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Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig

ABSTRACT: The inner cell mass in blastocyst is the origin of all the somatic and germ cells in mammals, and of pluripotent stem cells in vitro. As the conserved principles between pig and human, here we performed comprehensive single-cell RNA-seq for porcine early embryos from oocyte to early blastocyst. We show the specification of inner cell mass and trophectoderm in morula, and the molecular signature of the precursors. We demonstrate the existence of naïve pluripotency signature in morula and inner cell mass of early blastocyst, and the specific pluripotent genes and the activity of signalling pathways highlight the characteristics of the naïve pluripotency. We observe absence of dosage compensation with respect to X-chromosome in morula, and incomplete dosage compensation in early blastocyst. However, the dynamics of dosage compensation may be independent on the expression of XIST induced X-chromosome inactivation. Our study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine pluripotent stem cells and improve the research in regenerative medicine.

ORGANISM(S): Sus scrofa

PROVIDER: GSE139512 | GEO | 2019/10/29

REPOSITORIES: GEO

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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.

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 ERK-inhibited ICM and control-treated ICM were compared in a common reference experiment design using 4 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 treatment, sample versus common reference hybridizations were performed in balanced dye-swap.

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Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig

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