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J Rossant

Publications and source records attributed to J Rossant.

At least 91 records · Page 5Linked to original sources
PubMed1994

Essential role of Mash-2 in extraembryonic development.

The outer layer of the blastocyst, or trophectoderm, is the first cell lineage to differentiate in the mouse embryo, but little is known about the genetic control of its development. Lineage-specific transcription factors may be important in lineage specification, and the product of the Mash-2 gene fulfils the criteria for such a factor. Mash-2 is a mammalian member of the achaete-scute family which encodes basic-helix-loop-helix transcription factors and is strongly expressed in the extraembryonic trophoblast lineage. Mash-2 transcripts are found in the female germ line and in the embryo throughout preimplantation development, but are highly expressed later only in the ectoplacental cone, the chorion and their derivatives in the placenta. Mash-2 transcripts are not found in primary and secondary giant cells, yolk sac or allantois at any post-implantation stage, and are present only transiently and at low levels in the embryo during gastrulation. To analyse the role of Mash-2 in development, we have used gene targeting to generate mice having no Mash-2 function. We report here that Mash-2-/- embryos die from placental failure at 10 days postcoitum. In mutant placentas, spongiotrophoblast cells and their precursors are absent and chorionic ectoderm is reduced. We have rescued this placental mutant phenotype by constructing chimaeras with tetraploid wild-type embryos which contribute almost exclusively to extraembryonic tissues. Mash-2-/- embryos developed normally and adult Mash-2-/- mice were viable, demonstrating that Mash-2 has no major role in the embryo itself. Mash-2 is the first transcription factor shown to play a critical part in the development of the mammalian trophoblast lineage.

Animals↗
PubMed1994

HNF-3 beta is essential for node and notochord formation in mouse development.

HNF-3 beta, a member of the HNF-3/fork head family of transcription factors, is expressed in the node, notochord, floor plate, and gut in mouse embryos. A null mutation of this gene leads to embryonic lethality. The primary defect of HNF-3 beta -/- embryos is an absence of organized node and notochord formation, which leads to secondary defects in dorsal-ventral patterning of the neural tube. In contrast, patterning along the anterior-posterior axis was surprisingly little affected. Although HNF-3 beta is required for node and notochord formation, some organizer activity persists in the absence of these structures. HNF-3 beta is not required for the development of definitive endoderm cells, but foregut morphogenesis is severely affected in HNF-3 beta -/- embryos.

Animals↗
PubMed1994

brg1: a putative murine homologue of the Drosophila brahma gene, a homeotic gene regulator.

To identify potential regulators of Hox gene expression in mice, we have screened for genes highly related to brahma (brm), an activator of homeotic gene expression in Drosophila. We have cloned a murine gene, brg1, which, like brm, encodes a member of the DEGH protein family, suggesting that brg1 may be a DNA-dependent ATPase or a helicase. brg1 also contains a bromodomain which may be involved in transactivation. Although the sequences of a number of mammalian genes similar to Drosophila brm have been reported, they are related to brm only within specific portions of the putative helicase region, while brg1 is highly similar to brm throughout and outside of this region. A 5.8-kb brg1 transcript was detected throughout embryogenesis and in numerous adult tissues. RNA in situ hybridization revealed widespread expression of brg1 in embryonic tissues. At later stages of embryogenesis, differences in levels of brg1 expression were seen among different tissues. brg1 expression was highest in the spinal cord, the brain, parts of the peripheral nervous system, and the vertebral column. These expression domains within the spinal cord and vertebral column encompass major regions of Hox gene expression. Within the spinal cord, brain, and retina, mRNA levels were higher in regions consisting of differentiated cells than in regions consisting of undifferentiated, proliferating cells. These patterns of brg1 expression are consistent with a possible role for brg1 in Hox gene regulation as well as in other regulatory pathways.

Adenosine Triphosphatases↗
PubMed1994

The genomic structure of an insertional mutation in the dystonia musculorum locus.

We have previously identified a line of transgenic mice, Tg4, in which an hsp68-lacZ hybrid gene has inserted into the dystonia musculorum (dt) locus on chromosome 1. We have confirmed the localization of the Tg4 integration site to the proximal region of mouse chromosome 1 by interspecific backcross analysis. One end of the integration complex has been cloned and we have used single-copy probes from the flanking region to screen a mouse genomic library. Several overlapping lambda phage clones have been isolated and arranged into a contig spanning 75 kb of genomic DNA. Probes from the genomic contig have enabled us to characterize the wildtype and Tg4 loci. We report that the integration of the transgene was accompanied by a deletion of 45 kb of host genomic sequences with no other detectable rearrangement in the Tg4 genome.

