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Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

ABSTRACT: Proper lineage specification is extremely important for the embryo's implantation and development. Numerous findings of lineage specification emerged in the mouse embryo but remain unclear in the bovine embryo due to the limited techniques. Here, our study is the first to demonstrate that the single base editor (ABE7.10 and BE3) can be applied to bovine embryos for base conversion, which contributes us to further exploring the lineage specification of the bovine embryo. We further show SOX2 is not necessary for blastocyst formation but is required for ICM maintenance in bovine early embryo development. The underlying mechanism may be that SOX2 promoted the differentiation of ICM by maintaining the expression levels of OCT4 and NANOG. Meanwhile, the disappearance of SOX2 in TE of bovine late blastocyst depended on the expression of CDX2.

ORGANISM(S): Bos taurus

PROVIDER: GSE189318 | GEO | 2022/03/22

REPOSITORIES: GEO

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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.

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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Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

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