Project description:The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryo lethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. Affymetrix microarray analysis and quantitative RT-PCR validation of the relative gene expression profiles in E9.5 RFC1-/- vs. RFC1+/+ embryos indicates a dramatic downregulation of multiple genes involved in erythropoiesis, and upregulation of several genes that form the cubilin-megalin multiligand endocytic receptor complex. Megalin protein expression disappears from the visceral yolk sac of RFC1-/- embryos, and cubilin protein is widely misexpressed. Inactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate, vitamin B12, and other nutrients, lipids and morphogens required for normal embryogenesis. Comparison of RFC KO, wildtype normal embryos vs. RFC KO, nullizygous affected embryos Experiment Overall Design: 6 samples RFC KO mouse embryos, E9.5, folic acid treated: 3 Control, Wildtype, normal; 3 Affected, Nullizygous, CR/chorioallantoic defect; as paired-littermates with one normal and one affected embryo per set from each of three separate litters for RNA extraction and hybridization on Affymetrix microarrays.
Project description:The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryo lethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. Affymetrix microarray analysis and quantitative RT-PCR validation of the relative gene expression profiles in E9.5 RFC1-/- vs. RFC1+/+ embryos indicates a dramatic downregulation of multiple genes involved in erythropoiesis, and upregulation of several genes that form the cubilin-megalin multiligand endocytic receptor complex. Megalin protein expression disappears from the visceral yolk sac of RFC1-/- embryos, and cubilin protein is widely misexpressed. Inactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate, vitamin B12, and other nutrients, lipids and morphogens required for normal embryogenesis. Comparison of RFC KO, wildtype normal embryos vs. RFC KO, nullizygous affected embryos Keywords: reduced folate carrier knockout, folate receptor, cubilin, megalin, embryos, gene expression, neural tube defect, chorioallantoic fusion
Project description:Comparison of conserved TAD boundaries reveals that diverging CTCF sites is an evolutionary conserved signature associated with TAD borders 4C-seq samples for six gene family promoters in different samples: E9.5 and E14.5 mouse embryos ; 24hpf, 48hpf, 80%epiboly and Dome zebrafish embryos ; 48hpf S.purpuratus embryos
Project description:Transcriptional profiling of E9.5 mouse embryo tissue from the presomitic mesoderm (PSM) and somites I-IV. Tissue from embryos lacking a functional Paraxis gene (Paraxis-/-) was compared to identical tissue from E9.5 Wild Type embryos. The goal was to identify genes that had become deregulated in the absence of the transcription factor, Paraxis.
Project description:Purpose: The goal of this study is to identify the differential cardiac transcriptome profiling between WT and Smyd1 null (Smyd1-KO) hearts at E9.5 using RNA-seq. Methods: mRNA profiles of E9.5 WT and Smyd1-KO mouse hearts were generated by deep sequencing, n=3 for each genotype, using Illumina HiSeq2500. The sequence reads were aligned to the mm10 reference genome using STAR via the bcbio-nextgen RNA-sequencing pipeline. Differential gene expression was determined by DEseq2. Results: 1756 genes were differentially expressed between WT and Smyd1-KO hearts [adjusted P value <0.05, |log2(Fold Change)| > 0.5], with 1130 upregulated and 626 downregulated in E9.5 Smyd1-KO hearts.
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:TMT based quantification of protein abundance and phosphorylation state for developing mouse placenta. Timed-pregnant CD-1 mice were obtained from Charles Rivers Labs and dissected at e7.5 to extract the ectoplacental cones (EPCs) and at e9.5 to obtain the placenta as described by Martin and Cockroft (Martin, P.; Cockroft, D. L. Culture of Postimplantation Mouse Embryos). Staging of the mouse embryos was done according to Theiler criteria (Theiler, K. The House Mouse: Development and Normal Stages from Fertilization to 4 Weeks of Age; Springer-Verlag, 1972.)
Project description:This study was aimed at identifying Tbx1 dosage-dependent genes in vivo, so we performed a transcriptome analysis of Tbx1 mutants with nine different genotypes corresponding to different Tbx1 mRNA dosages. RNA isolated from whole E9.5 embryos with 9 different genotypes was hibridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays. For each genotype, we used two embryos (biological replicates), each hybridized to one array, thus we analyzed a total of 18 arrays. Tbx1 gene dosage
Project description:Regulation of neural stem cell (NSC) fate decisions is critical during the transition from a multicellular mammalian forebrain neuroepithelium to the multilayered neocortex. Forebrain development requires coordinated vascular investment alongside NSC differentiation. Vascular endothelial growth factor A (Vegf) has proven to be a pleiotrophic gene whose multiple protein isoforms regulate a broad range of effects in neurovascular systems. To test the hypothesis that the Vegf isoforms (120, 164, and 188) are required for normal forebrain development, we analyzed the forebrain transcriptome of mice expressing specific Vegf isoforms, Vegf120, VegfF188, or a combination of Vegf120/188. Transcriptome analysis identified differentially expressed genes in embryonic day (E) 9.5 forebrain, a time point preceding dramatic neuroepithelial expansion and vascular investment in the telencephalon. Meta-analysis identified gene pathways linked to chromosome-level modifications, cell fate regulation, and neurogenesis that were altered in Vegf isoform mice. Twelve E9.5 wildtype forebrain samples were compared to four E9.5 Vegf120 mouse forebrains, four E9.5 Vegf188 mouse forebrains, three E9.5 Vegf120/188 mouse forebrains, and four E11.5 wild type forebrains using the Mouse 430. 2.0 Affymetrix GeneChip. This study comprises of new samples and reanalysis of Samples from GSE30767 and GSE8091.