[show abstract][hide abstract] ABSTRACT: During the early development of the gastrointestinal tract, signaling through the receptor tyrosine kinase RET is required for initiation of lymphoid organ (Peyer's patch) formation and for intestinal innervation by enteric neurons. RET signaling occurs through glial cell line-derived neurotrophic factor (GDNF) family receptor α co-receptors present in the same cell (signaling in cis). It is unclear whether RET signaling in trans, which occurs in vitro through co-receptors from other cells, has a biological role. We showed that the initial aggregation of hematopoietic cells to form lymphoid clusters occurred in a RET-dependent, chemokine-independent manner through adhesion-mediated arrest of lymphoid tissue initiator (LTin) cells. Lymphoid tissue inducer cells were not necessary for this initiation phase. LTin cells responded to all RET ligands in trans, requiring factors from other cells, whereas RET was activated in enteric neurons exclusively by GDNF in cis. Furthermore, genetic and molecular approaches revealed that the versatile RET responses in LTin cells were determined by distinct patterns of expression of the genes encoding RET and its co-receptors. Our study shows that a trans RET response in LTin cells determines the initial phase of enteric lymphoid organ morphogenesis, and suggests that differential co-expression of Ret and Gfra can control the specificity of RET signaling.
[show abstract][hide abstract] ABSTRACT: The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.
The Journal of Immunology 08/2011; 187(7):3712-20. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: During cortical development, coordination of proliferation and differentiation ensures the timely generation of different neural progenitor lineages that will give rise to mature neurons and glia. Geminin is an inhibitor of DNA replication and it has been proposed to regulate cell proliferation and fate determination during neurogenesis via interactions with transcription factors and chromatin remodeling complexes. To investigate the in vivo role of Geminin in the maintenance and differentiation of cortical neural progenitors, we have generated mice that lack Geminin expression in the developing cortex. Our results show that loss of Geminin leads to the expansion of neural progenitor cells located at the ventricular and subventricular zones of the developing cortex. Early cortical progenitors lacking Geminin exhibit a longer S-phase and a reduced ability to generate early born neurons, consistent with a preference on self-renewing divisions. Overexpression of Geminin in progenitor cells of the cortex reduces the number of neural progenitor cells, promotes cell cycle exit and subsequent neuronal differentiation. Our study suggests that Geminin has an important role during cortical development in regulating progenitor number and ultimately neuron generation.
[show abstract][hide abstract] ABSTRACT: The CD8 gene is silent in CD4(-)CD8(-) double-negative thymocytes, expressed in CD4(+)CD8(+) double-positive cells, and silenced in cells committing to the CD4(+) single-positive (SP) lineage, remaining active in the CD8(+) SP lineage. In this study, we show that the chromatin of the CD8 locus is remodeled in C57BL/6 and B6/J Rag1(-/-) MOM double-negative thymocytes as indicated by DNaseI hypersensitivity and widespread bivalent chromatin marks. Pre-TCR signaling coincides with chromatin bivalency resolution into monovalent activating modifications in double-positive and CD8 SP cells. Shortly after commitment to CD4 SP cell lineage, monovalent repressive characteristics and chromatin inaccessibility are established. Differential binding of Ikaros, NuRD, and heterochromatin protein 1α on the locus during these processes may participate in the complex regulation of CD8.
The Journal of Immunology 06/2011; 186(11):6368-77. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thymic T cell differentiation to peripheral T cells aims to assist the generation of effector cells mediating adaptive immune responses. During this process, which takes place during embryogenesis and in adulthood, proliferation is coupled with changes in chromatin organization and transcription. Moreover, B and T lymphocytes start to proliferate and rapidly expand their numbers when activated following an encounter with an antigen. This expansion phase is accompanied by differentiation of naïve T cells and is followed by a period of population contraction, resulting in only a small fraction of the expanded population surviving and entering the memory cell pool. The kinetics of the expansion and contraction affect the speed of antigen clearance and the clinical course of disease. Molecules that are involved in the coordination of proliferation, chromatin reorganization, and transcriptional regulation are likely to play an important role in T cell generation, homeostasis, and disease. Here we review how cell cycle regulators affect lymphoid system development and homeostasis and discuss recent evidence implicating the cell cycle inhibitor Geminin in this process. Geminin has been shown to coordinate proliferation and differentiation by regulating cell cycle progression, chromatin organization, and transcription in the nervous system. In the immune system, progenitor T cell commitment and differentiation progresses normally in the absence of Geminin. However, Geminin is required for TCR response in vitro and T cell proliferation upon lymphopenia-induced proliferation, suggesting that Geminin might be an essential factor for T cell expansion during the immune response.
