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Janet Rossant

Publications and source records attributed to Janet Rossant.

At least 55 records · Page 3Linked to original sources
PubMed2004

Gene-trap expression screening to identify endothelial-specific genes.

The endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. In a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-beta type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.

Animals↗
PubMed2004

The Centre for Modeling Human Disease Gene Trap resource.

Gene trap mutagenesis of mouse embryonic stem cells generates random loss-of-function mutations, which can be identified by a sequence tag and can often report the endogenous expression of the mutated gene. The Centre for Modeling Human Disease is performing expression- and sequence-based screens of gene trap insertions to generate new mouse mutations as a resource for the scientific community. The gene trap insertions are screened using multiplexed in vitro differentiation and induction assays, and sequence tags are generated to complement expression profiles. Researchers may search for insertions in genes expressed in target cell lineages, under specific in vitro conditions, or based upon sequence identity via an online searchable database (http://www.cmhd.ca/sub/genetrap.asp). The clones are available as a resource to researchers worldwide to help to functionally annotate the mammalian genome and will serve as a source to test candidate loci identified by phenotype-driven mutagenesis screens.

Animals↗
PubMed2004

Implementing large-scale ENU mutagenesis screens in North America.

A step towards annotating the mouse genome is to use forward genetics in phenotype-driven screens to saturate the genome with mutations. The purpose of this article is to highlight the new projects in North America that are focused on isolating mouse mutations after ENU mutagenesis and phenotype screening.

Animals↗
PubMed2004

Emerging asymmetry and embryonic patterning in early mouse development.

Recent studies have revealed asymmetries in the mouse zygote and preimplantation embryo, well before the establishment of anterior-posterior polarity after implantation. Whether these asymmetries are causally related to embryonic patterning or are coincidental outcomes of the topology of normal development remains uncertain.

Animals↗
PubMed2004

Foxh1 is essential for development of the anterior heart field.

The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFbeta-Smad pathway. Here we demonstrate that Foxh1-/- mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c-/- mutant hearts. Further, we show that Mef2c is a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFbeta response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFbeta-like signals.

Animals↗
PubMed2004

Lineage development and polar asymmetries in the peri-implantation mouse blastocyst.

The early events of mouse embryogenesis lead to the formation of three distinct cell lineages by the blastocyst: the pluripotent epiblast and the two extraembryonic lineages, the trophoblast and primitive endoderm. Segregation of the lineages depends on the relative levels of expression of key transcription factors, whose localized expression must be controlled by the earlier events of compaction and polarization of the morula. Soon after lineage specification, the two extraembryonic lineages show evidence of early polarities that may relate to the polarity of the postimplantation embryo at gastrulation. The exact relationship between lineage segregation, preimplantation polarities and the postimplantation axes remain to be determined but are now open to molecular and cellular investigation.

Animals↗
PubMed2004

Characterization of mouse Rsk4 as an inhibitor of fibroblast growth factor-RAS-extracellular signal-regulated kinase signaling.

Receptor tyrosine kinase (RTK) signals regulate the specification of a varied array of tissue types by utilizing distinct modules of proteins to elicit diverse effects. The RSK proteins are part of the RTK signal transduction pathway and are thought to relay these signals by acting downstream of extracellular signal-regulated kinase (ERK). In this study we report the identification of ribosomal S6 kinase 4 (Rsk4) as an inhibitor of RTK signals. Among the RSK proteins, RTK inhibition is specific to RSK4 and, in accordance, is dependent upon a region of the RSK4 protein that is divergent from other RSK family members. We demonstrate that Rsk4 inhibits the transcriptional activation of specific targets of RTK signaling as well as the activation of ERK. Developmentally, Rsk4 is expressed in extraembryonic tissue, where RTK signals are known to have critical roles. Further examination of Rsk4 expression in the extraembryonic tissues demonstrates that its expression is inversely correlated with the presence of activated ERK 1/2. These studies demonstrate a new and divergent function for RSK4 and support a role for RSK proteins in the specification of RTK signals during early mouse development.

Animals↗
PubMed2003

Transcriptome analysis of mouse stem cells and early embryos.

Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

Animals↗
PubMed2003

Trophoblast expression of fms-like tyrosine kinase 1 is not required for the establishment of the maternal-fetal interface in the mouse placenta.

