Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

原肠胚形成是胚胎发育的关键阶段，在此期间胚层得以建立。测序技术的进步导致了控制胚层及其衍生物出现的基因调控程序的识别。然而，基于蛋白质组的早期哺乳动物发育研究很少。为了克服这个问题，我们利用原肠胚和基于多层质谱的蛋白质组学方法来研究原肠胚分化过程中（磷酸）蛋白质表达的整体动态。我们的研究结果揭示了许多蛋白质具有时间表达和每个胚层独特的表达谱，我们还使用单细胞蛋白质组学技术对其进行了验证。此外，我们使用 P300 邻近标记分析了增强子相互作用景观，揭示了许多原肠胚特异性转录因子和染色质重塑因子。随后基于降解决定子的扰动与单细胞 RNA 测序 (scRNA-seq) 相结合，确定了 ZEB2 在小鼠和人类体节发生中的关键作用。总体而言，这项研究为致力于了解哺乳动物胚胎发生的发育和合成生物学界提供了丰富的资源。