Animals↗
PubMed1994

Human homolog of a mouse sequence from the dystonia musculorum locus is on chromosome 6p12.

Dystonia musculorum is a hereditary neurodegenerative disease in mice that affects sensory neurons. In an effort to clone the gene responsible for this disorder, we have assembled a genomic contig spanning 75 kb of the dystonia musculorum (dt) locus. Within this genomic contig, we have identified a small restriction fragment that shows evolutionary conservation to rat, hamster, rabbit, and human genomic DNA. Using this mouse sequence, we have cloned the conserved human genomic fragment. Sequence analysis of the mouse and human genomic fragments revealed that they share a sequence similarity of 82% over 175 bp. A panel of human/rodent somatic cell hybrids was used to map the human genomic sequence to Chromosome (Chr) 6, and high-resolution in situ hybridization (FISH) allowed it to be sublocalized to 6p12. The human homolog of the mouse Bpag1 gene, a gene tightly linked to the mouse dt gene, also maps to Chr 6. Thus, this comparative mapping reveals a new region of conserved synteny between the chromosomes of mouse and human. Mapping the human homolog of the mouse dt gene enables us to initiate linkage studies to identify neurodegenerative disorders that may be caused by mutations in this gene.

Animals↗
PubMed1994

Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants.

Otx2, a mouse homolog of the Drosophila orthodenticle gene, is first widely expressed in the epiblast but becomes progressively restricted to the anterior third of the embryo by the headfold stage. This progressive restriction correlates with the anterior migration of mesoderm in the embryo, suggesting that interactions with mesoderm may be involved in setting up the anterior domain of Otx2 expression in vivo. Using explant-recombination assays, we have obtained evidence that a positive signal from anterior mesendoderm is required to stabilize expression of Otx2 in vivo, whereas a negative signal from the later-forming posterior mesendoderm represses Otx2 expression in the posterior part of the embryo. We have also found that exogenous retinoic acid can mimic the effect of this negative signal and reduces the anterior domain of Otx2 expression.

Animals↗
PubMed1993

Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.

Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell <==> tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.

Animals↗
PubMed1993

Genome manipulation in embryonic stem cells.

Embryonic stem (ES) cells derived from pluripotent cells of the early mouse embryo provide a powerful tool for genome manipulation in mammals. Dominantly acting effects can be achieved by introducing constructs to misexpress or ectopically express a gene product, express an altered product or express antisense constructs. Use of ES cell chimeras to analyse the effects of such alterations may provide information not readily available from transgenic mice. However, the most important use of ES cells, to date, is in the generation of recessive mutations, either in known genes by targeted mutagenesis or randomly by insertional mutagenesis. Examples of these approaches and possible future strategies are discussed.

Animals↗
PubMed1993

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro.

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5-18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro.

Animals↗
PubMed1993

Anterior mesendoderm induces mouse Engrailed genes in explant cultures.

We have developed germ layer explant culture assays to study the role of mesoderm in anterior-posterior (A-P) patterning of the mouse neural plate. Using isolated explants of ectodermal tissue alone, we have demonstrated that the expression of Engrailed-1 (En-1) and En-2 genes in ectoderm is independent of mesoderm by the mid- to late streak stage, at least 12 hours before their onset of expression in the neural tube in vivo at the early somite stage. In recombination explants, anterior mesendoderm from headfold stage embryos induces the expression of En-1 and En-2 in pre- to early streak ectoderm and in posterior ectoderm from headfold stage embryos. In contrast, posterior mesendoderm from embryos of the same stage does not induce En genes in pre- to early streak ectoderm but is able to induce expression of a general neural marker, neurofilament 160 x 10(3) M(r). These results provide the first direct evidence for a role of mesendoderm in induction and regionalization of neural tissue in mouse.

Animals↗
PubMed1993

flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors.

We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximal-lateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.

Amino Acid Sequence↗
PubMed1993

Defects in heart and lung development in compound heterozygotes for two different targeted mutations at the N-myc locus.

