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J Rossant

Publications and source records attributed to J Rossant.

At least 73 records · Page 4Linked to original sources
PubMed1996

A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain.

Mouse Otx2 is a bicoid-class homeobox gene, related to the Drosophila orthodenticle (otd) gene. Expression of this gene is initially widespread in the epiblast at embryonic day 5.5 but becomes progressively restricted to the anterior end of the embryo at the headfold stage. In flies, loss of function mutations in otd result in deletion of pre-antennal and antennal segments; which leads to the absence of head structures derived from these segments. To study the function of Otx2 in mice, we have generated a homeobox deletion mutation in this gene. Mice homozygous for this mutation show severe defects in gastrulation and in formation of axial mesoderm and loss of anterior neural tissues. These results demonstrate that Otx2 is required for proper development of the epiblast and patterning of the anterior brain in mice, and supports the idea of evolutionary conservation of the function of Otd/Otx genes in head development in flies and mice.

Animals↗
PubMed1996

Cell autonomous functions of the receptor tyrosine kinase TIE in a late phase of angiogenic capillary growth and endothelial cell survival during murine development.

TIE is a receptor tyrosine kinase expressed in both mature endothelial cells and their precursors, as well as in some hematopoietic cells. Mouse embryos homozygous for a disrupted Tie allele die at midgestation due to impaired endothelial cell integrity and resulting hemorrhage. Here we have performed chimeric analysis to study further the function of the murine TIE in the development of embryonic vasculature and in the hematopoietic system. Cells lacking a functional Tie gene (tie(lcz)/tie(lczn-) cells) contributed to the embryonic vasculature at E10.5 as efficiently as cells heterozygous for a targeted Tie allele (tie(lcz)/+ cells). Thus, TIE does not play a significant role in vasculogenesis or in early angiogenic processes, such as formation of the intersomitic arteries and limb bud vascularization. At E15.5 tie(lcz)/tie(lczn-) cells still readily contributed to major blood vessels and to endothelial cells of organs such as lung and heart, which have been suggested to be vascularized by angioblast differentiation. In contrast, the tie(lcz)/tie(lczn-) cells were selected against in the capillary plexuses of several angiogenically vascularized tissues, such as brain and kidney. Our results thus support a role for TIE in late phases of angiogenesis but not vasculogenesis. Furthermore, the results suggest that different mechanisms regulate early and late angiogenesis and provide support for a model of differential organ vascularization by vasculogenic or angiogenic processes. Analysis of adult chimeras suggested that TIE is required to support the survival or proliferation of certain types of endothelial cells demonstrating heterogeneity in the growth/survival factor requirements in various endothelial cell populations. Chimeric analysis of adult hematopoietic cell populations, including peripheral platelets and bone marrow progenitor cells, revealed that tie(lcz)/tie(lczn-) cells were able to contribute to these cell types in a way indistinguishable from tie(lcz)/+ or wild-type cells. Thus, the primary function of TIE appears to be restricted to the endothelial cell lineage.

Animals↗
PubMed1995

The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells.

Vascular endothelial cells are critical for the development and function of the mammalian circulatory system. We have analyzed the role of the endothelial cell-specific receptor tyrosine kinase TIE in the mouse vasculature. Mouse embryos homozygous for a disrupted Tie allele developed severe edema, their microvasculature was ruptured and they died between days 13.5 and 14.5 of gestation. The major blood vessels of the homozygous embryos appeared normal. Cells lacking a functional Tie gene were unable to contribute to the adult kidney endothelium in chimeric animals, further demonstrating the intrinsic requirement for TIE in endothelial cells. We conclude that TIE is required during embryonic development for the integrity and survival of vascular endothelial cells, particularly in the regions undergoing angiogenic growth of capillaries. TIE is not essential, however, for vasculogenesis, the early differentiation of endothelial cells.

Animals↗
PubMed1995

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

Animals↗
PubMed1995

Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.

The vascular endothelial growth factor (VEGF) and its high-affinity binding receptors, the tyrosine kinases Flt-1 and Flk-1, are thought to be important for the development of embryonic vasculature. Here we report that Flt-1 is essential for the organization of embryonic vasculature, but is not essential for endothelial cell differentiation. Mouse embryos homozygous for a targeted mutation in the flt-1 locus, flt-1lcz, formed endothelial cells in both embryonic and extra-embryonic regions, but assembled these cells into abnormal vascular channels and died in utero at mid-somite stages. At earlier stages, the blood islands of flt-1lcz homozygotes were abnormal, with angioblasts in the interior as well as on the periphery. We suggest that the Flt-1 signalling pathway may regulate normal endothelial cell-cell or cell-matrix interactions during vascular development.

