[Show abstract][Hide abstract] ABSTRACT: Reciprocal defects in signaling between the myotome and the sclerotome compartments of the somites in PDGFRalpha and Myf5 mutant embryos lead to alterations in the formation of the vertebrae and the ribs. To investigate the significance of these observations, we have examined the role of PDGF signaling in the developing somite. PDGFA ligand expression was not detected in the myotome of Myf5 null mutant embryos and PDGFA promoter activity was regulated by Myf5 in vitro. PDGFA stimulated chondrogenesis in somite micromass cultures as well as in embryos when PDGFA was knocked into the Myf5 locus, resulting in increased vertebral and rib development. PDGFA expression in the myotome was fully restored in embryos in which MyoD has been introduced at the Myf5 locus but to a lesser extent in similar myogenin knock-in embryos. These results underscore the importance of growth factor signaling within the developing somite and suggest an important role for myogenic determination factors in orchestrating normal development of the axial skeleton.
[Show abstract][Hide abstract] ABSTRACT: Signal transduction by the platelet-derived growth-factor receptor beta (PDGFR-beta) tyrosine kinase is required for proper formation of vascular smooth muscle cells (VSMC). However, the importance of individual PDGFR-beta signal transduction pathways in vivo is not known. To investigate the role of two of the pathways believed to be critical for PDGF signal transduction, we have generated mice that bear a PDGFR-beta that can no longer activate PI3kinase or PLCgamma. Although these mutant mice have normal vasculature, we provide multiple lines of evidence in vivo and from cells derived from the mutant mice that suggest that the mutant PDGFR-beta operates at suboptimal levels. Our observations indicate that although loss of these pathways can lead to attenuated PDGF-dependent cellular function, certain PDGFR-beta-induced signal cascades are not essential for survival in mice.
No preview · Article · Jan 2001 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.
Full-text · Article · Jul 2000 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel's cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.
[Show abstract][Hide abstract] ABSTRACT: Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development.
Full-text · Article · Jan 2000 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: The action of growth factors is thought to make a substantial contribution to the events leading to proliferative vitreoretinopathy (PVR). In this study, the importance of platelet-derived growth factor (PDGF) was tested in a rabbit model of PVR.
The approach was to compare the extent of PVR induced by cells that do or do not express the receptors for PDGF and therefore differ in their ability to respond to PDGF.
Mouse embryo fibroblasts derived from PDGF receptor knock-out embryos that do not express either of the two PDGF receptors induced PVR poorly when injected into the eyes of rabbits that had previously undergone gas vitrectomy. Re-expression of the PDGF beta receptor in these cells did not improve the ability of the cells to cause PVR. In contrast, injection of cells expressing the PDGF alpha receptor resulted in stage 3 or higher PVR in 8 of 10 animals.
These findings show that PDGF makes an important contribution to the development of PVR in this animal model. Furthermore, there is a marked difference between the two receptors for PDGF, and it is the PDGF alpha receptor that is capable of driving events that lead to PVR.
[Show abstract][Hide abstract] ABSTRACT: Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene. Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.
Preview · Article · Jul 1999 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Kidney glomerulus mesangial cells fail to develop in mice carrying targeted null mutations in the platelet-derived growth factor (PDGF)-B or PDGF-Rbeta genes. We have examined the pattern of expression of these genes and smooth muscle markers during kidney development, to address the possible mechanisms underlying the mutant phenotypes. In wild-type embryos, PDGF-B was expressed in vascular endothelial cells, particularly in capillary endothelial cells in the developing glomeruli, whereas PDGF-Rbeta was found in perivascular mesenchymal cells in the developing renal cortex. In the course of glomerular development, small groups of PDGF-Rbeta and desmin-expressing cells collected in the 'S'-shaped and early cup-shaped vesicles, and at later stages such cells were found in the glomerular mesangium. In PDGF-B or -Rbeta null embryos, some PDGF-Rbeta/desmin or desmin-positive cells, respectively, were seen in early cup-shaped vesicles, but fewer than in the wild type, and further development of the mesangium failed. In mouse chimeras composed of PDGF-Rbeta +/+ and -/- cells, the Rbeta-/- cells failed to populate the glomerular mesangium. Our results show that while the mesangial cell lineage is specified independently of PDGF-B/Rbeta, these molecules provide critical permissive signals in mesangial cell development. We propose a model in which mesangial cells originate from PDGF-Rbeta-positive progenitors surrounding the developing glomerular afferent and efferent arterioles, and are co-recruited in response to PDGF-B during angiogenic formation of the glomerular capillary tuft.