Two types of mutant allele, one leaky and one null, have been generated by gene targeting at the N-myc locus in embryonic stem cells and the phenotypes of mice homozygous for these mutations have been described. These mutations have shown that N-myc has a number of functions during development, including a role in branching morphogenesis in the lung, which manifests itself at birth in mice homozygous for the leaky allele, and roles in the development of the mesonephric tubules, the neuroepithelium, the sensory ganglia, the gut and the heart, which become evident at midgestation in embryos homozygous for the null allele. In an attempt to define roles for N-myc at other stages of development, we have combined the two types of N-myc mutant allele in a compound heterozygote that as a result contains approximately 15% of normal levels of N-Myc protein. Compound heterozygotes died during gestation at a time intermediate to the times of death of embryos homozygous for either mutation individually, and their death appeared to result from cardiac failure stemming from hypoplasia of the compact subepicardial layer of the myocardium. Investigation of the expression pattern of N-myc and various markers of differentiation in wild-type and compound heterozygote mutant hearts has suggested that N-myc may function in maintaining the proliferation and/or preventing the differentiation of compact layer myocytes. This study illustrates the importance of generating different mutations at a given locus to elucidate fully the function of a particular gene during development.

Animals↗
PubMed1993

The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins.

Little is known about genes that govern the development of the definitive endoderm in mammals; this germ layer gives rise to the intestinal epithelium and various other cell types, such as hepatocytes, derived from the gut. The discovery that the rat hepatocyte transcription factor HNF3 is similar to the Drosophila forkhead gene, which plays a critical role in gut development in the fly, led us to isolate genes containing the HNF3/forkhead (HFH) domain that are expressed in mouse endoderm development. We recovered mouse HNF3 beta from an embryo cDNA library and found that the gene is first expressed in the anterior portion of the primitive streak at the onset of gastrulation, in a region where definitive endoderm first arises. Its expression persists in axial structures derived from the mouse equivalent of Hensen's node, namely definitive endoderm and notochord, and in the ventral region of the developing neural tube. Expression of the highly related gene, HNF3 alpha, appears to initiate later than HNF3 beta and is first seen in midline endoderm cells. Expression subsequently appears in notochord, ventral neural tube, and gut endoderm in patterns similar to HNF3 beta. Microscale DNA binding assays show that HNF3 proteins are detectable in the midgut at 9.5 days p.c. At later stages HNF3 mRNAs and protein are expressed strongly in endoderm-derived tissues such as the liver. HNF3 is also the only known hepatocyte-enriched transcription factor present in a highly de-differentiated liver cell line that retains the capacity to redifferentiate to the hepatic phenotype. Taken together, these studies suggest that HNF3 alpha and HNF3 beta are involved in both the initiation and maintenance of the endodermal lineage. We also discovered a novel HFH-containing gene, HFH-E5.1, that is expressed transiently in posterior ectoderm and mesoderm at the primitive streak stage, and later predominantly in the neural tube. HFH-E5.1 is highly similar in structure and expression profile to the Drosophila HFH gene FD4, suggesting that HFH family members have different, evolutionarily conserved roles in development.

Amino Acid Sequence↗
PubMed1992

Close linkage of retinoic acid receptor genes with homeobox- and keratin-encoding genes on paralogous segments of mouse chromosomes 11 and 15.

Retinoic acid is essential for normal development and growth of structures such as head and limbs, and it can act as morphogen or teratogen. Retinoic acid induces expression of genes such as the homeobox genes and keratin type I and type II genes. Retinoic acid receptors are nuclear transcription factors that play a key role in retinoid physiology. As part of the characterization of retinoic acid receptor gene family, linkage of genes encoding the three receptors was determined by using interspecific backcross and recombinant inbred strain analysis of restriction fragment variants. Retinoic acid receptor alpha is located on mouse Chromosome (Chr) 11 near the homeobox-2 complex and the keratin type I gene complex, whereas retinoic acid receptor gamma is on mouse Chr 15 near the homeobox-3 complex and the keratin type II complex. Close genetic proximity of these functionally related genes may be significant. We confirmed assignment of retinoic acid receptor beta to the centromeric portion of Chr 14. These linkage assignments provide further evidence for duplicated segments in the mouse genome.

Animals↗
PubMed1992

Expression analysis of a Notch homologue in the mouse embryo.

The Drosophila Notch gene has been shown to be involved in the determination of fate in a number of different cell types. Similarly, Notch homologues in Caenorhabditis elegans are involved in cell decision-making steps. It is of interest to determine if a mammalian Notch homologue plays a role in cell fate determination. We have isolated cDNA from a mouse Notch gene using low-stringency hybridization with probes derived from the Xenopus Notch gene. Sequence analysis reveals that this gene possesses EGF repeats, Notch/lin-12 repeats, and CDC-10/SWI-6 repeats, characteristic of other Notch homologues. Northern analysis revealed that the transcript size was roughly 10 kb as has been found for the other Notch genes. We have studied the expression pattern of the gene by both conventional and whole mount in situ hybridization. Expression patterns were consistent with mouse Notch having a determinative role in the formation of mesoderm, somites, and the nervous system.

Amino Acid Sequence↗