Project description:Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin mono-allelic expression of a small subset of mammalian genes. Imprinted genes have been categorized into two groups: multi lineage (ML) imprinted genes that can show imprinted expression in both embryonic and extra-embryonic lineages, and genes that show extra-embryonic lineage (EXEL) specific imprinted expression restricted to tissues like the placenta and visceral yolk sac (VYS) endoderm. Many genes showing EXEL imprinted expression are silenced by lncRNAs that act over long distances. Thus the analysis of this form of gene silencing is likely to be pivotal for understanding new mechanisms of long range gene silencing by lncRNAs. It has been reported that ES cells differentiated into cystic embryoid bodies (cystic EBs) contain VYS endoderm and here we investigate if cystic EBs could serve as an in vitro model for the analysis of EXEL imprinted expression. Unexpectedly we found that cystic EBs lack EXEL imprinted expression, while retaining normal imprinted expression of ML genes. This shows that cystic EBs do not model VYS imprinted expression and also argues against previous claims that cystic EBs contain VYS endoderm. We further characterized cystic EBs by performing RNA-seq and whole genome bisulphite sequencing. By comparison to various embryonic tissues we found that cystic EBs more resemble embryonic liver, which contains definitive endoderm, than the extra-embryonic endoderm of the VYS. Examination of gene and retrotranssposone expression and DNA methylation in extraembryonic, embryonic tissues and ES cell diferentiated as cystic EBs
Project description:Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin mono-allelic expression of a small subset of mammalian genes. Imprinted genes have been categorized into two groups: multi lineage (ML) imprinted genes that can show imprinted expression in both embryonic and extra-embryonic lineages, and genes that show extra-embryonic lineage (EXEL) specific imprinted expression restricted to tissues like the placenta and visceral yolk sac (VYS) endoderm. Many genes showing EXEL imprinted expression are silenced by lncRNAs that act over long distances. Thus the analysis of this form of gene silencing is likely to be pivotal for understanding new mechanisms of long range gene silencing by lncRNAs. It has been reported that ES cells differentiated into cystic embryoid bodies (cystic EBs) contain VYS endoderm and here we investigate if cystic EBs could serve as an in vitro model for the analysis of EXEL imprinted expression. Unexpectedly we found that cystic EBs lack EXEL imprinted expression, while retaining normal imprinted expression of ML genes. This shows that cystic EBs do not model VYS imprinted expression and also argues against previous claims that cystic EBs contain VYS endoderm. We further characterized cystic EBs by performing RNA-seq and whole genome bisulphite sequencing. By comparison to various embryonic tissues we found that cystic EBs more resemble embryonic liver, which contains definitive endoderm, than the extra-embryonic endoderm of the VYS.
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: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:A major role of yolk sac endoderm is the uptake of lipids and other constituents from the yolk and transfer of these components into the embryonic circulation. The molecular basis of the initial step of this regionalization has largely remained unclear. Using chick as a model system, we generated high-quality transcriptomic datasets of different stages of the yolksac endoderm and analyzed their molecular heterogeneity. Two independent samples of total RNA were isolated from yolk sac endodermal tissues on embryonic day 1, 2, 3, and 4. 5 M-NM-<g of RNA from each sample (total of 8 samples) were used to screen Affymetrix Chicken Genome Array without an amplification step.
Project description:Here, we constructed monkey blastoids resembling blastocysts in morphology and transcriptomics using naïve ESCs and optimized protocol. The synthetic blastoids could develop to embryonic disk stage with the structure of yolk sac, chorionic cavity, amnion cavity, primitive streak, connecting stalk along the rostral–caudal axis by in-vitro prolonged culture (IVC). Primordial germ cells, gastrulating cells, visceral endoderm/yolk-sac endoderm, three germ layers and haemato-endothelial progenitors were identified in the monkey blastoid IVC embryo by single-cell transcriptomics or immunostaining. Besides, pregnancies with early gestation sacs were achieved by transferring monkey blastoids to surrogates. Our results revealed the in-vitro gastrulation and in-vivo early pregnancy of primate synthetic embryos, providing a powerful system to dissect primate embryonic development with less ethical concerns and restrict access.
Project description:A major role of yolk sac endoderm is the uptake of lipids and other constituents from the yolk and transfer of these components into the embryonic circulation. The molecular basis of the initial step of this regionalization has largely remained unclear. Using chick as a model system, we generated high-quality transcriptomic datasets of different stages of the yolksac endoderm and analyzed their molecular heterogeneity.
Project description:The human yolk sac (YS) is an extra-embryonic tissue critical for early prenatal life development. It is the first site of haematopoiesis where progenitors differentiate from endoderm within blood islands of the yolk sac contributing initially to primitive erythropoiesis and in subsequent waves to erythro-myeloid and lymphoid differentiation.
Project description:The human yolk sac (YS) is an extra-embryonic tissue critical for early prenatal life development. It is the first site of haematopoiesis where progenitors differentiate from endoderm within blood islands of the yolk sac contributing initially to primitive erythropoiesis and in subsequent waves to erythro-myeloid and lymphoid differentiation.
Project description:We report for the first time, the derivation and characterization of extra-embryonic endoderm (XEN) cells from primitive endoderm (PrE) of porcine (p) embryos. The pXEN cells can be reliably and reproducibly generated from parthenogenic, in vitro or in vivo derived embryos, and express canonical PrE or XEN cell genes (GATA4, GATA6, SOX17, SALL4, FOXA2, and HNF4A). Transcriptome analysis of pXEN cells revealed close resemblance to yolk sac than any other embryonic or extraembryonic tissue. When introduced into blastocyst stage embryo, the pXEN cells contributed to wide-spread chimerism including visceral yolk sac, chorion, as well as embryonic gut and liver primordium in the fetus. The pXEN cells were shown to be an efficient nuclear donor for generating cloned offspring. Taken together, pXEN cells fulfil a longstanding need for a stable, chimera-competent, and nuclear transfer-compatible porcine embryonic cells with applications for genome editing in livestock.