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J Rossant

Publications and source records attributed to J Rossant.

At least 55 records · Page 3Linked to original sources
PubMed1998

Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles.

The N-myc proto-oncogene is expressed in many organs of the mouse embryo, suggesting that it has multiple functions. A null mutation leads to mid-gestation lethality [1-4], obscuring the later roles of the gene in organogenesis. We have generated a multi-purpose gene alteration by combining the potential for homologous and site-specific recombination in a single targeting vector, and using the selectable marker for neomycin-resistance, neo, to downregulate gene activity. This allowed us to create a series of alleles that led to different levels of N-myc expression. The phenotypes revealed a spectrum of developmental problems. The hypomorphic allele produced can be repaired in situ by Cre-recombinase-mediated DNA excision. We show here for the first time the use of a single targeting vector to generate an allelic series. This, and the possibility of subsequent lineage-specific or conditional allele repair in situ, represent new genome modification strategies that can be used to investigate multiple functions of a single gene.

Alleles↗
PubMed1998

The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo.

Mutations in the SMAD4/DPC4 tumor suppressor gene, a key signal transducer in most TGFbeta-related pathways, are involved in 50% of pancreatic cancers. Homozygous Smad4 mutant mice die before day 7.5 of embryogenesis. Mutant embryos have reduced size, fail to gastrulate or express a mesodermal marker, and show abnormal visceral endoderm development. Growth retardation of the Smad4-deficient embryos results from reduced cell proliferation rather than increased apoptosis. Aggregation of mutant Smad4 ES cells with wild-type tetraploid morulae rescues the gastrulation defect. These results indicate that Smad4 is initially required for the differentiation of the visceral endoderm and that the gastrulation defect in the epiblast is secondary and non-cell autonomous. Rescued embryos show severe anterior truncations, indicating a second important role for Smad4 in anterior patterning during embryogenesis.

Alleles↗
PubMed1998

Chimeras and mosaics in mouse mutant analysis.

As the number of mouse mutants generated by gene targeting continues to grow exponentially, the challenge is not how to generate a mutant but how to analyse the phenotype. Genes might play multiple roles in development and act in cell-autonomous and cell non-autonomous modes, making phenotypic analysis complex. Genetic mosaic analysis is a powerful tool for dissecting complex gene functions. Classical preimplantation chimeras made between mutant and wild-type embryos can answer many questions, and new genetic techniques for generating restricted genetic mosaicism promise to enhance the future power of mosaic analysis in mammals.

Animals↗
PubMed1998

Mash2 is expressed in oogenesis and preimplantation development but is not required for blastocyst formation.

The basic helix-loop-helix transcription factor, Mash2, has been shown to be necessary for the development of the spongiotrophoblast of the mature chorioallantoic placenta of the mouse. Here we show that Mash2 is transcribed during oogenesis and expressed throughout preimplantation development, only becoming restricted to the diploid trophoblast around the time of implantation. This expression raised the possibility that Mash2 has earlier functions in the trophoblast lineage that were not detectable in mutant embryos because of the persistence of oogenetically derived protein. This was tested by generating viable Mash2-/- females by tetraploid rescue of the extraembryonic defect. Mutant embryos derived from such females showed no enhanced phenotype over embryos produced from heterozygous females, demonstrating unequivocally that neither maternal nor zygotic Mash2 is required for early trophoblast development. If Mash2 functions in other aspects of trophoblast development, it must act cooperatively with other factors.

Animals↗
PubMed1998

Diminished Sonic hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice.

Floor plate cells at the midline of the neural tube are specified by high-level activity of Sonic hedgehog (Shh) secreted by notochord, whereas motor neurons are thought to be specified by a lower level activity of Shh secreted in turn by floor plate cells. In Drosophila, the Gli zinc finger protein Cubitus interruptus functions as a transcription factor activating Hedgehog-responsive genes. We report that the expression of known Shh-responsive genes such as Ptc and Gli1 is downregulated in mutant mice lacking Gli2 function. Gli2 mutants fail to develop a floor plate yet still develop motor neurons, which occupy the ventral midline of the neural tube. Our results imply that Gli2 is required to mediate high level but not low level Shh activity and show that the development of motor neurons can occur in the absence of floor plate induction.

