Table 1_Screening for novel factors involved in mouse early embryonic development using inhibitor libraries.docx

Mammalian early embryonic development is regulated by numerous factors, yet not all have been identified. Although omics approaches such as next-generation sequencing and proteomics provide powerful tools, screening methods using inhibitor libraries remain highly effective for identifying novel factors involved in embryonic development. To this end, we developed a novel screening system that combines ultra-superovulation technology with one-cell stage embryos cryopreservation in mice. Using this system, we screened 95 inhibitors to identify factors essential for the development of mouse fertilized eggs and identified 16 factors, including 5 previously known ones. Among the known factors, two were ATPases, and our data confirmed that inhibition of different ATPase types arrested embryonic development at distinct stages. In addition, we discovered novel regulators affecting various developmental stages, including a p53 activator (PRIMA-1), cathepsin D, CXCR2, and potassium channels (SK2 and SK3). Genome editing experiments involving knockout of the cathepsin D and CXCR2 genes further verified the arrest of embryonic development. These results demonstrate that our developed screening method can effectively identify novel factors involved in embryonic development. Application of this approach to additional inhibitor libraries and other species may facilitate the discovery of further species-specific regulators of early embryonic development.

哺乳动物早期胚胎发育受诸多因子调控，但目前并非所有调控因子均已被鉴定。尽管下一代测序（next-generation sequencing）、蛋白质组学（proteomics）等组学方法已提供了强大的研究工具，但利用抑制剂文库的筛选方法仍是鉴定胚胎发育相关新型因子的高效手段。为此，我们开发了一套全新的筛选系统，该系统将超高阶超数排卵（ultra-superovulation）技术与小鼠单细胞阶段胚胎冷冻保存技术相结合。利用该系统，我们对95种抑制剂开展筛选，以鉴定小鼠受精卵发育所必需的调控因子，最终共鉴定出16种相关因子，其中5种为已知调控因子。在已知因子中，有2种为ATP酶（ATPases），我们的实验数据证实，抑制不同类型的ATP酶会使胚胎发育停滞于不同阶段。此外，我们还发现了影响不同发育阶段的新型调控因子，包括p53激活剂（PRIMA-1）、组织蛋白酶D（cathepsin D）、趋化因子受体CXCR2以及钾离子通道（SK2、SK3）。针对组织蛋白酶D和CXCR2基因的基因敲除基因组编辑实验，进一步验证了上述因子可诱导胚胎发育停滞。上述结果表明，我们开发的筛选方法可有效鉴定参与胚胎发育的新型调控因子。将该方法应用于更多抑制剂文库以及其他物种，将有助于进一步发现物种特异性的早期胚胎发育调控因子。