Animals↗
PubMed1995

Cardiac malformation in neonatal mice lacking connexin43.

Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

Animals↗
PubMed1995

Identification of two regulatory elements within the promoter region of the mouse connexin 43 gene.

To define the minimal sequences required for expression of the connexin 43 gene (cx43) in myometrial cells, we generated 5' deletion constructs of a fragment extending 1686 base pairs upstream and 162 base pairs downstream of the transcription start site and determined their ability to drive expression of the chloramphenicol acetyltransferase reporter gene in transfected myometrial cell lines. Our investigation revealed two cis-acting regulatory elements within this fragment. Deletion of a region extending from -102 to -92 led to an increase of the promoter activity by greater than 10-fold, indicating a presence of a repressor element. Deletion of a region extending from -72 to -62 caused a decrease of the promoter activity of a similar extent, implying the existence of a positive element. Electrophoretic mobility shift assays demonstrated that synthetic oligonucleotides derived from these two small regions can each bind with a nuclear protein(s) prepared from myometrial cells, and an introduction of three and two base substitutions into each of these oligomers was sufficient to abolish their protein binding capability. These same mutations, when incorporated in the chloramphenicol acetyltransferase constructs, diminished regulatory functions of the negative and positive elements, and the protein(s) that bind to these functional elements was found in several tissues known to express cx43 gene.

Animals↗
PubMed1995

Characterization of a gene trap insertion into a novel gene, cordon-bleu, expressed in axial structures of the gastrulating mouse embryo.

We have used a gene trap (GT) vector and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. GT vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. In this paper we describe a GT insertion which displays a very restricted pattern of expression in the gastrulating embryo. beta-Galactosidase activity was first detected at 7.5 days post-coitum (E7.5) in the node region of the embryo and extended to the midline structures at E8.0. At E9.5 expression was restricted to the floor plate, the notochord, the roof of the gut, and the liver anlage. Expression appeared in the somites at E10.0 and later became more widespread. We used rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to clone a partial 360 base pair (bp) cDNA representing an endogenous sequence and containing an open reading frame (ORF) fused in frame to the lacZ reporter gene. The sequence showed no homology to any known protein or protein domain. An overlapping 1,200 bp fragment from a wild-type cDNA library was cloned and it detected the same pattern of expression as the reporter gene in E7.5, E8.5, and E9.5 wild-type embryos. It hybridized to a 5.4 kb lacZ fusion transcript and to an endogenous transcript of 6.5 kb. The gene was mapped to chromosome 11 and was named cordon-bleu (cobl). No phenotype was detected in mice homozygous for the insertion. However, the insertion may not cause a complete disruption of the gene function. The pattern of expression of cobl is very similar to that of hepatic nuclear factor 3 beta (HNF3 beta) and sonic hedgehog (Shh), both of which are involved in axial patterning. Therefore, the product of the cobl gene may also prove to be an important component of the genetic pathway regulating vertebrate axis formation.

Amino Acid Sequence↗
PubMed1995

A novel regulatory region is required for trophoblast-specific transcription in transgenic mice.

The trophectoderm epithelium of the blastocyst is the first tissue to differentiate in the mammalian embryo and gives rise to all cells of the trophoblast lineage. These cells form the majority of the fetal component of the placenta, which is critical for intrauterine development. Our interest in the molecular determinants of trophoblast lineage development has led us to undertake an analysis of the regulatory region of a trophoblast-specific gene termed 4311. The transcription start site was mapped by both primer extension and RNase protection analyses. We show that cloned 4311 upstream sequences are able to confer the correct temporal and spatial expression pattern of 4311 on a reporter gene in transgenic mice. In addition, we have characterized a 340-bp region, 3.7 kb upstream of the transcription start, that is sufficient for the specific expression of lacZ in cells of the developing spongiotrophoblast in transgenic mice. Delineation of a diploid trophoblast-specific regulatory region will facilitate the identification of factors that may regulate the development of this lineage.

Animals↗
PubMed1995

Mice lacking all isoforms of retinoic acid receptor beta develop normally and are susceptible to the teratogenic effects of retinoic acid.

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RAR alpha, beta, and gamma. The expression of RAR beta gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RAR beta could play specific roles during morphogenesis. Four isoforms of the RAR beta gene (beta 1-beta 4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RAR beta 2 isoform is dispensable for normal development. To ascertain the function of all RAR beta isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RAR beta gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1/11 RAR beta mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RAR beta null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RAR beta may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RAR beta isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

Abnormalities, Drug-Induced↗
PubMed1995

Fibroblast growth factors in mammalian development.