Animals↗
PubMed1998

The transcription factor HNF3beta is required in visceral endoderm for normal primitive streak morphogenesis.

During early embryogenesis, the transcription factor HNF3beta is expressed in visceral and definitive endoderm, node, notochord and floorplate. A targeted mutation in the HNF3&bgr ; gene results in the lack of a definitive node and notochord. Furthermore, lack of HNF3beta results in failure of proper primitive streak elongation. To address whether HNF3beta is required in visceral endoderm, we have used tetraploid embryo-ES cell aggregations to generate chimeric mouse embryos with wild-type visceral endoderm and homozygous mutant HNF3beta embryonic ectoderm or vice versa. Replacing the visceral endoderm of mutant HNF3beta embryos rescued proper primitive streak elongation and, conversely, mutant visceral endoderm imposed a severe embryonic-extraembryonic constriction on wild-type embryonic ectoderm. Restoration of normal streak morphogenesis was not sufficient to allow formation of the node and notochord in HNF3beta mutant embryos. Thus, our results demonstrate that HNF3beta has two separate roles in primitive streak formation. One is to act within the visceral endoderm to promote proper streak morphogenesis. The second is autonomous to the node and its precursors and involves specification of node and notochord cell fates. HNF3beta mutant embryos rescued for the embryonic-extraembryonic constriction developed further than mutant embryos, allowing examination of later roles for HNF3beta. We show that such mutant embryos lack foregut and midgut endoderm. In addition, left-right asymmetry is affected in the mutant embryos.

Animals↗
PubMed1997

Mash2 acts cell autonomously in mouse spongiotrophoblast development.

The Mash2 gene, which encodes a basic helix-loop-helix transcription factor, is one of the mammalian homologues of the Drosophila achaete-scute genes. It is strongly expressed in diploid trophoblast cells of the postimplantation mouse embryo. Targeted mutagenesis of Mash2 revealed that loss of function results in embryonic lethality at midgestation, due to placental failure associated with a lack of spongiotrophoblast and reduced labyrinthine trophoblast layers. For the further study of Mash2 function in development of the trophoblast cell lineage, we have performed chimeric analysis combining Mash2 mutant and wild-type embryos. We have addressed the question of whether the phenotype of the Mash2 mutant embryo, which affects all of the three trophoblast cell layers, is caused by a cell autonomous or non-autonomous defect and whether Mash2 is required in both spongiotrophoblast and labyrinthine trophoblast development. Our results showed no contribution of Mash2 mutant cells to the spongiotrophoblast layer in chimeric placentae at 10.5 and 12.5 days postcoitum, suggesting that the product of the Mash2 gene is required cell autonomously during the development of the spongiotrophoblast. However, it seems that Mash2 is not required for development of labyrinthine trophoblast or giant cells, since high contributions of Mash2 mutant cells were observed in those trophoblast cell layers in the chimeric placentae analyzed. We can therefore conclude that the primary and cell-autonomous function of Mash2 appears to be an involvement in the development of diploid trophoblast cells in the ectoplacental cone to form the spongiotrophoblast cell layer of the mature chorioallantoic placenta.

Animals↗
PubMed1997

Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta.

Classical endocrine studies have shown that steroid hormones are required for the maintenance of pregnancy and placental viability. The oestrogen-receptor-related receptor beta (ERR-beta) is an orphan member of the superfamily of nuclear hormone receptors. Although ERR-beta is homologous to the oestrogen receptor and binds the oestrogen response element, it is not activated by oestrogens. Expression of ERR-beta during embryogenesis defines a subset of extra-embryonic ectoderm that subsequently forms the dome of the chorion, suggesting that ERR-beta may be involved in early placental development. Homozygous mutant embryos generated by targeted disruption of the Estrrb gene have severely impaired placental formation, and die at 10.5 days post-coitum. The mutants display abnormal chorion development associated with an overabundance of trophoblast giant cells and a severe deficiency of diploid trophoblast. The phenotype can be rescued by aggregation of Estrrb mutant embryos with tetraploid wild-type cells, which contribute exclusively to extra-embryonic tissues. Our results indicate that ERR-beta has an important role in early placentation, and suggest that an inductive signal originating from or modified by the chorion is required for normal trophoblast proliferation and differentiation.

