitive capacities and motor activity. To evaluate the impact of genetic backgrounds on behavioral phenotypes of Sept5 deficiency, we used mice on two genetic backgrounds. Our data show that Sept5 deficiency decreased affiliative active social interaction, but this phenotypic expression was influenced by genetic backgrounds. In contrast, Sept5 deficiency decreased anxiety-related behavior, increased prepulse inhibition and delayed acquisition of rewarded goal approach, independent of genetic background. These data suggest that Sept5 deficiency exerts pleiotropic effects on a select set of affective behaviors and cognitive processes and that genetic backgrounds could provide an epistatic influence on phenotypic expression. Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury. The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia. Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development. Notch molecules are well conserved from Drosophila melanogaster to mammals and regulate a broad spectrum of various cell lineage commitment processes. Recent studies using inhibitors, transgenic mice and conditional loss-of-function approaches have demonstrated essential roles for Notch signaling in the differentiation of thymocytes and peripheral T cells, as well as B cells. Here we highlight parallels in the developmental regulation of mammalian lymphocytes and the D. melanogaster nervous system through Notch cooperation with the transcriptional regulators RBP-J (Su(H)), MINT (Hairless) and E2A (Ac-Sc-Da). The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44(hi) memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44(hi) CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44(hi) memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses. Mammary alveoli are composed of luminal (secretory) and basal (myoepithelial) cells, which are descendants of a common stem cell. This study addressed the role of RBP-J-dependent Notch signaling in the formation, maintenance and cellular composition of alveoli during pregnancy. For this purpose, the genes encoding RBP-J, the shared transcriptional mediator of Notch receptors, and Pofut1, a fucosyltransferase required for the activity of Notch receptors, were deleted in mammary progenitor cells in the mouse using Cre-mediated recombination. Loss of RBP-J and Pofut1 led to an accumulation of basal cell clusters characterized by the presence of cytokeratins (K5) and K14 and smooth muscle actin (SMA) during pregnancy. Hormonal stimulation of mutant tissue induced the expression of the basal cell transcription factor p63 in luminal cells and excessive proliferation of basal cells. A transient enrichment of K6-positive luminal cells was observed upon hormonal treatment suggesting a temporary arrest at an immature stage prior to transdifferentiation and expansion as basal cells. Despite the extensive proliferation of RBP-J-null basal cells during pregnancy, hormonal withdrawal during involution resulted in complete remodeling and the restoration of normal tissue architecture. We propose that the Notch-RBP-J pathway regulates alveolar development during pregnancy by maintaining luminal cell fate and preventing uncontrolled basal cell proliferation. To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas. Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation. The Notch signaling pathway is essential for embryonic vascular development in vertebrates. Here we show that mouse embryos heterozygous for a targeted mutation in the gene encoding the DLL4 ligand exhibit haploinsufficient lethality because of defects in vascular remodeling. We also describe vascular defects in embryos homozygous for a mutation in the Rbpsuh gene, which encodes the primary transcriptional mediator of Notch signaling. Conditional inactivation of Rpbsuh function demonstrates that Notch activation is essential in the endothelial cell lineage. Notch pathway mutant embryos exhibit defects in arterial specification of nascent blood vessels and develop arteriovenous malformations. These results demonstrate that vascular remodeling in the mouse embryo is sensitive to Dll4 gene dosage and that Notch activation in endothelial cells is essential for embryonic vascular remodeling. Antigen-presenting cells (APC) tailor immune responses to microbial encounters by stimulating differentiation of CD4 T cells into the Th1 and Th2 lineages. We demonstrate that APC use the Notch pathway to instruct T cell differentiation. Strikingly, of the two Notch ligand families, Delta induces Th1, while Jagged induces the alternate Th2 fate. Expression of these different Notch ligands on APC is induced by Th1- or Th2-promoting stimuli. Th2 differentiation has been considered a default process as APC-derived instructive signals are unknown. We demonstrate that Jagged constitutes an instructive signal for Th2 differentiation, which is independent of IL4/STAT6. Th2 differentiation induced by APC is abrogated in T cells lacking the Notch effector RBPJkappa. Notch directs Th2 differentiation by