Fms-like tyrosine kinase 1 (Flt1)/vascular endothelial growth factor (VEGF) receptor 1, a receptor for VEGF-A and placental growth factor, is expressed in the spongiotrophoblast layer that segregates the maternal and fetal vasculature in the mouse placenta. A soluble form of Flt1 (sFlt1) produced in the mouse and human placenta can also be detected in the maternal blood. Levels of maternal sFlt1 are elevated in preeclampsia, suggesting that placental sFlt1 plays roles in regulating the maternal vasculature during pregnancy. However, it remains to be determined whether placental Flt1/sFlt1 serves as a regulator of VEGF-A activity in the placenta per se. Here, we investigated the placental development in Flt1-deficient mice. Flt1 is expressed in a subpopulation of ectoplacental cone cells and later marks the spongiotrophoblast cells, peri/endovascular trophoblast cells, and trophoblast glycogen cells. The labyrinth of Flt1lacZ/lacZ placentae lacked the fetal capillary network because of a defect in allantoic mesoderm invasion. To address whether the absence of Flt1 in the trophoblast alone affects placental development, we investigated chimeric placentae comprised of Flt1lacZ/lacZ trophoblast and Flt1+/+ mesoderm, generated by tetraploid aggregation. Fetal growth was supported normally, and no defect in the formation of placental circulation into the maternal spiral artery or invasion of peri/endovascular trophoblast was detected. These findings indicate that trophoblast-derived Flt1/sFlt1 is dispensable for the initial establishment of the maternal-fetal interface in the mouse placenta. Targeting maternal sFlt1 levels for treatment of preeclampsia may thus be possible without affecting the proper formation of the placenta.

Animals↗
PubMed2003

Control of effector CD8+ T cell function by the transcription factor Eomesodermin.

Activated CD8+ T cells play a critical role in host defense against viruses, intracellular microbes, and tumors. It is not clear if a key regulatory transcription factor unites the effector functions of CD8+ T cells. We now show that Eomesodermin (Eomes), a paralogue of T-bet, is induced in effector CD8+ T cells in vitro and in vivo. Ectopic expression of Eomes was sufficient to invoke attributes of effector CD8+ T cells, including interferon-gamma (IFN-gamma), perforin, and granzyme B. Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of CD8+ T cells. We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.

Amino Acid Sequence↗
PubMed2003

Activated Fps/Fes partially rescues the in vivo developmental potential of Flk1-deficient vascular progenitor cells.

Relatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A-mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A-independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell-derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

Animals↗
PubMed2003

Lineage allocation and asymmetries in the early mouse embryo.

The mouse blastocyst, at the time of implantation, has three distinct cell lineages: epiblast (EPI), trophoblast and primitive endoderm (PE). Interactions between these three lineages and their directional growth and migration are critical for establishing the initial asymmetries that result in anterior-posterior patterning of the embryo proper. We have re-investigated the timing of specification of the three lineages in relation to the differential allocation of progeny of the first two blastomeres to the embryonic versus abembryonic axis of the blastocyst. We find that the majority of cells of the inner cell mass (ICM) are specified to be EPI or PE by the mid 3.5 day blastocyst and that this is associated with localized expression of GATA-6 in the ICM. We propose a model for molecular specification of the blastocyst lineages in which a combination of cell division order, signal transduction differences between inner and outer cells and segregation of key transcription factors can produce a blastocyst in which all three lineages are normally set up in an ordered, lineage-dependent manner, but which can also reconstruct a blastocyst when division order or cell interactions are disturbed.

Animals↗
PubMed2003

FGFR1 is independently required in both developing mid- and hindbrain for sustained response to isthmic signals.

Fibroblast growth factors (FGFs) are signaling molecules of the isthmic organizer, which regulates development of the midbrain and cerebellum. Tissue-specific inactivation of one of the FGF receptor (FGFR) genes, Fgfr1, in the midbrain and rhombomere 1 of the hindbrain of mouse embryos results in deletion of the inferior colliculi in the posterior midbrain and vermis of the cerebellum. Analyses of both midbrain-hindbrain and midbrain-specific Fgfr1 mutants suggest that after establishment of the isthmic organizer, FGFR1 is needed for continued response to the isthmic signals, and that it has direct functions on both sides of the organizer. In addition, FGFR1 appears to modify cell adhesion properties critical for maintaining a coherent organizing center. This may be achieved by regulating expression of specific cell-adhesion molecules at the midbrain-hindbrain border.

Animals↗
PubMed2003

Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype.

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.

Animals↗
PubMed2003

The mouse genome sequence--the end of the tail, or just the beginning?

The recent flurry of papers on the mouse genome includes the description of the full genome assembly, analysis of the mouse transcriptome, the origin of interstrain variation, initial analysis of conserved non-coding regions and high-throughput expression analysis of a subset of genes. Each illustrates how the availability of the genome sequence will change the way mouse biologists do business in future.

Alternative Splicing↗