Critical Reviews in Immunology 01/2011; 31(3):209-31. · 3.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hox genes play important roles in haematopoietic development in mammals. ASH1 is a member of the trithorax group (trxG) that is required for proper expression of Hox genes and is preferentially expressed in haematopoietic stem cells. We have recently reported that ASH1 methylates histone H3 at lysine 36 (K36) but its biological function has remained elusive. Here we show that ASH1 regulates Hox gene expression positively and negatively in a leukemic cell line K562 and is required for myelomonocytic differentiation of murine haematopoietic stem cells. ASH1 binds to endogenous Hox loci in K562 cells and its knockdown causes reduced expression of Hox genes. In addition, ASH1 and MLL1 induce more than 100-fold activation of Hox promoters in HeLa cells if expressed simultaneously but not individually. Notably, ASH1 harbouring a point mutation that kills methyltransferase activity is more efficient than wild type ASH1 in Hox gene activation, indicating that K36 methylation is not a prerequisite for Hox gene expression. Moreover, tethering wild type or catalytically inactive methyltransferase domain of ASH1 to a heterologous promoter causes downregulation or upregulation, respectively, of transcription, supporting a hypothesis that K36 methylation imparts repression. Knockdown of ASH1 in K562 cells in vitro causes increased expression of ε-globin gene and reduced expression of myelomonocytic markers GPIIb and GPIIIa, whereas knockdown of ASH1 in murine haematopoietic stem cells in vivo results in decreased number of macrophages and granulocytes, a phenotype similar to that induced by loss of mll1 function. Taken together, our data suggest that ASH1 and MLL1 synergize in activation of Hox genes and thereby regulate development of myelomonocytic lineages from haematopoietic stem cells.
PLoS ONE 01/2011; 6(11):e28171. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To study the influence of a locus control region (LCR) on the expression of a highly characterized, developmentally regulated locus, we have targeted the hCD2-LCR as a single copy into the endogenous mouse CD8 gene complex. Two knock-in mouse lines that differ in the integration site of the hCD2-LCR within the mCD8 gene complex were generated, and the influence on expression of the CD8 coreceptor was assessed. In these mice the normal developmental silencing of the CD8 genes in the CD4 lineage is deregulated, and the mice develop CD4(+) cells that also express the CD8 genes. This is accompanied by the physical maintenance of the CD8 genes within an extended loop away from their subchromosomal territory. Further analysis of these mice revealed unexpected fluid chromatin dynamics, whereby the LCR can be initially dominant over the endogenous CD8 gene-repressive regulatory processes present in CD4(+) cells but is continuously contested by them, resulting in the eventual inactivation of the inserted LCR, probably as a result of multiple rounds of replication.
Proceedings of the National Academy of Sciences 09/2010; 107(39):16928-33. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The interplay of proliferation and differentiation is essential for normal development and organogenesis. Geminin is a cell cycle regulator which controls licensing of origins for DNA replication, safeguarding genomic stability. Geminin has also been shown to regulate cellular decisions of self-renewal versus commitment of neuronal progenitor cells. We discuss here our recent analysis of mice with conditional inactivation of the Geminin gene in the immune system. Our data indicate that Geminin is not indispensable for every cell division: in the absence of Geminin, development of progenitor T cells appears largely unaffected. In contrast, rapid cell divisions, taking place in vitro upon TCR receptor activation or in vivo during homeostatic proliferation, are defective.
[show abstract][hide abstract] ABSTRACT: Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.
Proceedings of the National Academy of Sciences 07/2010; 107(30):13414-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.
The Journal of Immunology 05/2010; 184(10):5686-95. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Stem/progenitor cells coordinate proliferation and differentiation, giving rise to appropriate cell numbers of functionally specialized cells during organogenesis. In different experimental systems, Geminin was shown to maintain progenitor cells and participate in fate determination decisions and organogenesis. Although the exact mechanisms are unclear, Geminin has been postulated to influence proliferation versus differentiation decisions. To gain insight into the in vivo role of Geminin in progenitor cell division and differentiation, we have generated mice that specifically lack Geminin in cells of lymphoid lineage through Cre-mediated recombination. T cells lacking Geminin expression upregulate early activation markers efficiently upon TCR stimulation in vitro and are able to enter the S phase of cell cycle, but show a marked defect in completing the cycle, leading to a large proportion of T cells accumulating in S/G2/M phases. Accordingly, T cells deficient in Geminin show a reduced ability to repopulate lymphopenic hosts in vivo. Contrary to expectations, Geminin deficiency does not alter development and differentiation of T cells in vivo. Our data suggest that Geminin is required for the proliferation events taking place either in vitro upon TCR receptor activation or during homeostatic expansion, but appears to be redundant for the proliferation and differentiation of the majority of progenitor T cell populations.
The Journal of Immunology 03/2010; 184(5):2432-41. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: A population of cells that expresses the NK cell receptor NKp46 and produces interleukin (IL)-22 have recently attracted considerable attention. The identity of these cells is still the subject of speculation, being variably defined as a novel NK cell subset or as a population containing conventional NK (cNK) cell precursors. In this issue, two studies shed light on this conundrum, demonstrating that NKp46(+) IL-22(+) cells and cNK cells belong to distinct lineages.
