Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency.
Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency.
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牛胚胎中的碱基编辑揭示了 SOX2 在多能性调节中的物种特异性作用
DOI：
10.1371/journal.pgen.1010307
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发表时间：
2022-07
期刊：
影响因子：
4.5
通讯作者：
中科院分区：
文献类型：
--
作者：
在发育过程中，前三个谱系的出现是由一个转录因子网络精心调控的，这在小鼠中得到了最好的阐释。然而，这些因子的作用和调控在包括人类和牛在内的其他哺乳动物中并不完全保守。在此，我们通过在牛早期胚胎中利用胞嘧啶碱基编辑器引入提前终止密码子，建立了一个高效的基因失活系统。利用这种方法，我们确定了三个关键的谱系特异性基因（SOX2、OCT4和CDX2）在牛胚胎中的功能影响。特别是，SOX2基因敲除导致囊胚多能性建立失败。实际上，OCT4水平显著降低，NANOG几乎检测不到。此外，原始内胚层的形成受到损害，只有少量SOX17阳性细胞。对单个囊胚（第7.5天）的RNA - seq分析显示2074个基因失调，其中90%在SOX2缺失的囊胚中上调。有趣的是，包括OCT4和NANOG在内的十几个谱系特异性基因下调。此外，在没有CDX2的情况下，SOX2水平在滋养外胚层中得以维持。然而，OCT4基因敲除并不影响SOX2的表达。总体而言，我们提出在牛中SOX2对OCT4和NANOG的表达是必不可少的，并且CDX2抑制滋养外胚层中SOX2的表达，这与在小鼠中的结果形成鲜明对比。
一个新生命的第一次和第二次细胞命运决定对后续的胚胎和胎盘发育很重要。这些事件是由一个转录因子网络精细调控的，这在小鼠中已有广泛的研究。这些蛋白质的物种特异性作用在哺乳动物中逐渐显现。在此，我们利用牛早期胚胎中的胞嘧啶碱基编辑器开发了一个基因功能缺失系统。我们发现SOX2的表达模式、功能作用和调控在小鼠和牛胚胎之间都有所不同。值得注意的是，SOX2对OCT4和NANOG这两个已确定的多能性因子极其重要。此外，需要CDX2来抑制滋养外胚层中的SOX2。鉴于人囊胚和牛囊胚之间SOX2的相似表达模式，牛胚胎代表了一个在体内研究人类多能性调控的潜在模型。
The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice. The first and second cell fate decisions of a new life are important for subsequent embryonic and placental development. These events are finely controlled by a network of transcriptional factors, which are extensively characterized in mice. Species-specific roles of these proteins are emerging in mammals. Here, we develop a gene loss-of-function system by using cytosine base editors in bovine early embryos. We find that expression pattern, functional roles, and regulation of SOX2 are all different between mouse and bovine embryos. Remarkably, SOX2 is extremely important for OCT4 and NANOG, two well-established pluripotency factors. Furthermore, CDX2 is required to repress SOX2 in the trophectoderm. Given similar expression pattern of SOX2 between human and bovine blastocysts, bovine embryo represents a putative model to investigate human pluripotency regulation in vivo.
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