Animals↗
PubMed1997

A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.

Mouse embryos lacking the receptor tyrosine kinase, Flk1, die without mature endothelial and hematopoietic cells. To investigate the role of Flk1 during vasculogenesis and hematopoiesis, we examined the developmental potential of Flk1-/- embryonic stem cells in chimeras. We show that Flk1 is required cell autonomously for endothelial development. Furthermore, Flk1-/- cells do not contribute to primitive hematopoiesis in chimeric yolk sacs or definitive hematopoiesis in adult chimeras and chimeric fetal livers. We also demonstrate that cells lacking Flk1 are unable to reach the correct location to form blood islands, suggesting that Flk1 is involved in the movement of cells from the posterior primitive streak to the yolk sac and, possibly, to the intraembryonic sites of early hematopoiesis.

Amnion↗
PubMed1997

Elements both 5' and 3' to the murine Hoxd4 gene establish anterior borders of expression in mesoderm and neurectoderm.

In this report, we show that a lacZ reporter spanning 12.5 kb of murine Hoxd4 genomic DNA contains the major regulatory elements controlling Hoxd4 expression in the mouse embryo. Mutational analysis revealed multiple regulatory regions both 5' and 3' to the coding region. These include a 3' enhancer region required for expression in the central nervous system (CNS) and setting the anterior border in the paraxial mesoderm, and a 5' mesodermal enhancer that directs expression in paraxial and lateral plate mesoderm. A previously defined retinoic acid response element (RARE) is a component of the 5' mesodermal enhancer. Our results support a model in which retinoic acid receptors (RARs) and HOX proteins mediate the initiation and maintenance of Hoxd4 expression.

Animals↗
PubMed1997

Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement through the primitive streak.

Fibroblast growth factor (FGF) signaling has been implicated in the patterning of mesoderm and neural lineages during early vertebrate development. In the mouse, FGF receptor-1 (FGFR1) is expressed in an appropriate spatial and temporal manner to be orchestrating these functions. Mouse embryos homozygous for a mutated Fgfr1 allele (fgfr1(delta tmk)) die early in development, show abnormal growth and aberrant mesodermal patterning. We have performed a chimeric analysis to further study FGFR1 function in the morphogenesis and patterning of the mesodermal germ layer at gastrulation. At E9.5, fgfr1(delta tmk)/fgfr1(delta tmk) cells showed a marked deficiency in their ability to contribute to the extra-embryonic, cephalic, heart, axial and paraxial mesoderm, and to the endoderm of chimeric embryos. Analysis at earlier stages of development revealed that fgfr1(delta tmk)/fgfr1(delta tmk) cells accumulated within the primitive streak of chimeric embryos, and consequently failed to populate the anterior mesoderm and endodermal lineages at their inception. We suggest that the primary defect associated with the fgfr1(delta tmk) mutation is a deficiency in the ability of epiblast cells to traverse the primitive streak. fgfr1(delta tmk)/fgfr1(delta tmk) cells that accumulated within the primitive streak of chimeric embryos tended to form secondary neural tubes. These secondary neural tubes were entirely fgfr1(delta tmk)/fgfr1(delta tmk) cell derived. The adoption of ectopic neural fate suggests that normal morphogenetic movement through the streak is essential not only for proper mesodermal patterning but also for correct determination of mesodermal/neurectodermal cell fates.

Animals↗
PubMed1997

Conservation of the Notch signalling pathway in mammalian neurogenesis.

