Deciphering lineage specification during early embryogenesis in mouse gastruloids using integrative multi-layered proteomics [ChIP-seq]. Deciphering lineage specification during early embryogenesis in mouse gastruloids using integrative multi-layered proteomics [ChIP-seq]

Gastrulation is a critical stage of embryonic development during which the three germ layers are established. Deciphering the molecular mechanisms underlying this proces from a protein perspective remains a significant challenge. To address this, we employed a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho)protein expression during differentiation of ESCs towards gastruloids – an in vitro model of gastrulation-stage embryogenesis. Our findings revealed that many proteins exhibited temporal expression with unique expression profiles corresponding to the three germ layers. Additionally, we profiled enhancer interaction landscapes in ESCs and gastruloids using p300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron based perturbations combined with scRNA-seq revealed a critical role for Zeb2 in regulating mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis. Overall design: mouse gastruloids were generated as previously described (Protoc. Exch. https://doi.org/10.1038/protex.2018.094). ChIP-seq was performed on undifferentiated mESCs and 120h gastruloids.

原肠胚形成（Gastrulation）是胚胎发育的关键阶段，此阶段确立三个胚层的形成。从蛋白质组视角解析该过程背后的分子机制仍是一项重大挑战。为解决这一难题，我们采用基于多层质谱的蛋白质组学方法，探究胚胎干细胞（Embryonic Stem Cells, ESCs）向类原肠胚（gastruloids）——一种模拟原肠胚阶段胚胎发生的体外模型——分化过程中（磷酸化）蛋白质表达的全局动态变化。

研究结果显示，诸多蛋白质呈现出时间依赖性表达模式，其独特的表达谱与三个胚层一一对应。此外，我们利用p300邻近标记技术，绘制了胚胎干细胞与类原肠胚的增强子互作图谱，鉴定出大量类原肠胚特异性转录因子与染色质重塑因子。后续基于降解子（degron）的扰动实验结合单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）结果表明，Zeb2在调控小鼠与人类体节发生过程中发挥关键作用。

综上，本研究为致力于解析哺乳动物胚胎发生机制的发育生物学与合成生物学研究群体提供了丰富的资源库。

整体实验设计：按照此前报道的方法制备小鼠类原肠胚（Protoc. Exch. https://doi.org/10.1038/protex.2018.094）。对未分化的小鼠胚胎干细胞以及培养120小时的类原肠胚进行染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）。