Journal of Experimental Medicine 02/2010; 207(2):269-72. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lymphoid tissues have a unique role in the organization and function of the adaptive immune system. Mechanisms driving the development of these tissues have fascinated immunologists for the last 175 years. In this review, we will initially focus on historical literature describing lymph node (LN) anlage development and then on the contemporary understanding of the molecular mechanisms driving LN and Peyer's patch (PP) formation. Utilizing transgenic reporters and gene knockout mice, the interplay between hematopoietic inducer cells and stromal organizer cells has been shown to have a key role in the development and organization of the lymphoid tissues. Although PPs and LNs share many similarities in their development, key differences in the molecular requirements for their development have recently emerged.
Progress in molecular biology and translational science 01/2010; 92:177-205. · 2.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: This chapter provides information on imaging tools that can be employed to visualise and study lymphoid organ development. We focus on the use of genetically modified mouse models that take advantage of fluorescent protein expression in discrete cell populations, thus allowing live cell imaging during lymphoid organogenesis. We discuss approaches that allow characterisation of the cell types involved in the formation of lymphoid organs, including (i) functional assays in explant organ cultures and (ii) high-resolution whole-mount immunostaining methods, which are useful for the characterisation of specific cell populations in the context of the whole developing organ.
Methods in molecular biology (Clifton, N.J.) 01/2010; 616:161-79.
[show abstract][hide abstract] ABSTRACT: In addition to progressive CD4(+) T cell immune deficiency, HIV infection is characterized by generalized immune activation, thought to arise from increased microbial exposure resulting from diminishing immunity.
Here we report that, in a virus-free mouse model, conditional ablation of activated CD4(+) T cells, the targets of immunodeficiency viruses, accelerates their turnover and produces CD4(+) T cell immune deficiency. More importantly, activated CD4(+) T cell killing also results in generalized immune activation, which is attributable to regulatory CD4(+) T cell insufficiency and preventable by regulatory CD4(+) T cell reconstitution. Immune activation in this model develops independently of microbial exposure. Furthermore, microbial translocation in mice with conditional disruption of intestinal epithelial integrity affects myeloid but not T cell homeostasis.
Although neither ablation of activated CD4(+) T cells nor disruption of intestinal epithelial integrity in mice fully reproduces every aspect of HIV-associated immune dysfunction in humans, ablation of activated CD4(+) T cells, but not disruption of intestinal epithelial integrity, approximates the two key immune alterations in HIV infection: CD4(+) T cell immune deficiency and generalized immune activation. We therefore propose activated CD4(+) T cell killing as a common etiology for both immune deficiency and activation in HIV infection.
[show abstract][hide abstract] ABSTRACT: Strong evidence is emerging that the nervous and immune systems share mechanisms of gene regulation, signaling, cell communication, and supracellular organization. This brings to the fore many questions, not least of which is the developmental and evolutionary origin of the commonalities between the two systems. By providing answers to these questions, immunologists and neurobiologists increasingly expose the mechanistic and conceptual affinities of their respective fields and facilitate the understanding of fundamental principles that govern the organization of complex cellular systems. The current essay and reviews in Immunity and Neuron attempt to communicate to the wider scientific community a series of examples relating to commonalities between the immune and nervous system and enhance the dialog and exchange of ideas between the two fields.
[show abstract][hide abstract] ABSTRACT: Glucocorticoids have significant immunoregulatory actions on thymocytes and T cells and act by binding and activating cytosolic glucocorticoid receptors, which translocate to the nucleus and control gene expression through binding to specific response elements in target genes. Glucocorticoids promote cell death by activating an apoptotic program that requires transcriptional regulation. We set out to identify genes that are crucial to the process of glucocorticoid-mediated thymocyte apoptosis. Freshly isolated murine primary thymocytes were treated with dexamethasone, mRNA isolated and used to screen DNA microarrays. A set of candidate genes with upregulated expression was identified and selected members assayed in reconstituted fetal thymic organ culture (FTOC). Fetal liver-derived hematopoietic progenitor cells (HPCs) were infected with retroviruses expressing individual genes then used to repopulate depleted fetal thymic lobes. Reconstituted FTOCs expressing the gene Tnfaip8 were treated with dexamethasone and shown to be greatly sensitized to dexamethasone. Retrovirus-mediated RNA interference was applied to knock down Tnfaip8 expression in HPCs and these were used to reconstitute FTOCs. We observed that downregulating the expression of Tnfaip8 alone was sufficient to effectively protect thymocytes against glucocorticoid-induced apoptosis. We propose that Tnfaip8 is crucial in regulating glucocorticoid-mediated apoptosis of thymocytes.
Cell death and differentiation 10/2009; 17(2):316-23. · 8.24 Impact Factor