inducing GATA3 and by directly regulating il4 gene transcription through RBPJkappa sites in a 3' enhancer. RBP-J is a key mediator of Notch signaling that regulates a large spectrum of cell fate determinations. To elucidate the functions of Notch signaling in T cell development, we inactivated RBP-J specifically at two stages of T cell development by crossing RBP-J floxed mice with lck-cre or CD4-cre transgenic mice. The loss of RBP-J at an earlier developmental stage resulted in enhanced generation and accelerated emigration of gammadelta T cells, whereas alphabeta T cell development was arrested at the double-negative 3 stage. The loss of RBP-J at a later stage did not affect the absolute number or the production rate of CD4 or CD8-positive mature T cells but enhanced Th1 cell response and reduced CD4(+) T cell proliferation. Our data demonstrated that Notch/RBP-J signaling regulates gammadelta T cell generation and migration, alphabeta T cell maturation, terminal differentiation of CD4(+) T cells into Th1/Th2 cells, and activation of T cells. The DNA-binding protein recombination signal binding protein-Jkappa (RBP-J) mediates transcriptional activation of the Notch intracellular domain (NIC). In the absence of transcriptional activators, RBP-J suppresses transcription by recruiting co-suppressors. KyoT2 is a LIM domain protein that inhibits the RBP-J-mediated transcriptional activation. Here we provide evidence that the polycomb group protein RING1 interacts with the LIM domains of KyoT2 in yeast and mammalian cells. The interaction between KyoT2 and RING1 was detected both in vitro and in vivo. By using a co-immunoprecipitation assay, we also showed that, though RING1 and RBP-J did not associate directly, the two molecules could be co-precipitated simultaneously by KyoT2, probably through the LIM domains and the RBP-J-binding motif of KyoT2, respectively. These results suggested the formation of a three-molecule complex consisting of RBP-J, KyoT2 and RING1 in cells. Moreover, we found that overexpression of RING1 together with KyoT2 in cells inhibited transactivation of RBP-J by NIC. Suppression of the NIC- mediated transactivation of RBP-J by RING1 was abrogated by overexpression of KBP1, a molecule that competed with RING1 for binding to LIM domains of KyoT2, suggesting that suppression of RBP-J by RING1 was dependent on its associating with KyoT2. Taken together, our data suggested that there might be at least two ways of the KyoT2-mediated suppression of RBP-J, namely competition for binding sites with transactivators, and recruitment of suppressors such as RING1. RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Notch signaling is involved in the cell fate determination of various cell lineages. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J to transactivate transcription of target genes. All four Notches in mammals bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs, inhibits the terminal differentiation of the epidermis, and regulates hair differentiation. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region. We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless. The transcription factor recombination signal binding protein-J (RBP-J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP-J, we introduced loxP sites on both sides of the RBP-J exons encoding its DNA binding domain. Mice bearing the loxP-flanked RBP-J alleles, RBP-J(f/f), were mated with Mx-Cre transgenic mice and deletional mutation of the RBP-J gene in adult mice was induced by injection of the IFN-alpha inducer poly(I)-poly(C). Here we show that inactivation of RBP-J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP-J differentiate into B but not T cells when cultured in 2'-deoxyguanosine-treated fetal thymic lobes by hanging-drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP-J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP-J-inactivated mice. These results suggest that RBP-J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors. RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria. A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins. Notch1 has been shown to induce glia in the peripheral nervous system. However, it has not been known whether Notch can direct commitment to glia from multipotent progenitors of the central nervous system. Here we present evidence that activated Notch1 and Notch3 promotes the differentiation of astroglia from the rat adult hippocampus-derived multipotent progenitors (AHPs). Quantitative clonal analysis indicates that the action of Notch is likely to be instructive. Transient activation of Notch can direct commitment of AHPs irreversibly to astroglia. Astroglial induction by Notch signaling was shown to be independent of STAT3, which is a key regulatory transcriptional factor when ciliary neurotrophic factor (CNTF) induces astroglia. These data suggest that Notch provides a CNTF-independent instructive signal of astroglia differentiation in CNS multipotent progenitor cells.
    Eur J Immunol. 41(5):1309-20.