Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency
Ontology highlight
ABSTRACT:
ORGANISM(S): Bos taurus
PROVIDER: GSE189318 | GEO | 2022/03/22
REPOSITORIES: GEO
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Project description:Current understandings of the initiation of the trophectoderm (TE) program in mammalian embryonic development lacks evidence of how TE-associated factors such as CDX2 and GATA3 participate in bovine lineage specification. In this study, we describe the effects of TE-associated factors on lineage specification marker genes such as SOX2, OCT4, NANOG, GATA6 and SOX17, assisted by a cytosine base editor system. Interestingly, GATA3 downregulates the NANOG expression in bovine blastocysts. Further analysis of the blastocyst mosaic shows that GATA3 is required for NANOG in TE cells of bovine blastocysts. It is worthy to notice that, unlike mouse embryos where GATA3 and CDX2 did not reciprocally affect each other in bovine embryos.
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Project description:Preimplantation stages of mouse embryo development involve temporal and spatial specification and segregation of three late blastocyst cell lineages; trophectoderm (TE), primitive endoderm (PrE) and epiblast (EPI). Spatial separation of the outer TE lineage from the two inner-cell-mass (ICM) lineages (PrE and EPI) starts with the 8- to 16-cell transition and concludes following transit through the 16- to 32-cell stages. This results in a nascent early blastocyst ICM derived from descendants of primary founding inner cells and a secondarily contributed population; of which subsequent relative EPI versus PrE potencies are subject to debate. We showed that generation of primary but not the secondary ICM populations is highly dependent on temporally discreet activation of the mammalian target of rapamycin (mTOR – specifically mTORC1) around 8-cell stage M-phase entry. Moreover, that this role is mediated via the regulated function of the 7-methylguanosine- (7mG) cap binding initiation complex (EIF4F) and potentiating the translation of a subset of key but otherwise intransigent mRNAs containing 5’ UTR terminal oligopyrimidine (TOP-) sequence motifs. To find out translation of which mRNAs is regulated by mTOR during 8- to 16-cell transition, we analysed proteomes of control and mTOR-inhibited embryos during this time window.
2023-08-18 | PXD039423 | Pride
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Project description:In the preimplantation mouse embryo TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed both in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP-seq to define genome-wide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation towards the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification. ChIPseq profiles of TEAD4, IgG, Input in Mouse trophoblast stem cells using Illumina HiSeq 2000 and Illumina Genome Analyzer IIx
2012-04-18 | E-GEOD-37350 | biostudies-arrayexpress
Project description:p38-MAPKs are stress activated kinases necessary for placental development and nutrient and oxygen transfer during murine post-implantation development. In preimplantation development, p38-MAPK activity is required for blastocyst formation. Additionally, we have previously reported its role in regulating specification of inner cell mass (ICM) towards primitive endoderm (PrE), although a comprehensive mechanistic understanding is currently limited. Adopting live embryo imaging, proteomic and transcriptomic approaches, we report experimental data that directly address this deficit. Chemical inhibition of p38-MAPK activity during blastocyst maturation causes impaired blastocyst cavity expansion, most evident between the third and tenth hours post inhibition onset. We identify an overlapping minimal early blastocyst maturation window of p38-MAPKi inhibition (p38-MAPKi) sensitivity, that is sufficient to impair PrE cell fate by the late blastocyst (E4.5) stage. Comparative proteomic analyses reveal substantial downregulation of ribosomal proteins, the mRNA transcripts of which are also significantly upregulated. Ontological analysis of the differentially expressed transcriptome during this developmental period reveals “translation” related gene transcripts as being most significantly, yet transiently, affected by p38-MAPKi. Moreover, combined assays consistently report concomitant reductions in de novo translation that are associated with accumulation of unprocessed rRNA precursors. Using a phospho-proteomic approach, ± p38-MAPKi, to potentially identify p38-MAPK effectors, we report that clonal siRNA mediated knockdown of Mybpp1a, an rRNA transcription and processing regulator gene, is associated with significantly diminished PrE contribution. Lastly, we show that defective PrE specification caused by p38-MAPKi (but not MEK/ERK signalling inhibition) can be partially rescued by activating the archetypal mTOR mediated translation regulatory pathway. Activated p38-MAPK controls blastocyst maturation in an early and distinctly transient developmental window by regulating gene functionalities related to translation, that creates a permissive environment for appropriate specification of ICM cell fate.
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Project description:Genes and signaling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analyzed. Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE the expression of NANOG, SOX2 and POU5F1 was indeed increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was affected. The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent but epiblast-like state. These data indicate that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells. Microarrays used were bovine whole genome gene expression microarrays V2 (Agilent Technologies) representing 43,653 Bos taurus 60-mer oligos in a 4x44K layout. RNA samples from morula, blastocyst and dissected inner cell mass (ICM) and trophectoderm (TE) were compared in a common reference experiment design using 8 dual channel microarrays with each sample hybridized against an identical sample consisting of a pool of blastocysts total RNA. Within each group of two microarrays for each stage/tissue type, sample versus common reference hybridizations were performed in balanced dye-swap.
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Project description:In the preimplantation mouse embryo TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed both in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP-seq to define genome-wide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation towards the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.
2012-04-18 | GSE37350 | GEO