Regulation of mRNA polyadenylation governs body plan formation in gastruloids

Gastrulation marks a landmark event in early embryogenesis, laying the foundation for the three germ layers and body plan formation. While transcriptional and epigenetic regulation during gastrulation has been extensively studied, post-transcriptional mechanisms remain underexplored, partly due to technical constraints and limited access to mammalian embryos. Here, we combine the scalability of gastruloids, an in vivo model of early embryonic development, with a single-cell CRISPR screening platform to systematically dissect post-transcriptional regulators of germ layer differentiation. Our screen uncovers previously unrecognized regulators of mesodermal and endodermal fate and enables cell-type-specific functional characterization with single-cell transcriptomic readout. Focusing on the de-adenylase Cnot8, we demonstrate that Cnot8-mediated poly(A) tail regulation is essential for mesodermal differentiation and proper body axis formation, underscoring the critical role of post-transcriptional control in early mammalian development. Together, our findings support a critical role of poly(A) tail regulation in the patterning of the mammalian embryo and establish gastruloid based single cell CRISPR screens as tool to uncover novel regulators and mechanisms of embryogenesis.