p38-MAPK mediated rRNA processing and translation regulation enables PrE differentiation during mouse blastocyst maturation

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. 3 timepoints of mouse preimplantation development (E3.5+4 hours, E3.5+7 hours, E3.5+10 hours), for each timepoint there are two replicates of control (DMSO) dataset and two replicates of p38-inhibited dataset.

p38丝裂原活化蛋白激酶（p38-MAPKs）是一类应激激活的激酶，在小鼠植入后发育过程中，对于胎盘发育以及营养与氧气转运必不可少。在植入前发育阶段，p38-MAPK活性是囊胚形成的必需条件。此外，我们此前已有研究报道，其可调控内细胞团（ICM）向原始内胚层（PrE）的命运特化，但目前对其背后的分子机制仍缺乏全面深入的解析。本研究采用活胚胎成像、蛋白质组学与转录组学手段，报道了可填补这一研究空白的实验数据。在囊胚成熟阶段对p38-MAPK活性进行化学抑制，会导致囊胚腔扩张受损，该缺陷在抑制剂处理后3至10小时最为显著。我们鉴定出p38-MAPK抑制剂（p38-MAPKi）发挥作用的早期囊胚成熟关键窗口期，在此窗口期施加抑制即可导致晚期囊胚（E4.5）阶段的原始内胚层细胞命运异常。比较蛋白质组学分析显示，核糖体蛋白的表达显著下调，而其对应的mRNA转录本则显著上调。对该发育阶段差异表达转录组的功能本体分析显示，与"翻译过程"相关的基因转录本是受p38-MAPKi影响最显著、但仅呈瞬时变化的一类基因。此外，多组联合实验持续观测到，新生蛋白质合成量显著下降，这一现象与未加工rRNA前体的积累相关。为了潜在鉴定p38-MAPK的效应因子，我们采用了添加或不添加p38-MAPKi的磷酸化蛋白质组学策略。研究发现，靶向rRNA转录与加工调控基因Mybpp1a的克隆化小干扰RNA（siRNA）介导的基因敲低，会导致原始内胚层的细胞贡献显著减少。最后，我们证实，由p38-MAPKi（而非丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶（MEK/ERK）信号通路抑制）引发的原始内胚层特化缺陷，可通过激活经典的雷帕霉素靶蛋白（mTOR）介导的翻译调控通路得到部分挽救。本研究包含小鼠植入前发育的3个时间点（E3.5+4小时、E3.5+7小时、E3.5+10小时），每个时间点均设置2个二甲基亚砜（DMSO）对照组生物学重复样本与2个p38抑制组生物学重复样本。