Project description:Rudhira is essential for mouse developmental angiogenesis and tissue morphogenesis. Embryos lacking endothelial rudhira die at mid-gestation with vascular patterning defects. Rudhira mutant yolk sac endothelial cells show slow and random migration. So to identify key signaling pathways perturbed in the absence of rudhira, we undertook whole transcriptome based analysis of gene expression in rudhira null yolk sac and embryo. Transcriptome analysis shows that key mediators of angiogenesis, cell adhesion, migration and extracellular matrix degradation as well as several components of the TGFβ pathway are perturbed in rudhira null mutant yolk sacs at 9.5 dpc.
Project description:Rudhira is essential for mouse developmental angiogenesis and tissue morphogenesis. Embryos lacking endothelial rudhira die at mid-gestation with vascular patterning defects. Rudhira mutant yolk sac endothelial cells show slow and random migration. So to identify key signaling pathways perturbed in the absence of rudhira, we undertook whole transcriptome based analysis of gene expression in rudhira null yolk sac and embryo. Transcriptome analysis shows that key mediators of angiogenesis, cell adhesion, migration and extracellular matrix degradation as well as several components of the TGFβ pathway are perturbed in rudhira null mutant yolk sacs at 9.5 dpc. We used two embryo samples ( 2E- wild type; 5E- rudhira mutant). Repetition was done on each sample. We used 4 yolk sac samples (3Y,4Y- wild type; 7Y,8Y-rudhira mutant) for the analysis. Repetition was done on each sample.
Project description:KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell, and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. Embryonic -like globin gene expression is reduced in KLF2-/- embryos compared to wildtype (WT), and E10.5 erythroid cells exhibit abnormal morphology. Other KLF2 target genes were identified by comparing E9.5 KLF2-/- and WT yolk sac erythroid cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression; eighty-nine of these are downregulated in KLF2-/- compared to WT. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. Previously identified erythroid-enriched regulatory genes such as reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT. SOX2, a pluripotency factor in ES cells, is also a KLF2 target in embryonic erythroid cells. We investigated whether reelin, which has an established role in neuronal migration and proliferation, has a role in embryonic erythropoiesis. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter, but reelin mutant mice have no apparent abnormalities in embryonic erythroid morphology or globin gene expression. Timed-pregnant KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron KLF2-/- frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 4 different KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays
Project description:The mouse embryonic yolk sac consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and may function as a materno-fetal exchange system for nutrients, waste and gas, and physical protector for the embryo/fetus. The present study was undertaken to characterize gene expression of the VYS and PYS endodermal cells, and to identify their novel genetic markers from microarray data with RT-PCR and in situ hybridization analyses. As a result, Apoa4, Lrp2, Fxyd2, Slc34a3 and Entpd2 were selected as genetic markers of VYS epithelial cells. These markers were predominantly expressed in VYS epithelial cells, and may involve nutrient uptake and transport of various substances across the VYS membrane. Gkn2 and Pga5 were selected as markers for PYS cells. Gkn2 might be involved in the migration of PYS cells on Reichert’s membrane. Pga5 belongs to the aspartic proteinase family, and is known as ”pepsinogen F.” It may act on degradation of maternal proteins for the nutrient supply to the fetus. The present study also indicated that Mmp1a and Prl7b1 can be used as markers for placenta tissue. The markers reported may be useful for characterization of murine extraembryonic tissues, including yolk sacs and placenta, during development and in culture.
Project description:Transcriptional profiles of the embryonic yolk sac from embryos with ectopic Norrin expression were compared to their wild type littermate controls. The goal is to identify the transcriptional response to Norrin-Frizzled 4 signaling during embryonic angiogenesis. Experiment Overall Design: Ectopic Norrin expression was achieved using a conditional over-expression strategy. Yolk sacs from 3-5 embryos were pooled for each sample and 3 replicates of both control and experimental groups were analyzed.
Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in the yolk sac endoderm, and that gene-trap disruption of GW182 leads to growth arrest of yolk sac endoderm, impaired hematopoiesis and embryonic lethality. To investigate roles of GW182 in the yolk sac endoderm, we assessed changes in mRNA expression in the yolk sac of E9.5 GW182gt/gt embryos using microarrays (Affymetrix). Yolk sac of wild type littermates and GW182gt/gt embryos at E9.5 was collected for total RNA isolation using Trizol (Invitrogen). RNAs were purified according to the manufacturer’s protocol before subjected to Mouse Gene 1.0 ST Whole Genome Array (Affymetrix) for mRNA expression profiling. Experiments were performed in triplicate. Differentially expressed mRNAs were identified using a two-sample t-test (P<0.05 considered significant).
Project description:Transcriptomic analyses of yolk sacs from mouse embryos at E8.5 was performed to assess the dosage dependent effects of varying Etv2 dosage on early endothelial and hematopoietic development.
Project description:To identify genes critical for vascular development, we generated mice where ETV2 is inactivated in FLK1+ cells by a loxP-Cre recombination approach. Results provide a detailed insight into the function of ETV2 in emrbyonic vasculare formation. Total RNA obtained from E9.5 yolk sacs from Flk1Cre;ETV2 CKO and control mice.
Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in the yolk sac endoderm, and that gene-trap disruption of GW182 leads to growth arrest of yolk sac endoderm, impaired hematopoiesis and embryonic lethality. To investigate roles of GW182 in the yolk sac endoderm, we assessed changes in mRNA expression in the yolk sac of E9.5 GW182gt/gt embryos using microarrays (Affymetrix).
Project description:Blood and endothelial cells arise from hemangiogenic progenitors that are specified from FLK1-expressing mesoderm by the transcription factor ETV2. FLK1 mesoderm also contributes to other tissues, including vascular smooth muscle (VSM) and cardiomyocytes. However, the developmental process of FLK1 mesoderm generation and its derivatives and the lineage relationship among FLK1 mesoderm derivatives these tissues remain obscure. Recent single cell RNA-sequencing (scRNA-seq) studies of early stages of embryogenesis embryos, or in vitro differentiated human embryonic stem (ES) cells have differentiation provided unprecedented information on the spatiotemporal resolution of cells in embryogenesis. Nonetheless, these snapshots still nonetheless offer insufficient information on dynamic developmental processes due to inadvertently missing intermediate states and unavoidable batch effects. Here we performed scRNA-seq of mouse ES cells in asynchronous embryoid bodies (EBs), in vitro differentiated embryonic stem (ES) cells containing undifferentiated ES cells and its differentiated hemangiogenic progeny, as well as yolk sacs, the first hematopoietic extraembryonic tissue in developing embryo that contains hemangiogenic and VSM lineages. We captured a continuous developmental process from undifferentiated pluripotent cells to FLK1 mesoderm-derived tissues involved in hemangiogenesis. This continuous transcriptome map will benefit both basic and applied studies of mesoderm and its derivatives.