Polypeptide growth factors are secreted signalling molecules that function as intercellular communicators. Detailed analyses of the expression and function of members of the fibroblast growth factor (FGF) family and their recepotors have demonstrated that the FGF signalling pathways play essential roles in regulating cellular proliferation, differentiation and tissue patterning during vertebrate embryogenesis. Recent studies on the molecular basis of human dysmorphic syndromes have revealed that aberrant FGF signalling during limb and skeletal development can lead to pathogenesis.

Achondroplasia↗
PubMed1995

Genomic imprinting of Mash2, a mouse gene required for trophoblast development.

The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2-homozygous mutant embryos die at 10 days postcoitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/- embryos inheriting a wild-type allele from their father die at the same stage as -/- embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post-coitum, but appears almost completely repressed by 8.5 days post-coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2 and H19.

Alleles↗
PubMed1995

The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1.

Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We describe cloning of a candidate dt gene, dystonin, that is predominantly expressed in the dorsal root ganglia and other sites of neurodegeneration in dt mice. Dystonin encodes an N-terminal actin binding domain and a C-terminal portion comprised of the hemidesmosomal protein, bullous pemphigoid antigen 1 (bpag1). dt and bpag1 are part of the same transcription unit which is partially deleted in a transgenic strain of mice, Tg4, that harbours an insertional mutation at the dt locus, and in mice that carry a spontaneous dt mutation, dtAlb. We also demonstrate abnormal dystonin transcripts in a second dt mutant, dt24J. We conclude that mutations in the dystonin gene are the primary genetic lesion in dt mice.

Amino Acid Sequence↗
PubMed1995

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.

We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.

Animals↗
PubMed1995

Postaxial polydactyly in forelimbs of CRABP-II mutant mice.

The cytoplasmic retinoic acid (RA)-binding protein CRABP-II is expressed widely throughout early morphogenesis in mouse embryo, but its expression becomes more restricted as organogenesis progresses. CRABP-II expression remains strong in the developing limb bud suggesting a role for this protein in limb patterning. Here, we show that the CRABP-II promoter can direct expression of a lacZ transgene in a specific posterior domain during limb bud development. In order to investigate in more detail the role played by CRABP-II in RA signal transduction, we have also generated mice homozygous for a null mutation of this gene. CRABPII-/- mice are viable and fertile but show a developmental defect of the forelimb, specifically an additional, postaxial digit. This digit is generally, but not exclusively, limited to a single forepaw of an individual animal. The penetrance of the phenotype varies according to the genetic background, occurring most frequently on the inbred 129Sv background (50%), less frequently on the C57Bl/6 background (30%) and rarely on the outbred CD1 background (10%). This developmental abnormality implies a role for CRABP-II in normal patterning of the limb.

Animals↗
PubMed1995

Notch1 is required for the coordinate segmentation of somites.

Members of the Notch family of transmembrane receptors mediate a number of developmental decisions in invertebrates. In order to study Notch function in a vertebrate organism, we have mutated the Notch1 gene of the mouse. Notch1 gene function is required for embryonic survival in the second half of gestation. In the first half of gestation, we have found no effect of the mutation on the normal programs of neurogenesis, myogenesis or apoptosis. We conclude that Notch1 function is not essential for these processes, at least in early postimplantation development. However, we have found that somitogenesis is delayed and disorganized in Notch1 mutant embryos. We propose that Notch1 normally coordinates the process of somitogenesis, and we provide a model of how this might occur.

Animals↗
PubMed1994

fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation.

Experiments in amphibians have implicated fibroblast growth factors (FGFs) in the generation and patterning of mesoderm during embryogenesis. We have mutated the gene for fibroblast growth factor receptor 1 (fgfr-1) in the mouse to genetically dissect the role of FGF signaling during development. In the absence of fgfr-1 signaling, embryos displayed early growth defects; however, they remained capable of gastrulating and generating mesoderm. The nascent mesoderm of fgfr-1 homozygous mutant embryos differentiated into diverse mesodermal subtypes, but mesodermal patterning was aberrant. Somites were never generated and axial mesoderm was greatly expanded at the expense of paraxial mesoderm. These results suggest that FGFR-1 transduces signals that specify mesodermal cell fates and regional patterning of the mesoderm during gastrulation.

Animals↗