Role of OCT4 during preimplantation development of bovine embryos

文献中的证据表明，牛胚胎的植入前发育与广泛研究的小鼠模型有很大的不同。不同之处包括滋养外胚层中OCT4和CDX2的相互作用，以及成纤维细胞生长因子/丝裂原活化蛋白K信号在次母细胞二级谱系分化中的作用。作为一种补充小鼠胚胎发育的模型，加强对牛着床前发育中第一次谱系分化的机制及其调控的了解，将有助于我们全面理解哺乳动物植入前胚胎发育过程中早期谱系指定和选择性维持多能性的保守和物种特异性机制。由于OCT4同时参与多能性和分化的调节，破译其在牛体内的功能具有特殊的意义。因此，我们利用CRISPR/Cas9诱导的OCT4基因敲除的成年成纤维细胞和体细胞核移植(SCNT)，旨在阐明OCT4在牛着床前发育中的作用。我们特别假设，OCT4在第二次谱系分化中的参与在牛胚胎和已发表的小鼠机制中是不同的。此外，我们认为来自体细胞核移植的胚胎反映了受精胚胎早期谱系发育的主要机制，并且在胚胎嵌合体的两个聚集伙伴之一中OCT4的缺失导致了细胞谱系分配的偏差。我们的工作计划将包括在早期胚胎细胞系特异的标记上进行免疫荧光，即低分化的GATA6，滋养外胚层的NANOG和CDX2，以及通过RNA测序进行转录组分析。为了证实通过SCNT产生的胚胎的研究结果，我们希望通过将CRISPR/Cas9直接注射到体外受精的受精卵中来产生OCT4 KO胚胎。此外，我们的目标是通过在第7天将胚胎移植到受体牛以及在第12天恢复胚胎来阐明OCT4在牛胚胎二次世系分化过程中的作用。据我们所知，这是第一次使用反向遗传学方法研究OCT4在牛着床前发育过程中的功能，并首次全面地研究了牛胚胎的第二次世系分化。

Evidence in literature suggests substantial differences in preimplantation development of bovine embryos to the extensively studied mouse model. Differences include the interaction of OCT4 and CDX2 in the trophectoderm as well as the role of FGF/MAPK signaling during the second lineage differentiation of the hypoblast. Enhanced knowledge of the mechanisms and their regulation involved in the first lineage differentiations during preimplantation development in bovine, as a model complementary to mouse, would increase our overall understanding of conserved and species-specific mechanisms of early lineage specification and selective maintenance of pluripotency during mammalian preimplantation embryo development. With OCT4 being involved in both regulation of pluripotency and differentiation, deciphering its function in bovine is of special interest. Therefore, using adult fibroblasts with a CRISPR/Cas9 induced knockout of OCT4 and somatic cell nuclear transfer (SCNT), we aim to elucidate the role of OCT4 during bovine preimplantation development. We specifically hypothesize, that the involvement of OCT4 during the second lineage differentiation differs between bovine embryos and published mechanisms in the mouse. Furthermore, we suggest that embryos from somatic cell nuclear transfer reflect the principle mechanisms of early lineage development observed in fertilized embryos and that loss of OCT4 in one of the two aggregation partners in embryonic chimeras induces bias in cell lineage allocation. Our work program will include immunofluorescence on markers specific to the cell lineages in the early embryo, i.e. GATA6 for hypoblast, NANOG for epiblast and CDX2 for trophectoderm, and transcriptome analysis by RNA sequencing. To confirm findings from embryos produced via SCNT, we want to produce OCT4 KO embryos through direct injection of CRISPR/Cas9 into in vitro fertilized zygotes. Additionally, we aim to elucidate the second lineage differentiation and the role of OCT4 during by transfer of embryos to recipient cows at day 7 and their recovery at day 12. This is to our knowledge the first study on the function of OCT4 during bovine preimplantation development using a reverse genetics approach and the first comprehensive investigation of the second lineage differentiation in bovine embryos.