[Show abstract][Hide abstract] ABSTRACT: L1 is a neural cell adhesion molecule mainly involved in axon guidance and neuronal migration during brain development. Mutations in the human L1 gene give rise to a complex clinical picture, with mental retardation, neurologic abnormalities and a variable degree of hydrocephalus. Recently, a transgenic mouse model with a targeted null mutation in the L1 gene was generated. These knockout (KO) mice show hypoplasia of the corticospinal tract. Here we have performed further studies of these KO mice including magnetic resonance imaging of the brain, neuropathological analysis and behavioral testing. The ventricular system was shown to be abnormal with dilatation of the lateral ventricles and the 4th ventricle, and an altered shape of the Sylvius aqueduct. Additionally, the cerebellar vermis of the KO mice is hypoplastic. Their exploratory behavior is characterized by stereotype peripheral circling reminiscent of that of rodents with induced cerebellar lesions.
Full-text · Article · Jul 1998 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, we disrupted the Mad1 gene by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals revealed that cell cycle exit of granulocytic precursors was inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes were essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes was correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. In addition, recovery of the peripheral granulocyte compartment following bone marrow ablation was significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, were found to be expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation.
[Show abstract][Hide abstract] ABSTRACT: Neural cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been implicated in both the fasciculation and guidance of axons, but direct genetic evidence of a role for neural IgCAMs in axon guidance in vertebrates is lacking. The L1 subfamily of vertebrate neural IgCAMs function as both homophilic and heterophilic receptors for a variety of cell-surface and extracellular ligands and may signal through intracellular kinases or by recruitment of the fibroblast growth factor receptor. L1 itself has been implicated in many neural processes and is expressed widely in the embryonic and adult nervous systems. In humans, mutations in the L1 gene are linked with a spectrum of brain disorders, including loss of the corticospinal tract, but the mechanistic basis for these disorders is unknown.
We show that mice that do not express L1 have defects in the guidance of axons of the corticospinal tract, a major motor control pathway projecting from the cortex to the spinal cord. Although the pathway to the caudal medulla appears normal, a substantial proportion of axons fail to cross the midline to the opposite dorsal column as normal. In adults, this results in a reduced decussation and in large numbers of axons projecting ipsilaterally. There is also a varying, but reduced, number of corticospinal axons in the dorsal columns of the spinal cord. These do not project beyond cervical levels. We show that these are defects in axon guidance, because they arise during the early stages of the development of the decussation. The presence of a ligand for L1, CD24, specifically at the point of decussation suggests a mechanism in which L1 functions to guide corticospinal axons across the midline.
L1 function is necessary for the guidance of corticospinal axons across the pyramidal decussation in mice. Some of the defects in the corticospinal tract of humans with mutations in L1 could be due to errors in axon guidance at the pyramidal decussation.
[Show abstract][Hide abstract] ABSTRACT: Platelet-derived growth factors (PDGFs) have been implicated in the control of cell proliferation, survival and migration. Patch mutant mice harbor a deletion including the PDGF alpha receptor gene and exhibit defects of neural crest origin which affect pigmentation in heterozygotes and cranial bones in homozygotes. To verify the role of the PDGF alphaR gene during development, mice carrying a targeted null mutation were generated. No pigmentation phenotype was observed in heterozygotes. Homozygotes die during embryonic development and exhibit incomplete cephalic closure similar to that observed in a subset of Patch mutants. In addition, increased apoptosis was observed on pathways followed by migrating neural crest cells. However, alterations in mutant vertebrae, ribs and sternum were also observed, which appear to stem from a deficiency in myotome formation. These results indicate that PDGFs may exert their functions during early embryogenesis by affecting cell survival and patterning.