The Notch pathway functions in multiple cell fate determination processes in invertebrate embryos, including the decision between the neuroblast and epidermoblast lineages in Drosophila. In the mouse, targeted mutation of the Notch pathway genes Notch1 and RBP-Jk has demonstrated a role for these genes in somite segmentation, but a function in neurogenesis and in cell fate decisions has not been shown. Here we show that these mutations lead to altered expression of the Notch signalling pathway homologues Hes-5, Mash-1 and Dll1, resulting in enhanced neurogenesis. Precocious neuronal differentiation is indicated by the expanded expression domains of Math4A, neuroD and NSCL-1. The RBP-Jk mutation has stronger effects on expression of these genes than does the Notch1 mutation, consistent with functional redundancy of Notch genes in neurogenesis. Our results demonstrate conservation of the Notch pathway and its regulatory mechanisms from fly to mouse, and support a role for the murine Notch signalling pathway in the regulation of neural stem cell differentiation.

Animals↗
PubMed1997

Normal development of preimplantation mouse embryos deficient in gap junctional coupling.

The connexin multigene family (13 characterized members in rodents) encodes the subunits of gap junction channels. Gap junctional intercellular coupling, established during compaction of the preimplantation mouse embryo, is assumed to be necessary for development of the blastocyst. One member of the connexin family, connexin43, has been shown to contribute to the gap junctions that form during compaction, yet embryos homozygous for a connexin43 null mutation develop normally, at least until implantation. We show that this can be explained by contributions from one or more additional connexin genes that are normally expressed along with connexin43 in preimplantation development. Immunogold electron microscopy confirmed that roughly 30% of gap junctions in compacted morulae contain little or no connexin43 and therefore are likely to be composed of another connexin(s). Confocal immunofluorescence microscopy was then used to demonstrate that connexin45 is also assembled into membrane plaques, beginning at the time of compaction. Correspondingly, embryos homozygous for the connexin43 null mutation were found to retain the capacity for cell-to-cell transfer of fluorescent dye (dye coupling), but at a severely reduced level and with altered permeability characteristics. Whereas mutant morulae showed no evidence of dye coupling when tested with 6-carboxyfluorescein, dye coupling could be demonstrated using 2',7'-dichlorofluorescein, revealing permeability characteristics previously established for connexin45 channels. We conclude that preimplantation development in the mouse can proceed normally even though both the extent and nature of gap junctional coupling have been perturbed. Despite the distinctive properties of connexin43 channels, their role in preimplantation development can be fulfilled by one or more other types of gap junction channels.

Animals↗
PubMed1996

The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse.

Mutations of the BRCA1 gone in humans are associated with predisposition to breast and ovarian cancers. We show here that Brca1+/- mice are normal and fertile and lack tumors by age eleven months. Homozygous Brca1(5-6) mutant mice die before day 7.5 of embryogenesis. Mutant embryos are poorly developed, with no evidence of mesoderm formation. The extraembryonic region is abnormal, but aggregation with wild-type tetraploid embryos does not rescue the lethality. In vivo, mutant embryos do not exhibit increased apoptosis but show reduced cell proliferation accompanied by decreased expression of cyclin E and mdm-2, a regulator of p53 activity. The expression of cyclin-dependent kinase inhibitor p21 is dramatically increased in the mutant embryos. Buttressing these in vivo observations is the fact that mutant blastocyst growth is grossly impaired in vitro. Thus, the death of Brca1(5-6) mutant embryos prior to gastrulation may be due to a failure of the proliferative burst required for the development of the different germ layers.

Alternative Splicing↗
PubMed1996

Targeted mutagenesis: analysis of phenotype without germ line transmission.

The available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient, rapid, and complete. Genetic chimeras and mosaics, in which mutant cells are mixed with wild-type cells, can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice.

Animals↗
PubMed1996

Regulation of flt-1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium.

Flt-1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt-1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt-1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt-1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up-regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt-1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt-1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt-1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt-1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt-1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.

Animals↗
PubMed1996

Mouse mutants and cardiac development: new molecular insights into cardiogenesis.

Understanding the genetic control of cardiac development has been greatly assisted by the newly acquired ability to generate targeted mutations in the mouse. A number of mutations in genes that directly or indirectly affect cardiac development have now been reported, and the phenotypes of these mutations have suggested that cardiac development is under complex genetic control. Further analysis of the mouse model system should help elucidate the etiology of human congenital cardiovascular anomalies.

Animals↗