NOTCH signaling pathway is required for bovine early embryonic development. NOTCH signaling pathway is required for bovine early embryonic development

The NOTCH signaling pathway plays an important role in regulating various biological processes, including cell proliferation, lineage specification and apoptosis. Multiple components of the NOTCH pathway have been identified in mammalian preimplantation embryos. However, the precise role of NOTCH pathway in early embryonic development is poorly understood, especially in domestic animals. Here, we show that key transcripts of the NOTCH pathway exhibit a dynamic pattern throughout early embryonic development with an abundant level before embryonic genome activation in cattle. We also confirmed the presence of active NOTCH1 and RBPJ. By using pharmacological and RNAi tools, we demonstrated that the NOTCH pathway is required for the proper development of bovine early embryos. This functional consequence could be partly attributed to the major transcriptional mediator-RBPJ, whose deficiency also compromised the embryo quality. Indeed, we observed a significant increase of histone H3 serine 10 phosphorylation (pH3S10, a mitosis marker) positive blastomeres in not only NOTCH1 depleted embryos but RBPJ depleted ones. Importantly, RNA-seq analysis revealed that either NOTCH1 or RBPJ depletion triggers a reduction in H1FOO that encodes the oocyte-specific linker histone H1 variant. Interestingly, depleting H1FOO also results in detrimental effects on the developmental competence of early embryos, similar with NOTCH1 inhibition. Overall, our results reveal a crucial role for NOTCH pathway in regulating bovine preimplantation development, likely by controlling cell proliferation and maintaining H1FOO expression. Overall design: Late morula (D6) were harvested from nonspecific siRNA-injected control and NOTCH1 or RBPJ KD groups (n = 2; 15 embryos/group/replicate). Total RNA extraction was performed with a PicoPure RNA Isolation Kit based on the manufacturer’s manual. Before RNA isolation, equal amounts of GFP and RFP mRNAs (2×106 copies) were added into each group as spike-in controls. mRNA separation was achieved using oligo(dT)25 beads. Sequencing libraries were constructed with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) based on the manufacturer’s instructions.

NOTCH信号通路（NOTCH signaling pathway）在调控多种生物学过程中发挥关键作用，涵盖细胞增殖、细胞谱系特化与细胞凋亡。目前已在哺乳动物着床前胚胎中鉴定出NOTCH通路的多种组分。然而，NOTCH通路在早期胚胎发育中的精准功能仍有待阐明，在家畜中相关研究尤为匮乏。本研究发现，牛的NOTCH通路关键转录本在早期胚胎发育过程中呈现动态表达模式，且在胚胎基因组激活（embryonic genome activation）前呈现较高丰度。我们同时证实了活化型NOTCH1与RBPJ的存在。通过药理学手段与RNA干扰（RNAi）工具，我们证明NOTCH通路对于牛早期胚胎的正常发育不可或缺。这一功能效应可部分归因于核心转录介导因子RBPJ，其敲低同样会降低胚胎质量。值得注意的是，我们观察到，无论是NOTCH1敲低胚胎还是RBPJ敲低胚胎，其组蛋白H3丝氨酸10磷酸化（histone H3 serine 10 phosphorylation, pH3S10，有丝分裂标志物）阳性的卵裂球数量均显著增加。重要的是，RNA测序（RNA-seq）分析显示，敲低NOTCH1或RBPJ都会导致编码卵母细胞特异性连接组蛋白H1变体的H1FOO基因表达水平下调。有趣的是，敲低H1FOO同样会对早期胚胎的发育能力产生不利影响，其效果与抑制NOTCH1相似。综上，本研究结果揭示了NOTCH通路在调控牛着床前胚胎发育中的关键作用，其潜在机制可能是通过控制细胞增殖并维持H1FOO的表达。

整体实验设计：从非特异性小干扰RNA（small interfering RNA, siRNA）注射的对照组以及NOTCH1或RBPJ敲低（KD）组中收集发育第6天的晚期桑椹胚，每组设置2个生物学重复，每个重复包含15枚胚胎。总RNA提取采用PicoPure RNA提取试剂盒，严格按照制造商说明书操作。在RNA提取前，向每组样品中加入等量的GFP与RFP mRNA（2×10^6拷贝）作为外参对照。使用oligo(dT)25磁珠完成mRNA的分离纯化。测序文库的构建基于Illumina平台，采用NEBNext Ultra RNA文库制备试剂盒（New England Biolabs），并严格遵循制造商的操作指南。