[Show abstract][Hide abstract] ABSTRACT: Expression of the Src-family kinases--Src, Hck, and Fgr--increases dramatically during myeloid cell development. Src-deficient mice exhibit functional abnormalities in only one myeloid cell type, the osteoclast, resulting in impaired bone remodeling and osteopetrosis, while hck-/- or fgr-/- mice have few and subtle myeloid cell deficiencies. To determine whether these limited phenotypes are due to the coexpression of multiple Src-family kinases with overlapping functions, we have intercrossed src-/- mice to hck-/- and fgr-/- mutants to produce double mutants. Two thirds of hck-/- src-/- double mutants die at birth; surviving animals develop a severe form of osteopetrosis, resulting in extreme levels of splenic extramedullary hematopoiesis, anemia, and leukopenia. These hematopoietic defects are caused by abnormalities in the bone marrow environment because hck-/- src-/- mutant stem cells reconstitute a normal hematopoietic system in irradiated wild-type mice. In contrast, fgr-/- src-/- double mutants have no defects beyond those observed in src-/- animals. Cultured normal murine osteoclasts express abundant amounts of Src, Hck, and Fgr and Hck levels are increased in src-/- osteoclasts. These observations suggest that Hck and Src serve partially overlapping functions in osteoclasts and that the expression of Hck in src-/- osteoclasts ameliorates their functional defects.
[Show abstract][Hide abstract] ABSTRACT: Mouse primordial germ cells express tissue non-specific alkaline phosphatase (TNAP) during development, but the widespread expression of another alkaline phosphatase gene in the early embryo limits the potential use of this marker to trace germ cells. To attempt to identify germ cells at all stages during embryonic development and to understand the role of TNAP in germ cell ontogeny, mice carrying a beta geo (lacZ/neor) disrupted allele of the TNAP gene were generated by homologous recombination in embryonic stem cells. Using beta-galactosidase activity, the embryonic pattern of TNAP expression was examined from the blastocyst stage to embryonic day 14. Results indicate that primordial germ cell progenitors do not express TNAP prior to gastrulation although at earlier times TNAP expression is found in an extraembryonic lineage destined to form the chorion. In homozygous mutants, primordial germ cells appear unaffected indicating that TNAP is not essential for their development or migration.
[Show abstract][Hide abstract] ABSTRACT: Src-related nonreceptor protein tyrosine kinases in nerve growth cones (p59fyn, pp60c-src, and pp62c-yes) are potential intracellular signaling molecules for cell adhesion molecule-directed axonal growth. To determine whether src-related tyrosine kinases mediate NCAM-dependent neurite outgrowth, cultures of cerebellar and sensory neurons from fyn-, src-, and yes- minus mice were analyzed for neurite outgrowth on monolayers of NCAM140-transfected L fibroblasts. NCAM-dependent neurite outgrowth was selectively inhibited in cultures of cerebellar and dorsal root ganglion neurons from fyn-, but not src- or yes- mice. Neurite outgrowth by fyn-, src-, or yes- neurons on untransfected fibroblast monolayers was unaffected, indicating that these kinases do not contribute significantly to axon growth on at least some integrins or other adhesive substrates present on fibroblasts. This study demonstrates that p59fyn is an essential component of the NCAM signaling pathway leading to axonal growth.
Preview · Article · Dec 1994 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: We have used a retroviral gene trap in embryonic stem (ES) cells to derive a recessive embryonic lethal mouse strain, ROSA beta-geo5. Mutant embryos display an enlarged pericardial cavity, bradycardia, a dilated fourth ventricle in the brain, and die between embryonic days 11 and 12. Whereas heart development in the mutant embryos is extensive, the ventricular wall is abnormally thin with a reduced number of trabeculae. Cloning of the trapped gene indicates that proviral insertion creates a null mutation in the transcriptional enhancer factor 1 (TEF-1) gene. Although transcription of a number of muscle-specific genes believed to be TEF-1 targets appears normal, the defect in cardiogenesis is likely attributable to diminished transcription of one or several cardiac-specific genes.
Preview · Article · Nov 1994 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Three members of the Src family of tyrosine kinases, src, fyn, and yes, are broadly expressed throughout mouse development. Mutations in the c-src and fyn genes were shown previously to lead to restricted nonoverlapping phenotypes only in a subset of cells in which these kinases are expressed. In this work we show that a mutation in the yes gene does not lead to an overt phenotype. Except for brain, the level or distribution of related kinases is not altered in major tissues. To gain further insight into the possibility that these kinases compensate for each other, animals deficient in multiple src-kinases were generated. Whereas most of the src/fyn or src/yes double mutants die perinatally, a substantial proportion of fyn/yes double mutants are viable but undergo degenerative renal changes leading to diffuse segmental glomerulosclerosis. Taken together, these data are consistent with the hypothesis that, at least in some cells, these kinases are able to compensate for the loss of the other related kinases.
Full-text · Article · Oct 1994 · Genes & Development