Many thanks to all members of our lab and to our collaborators in the Lutolf and Zavolan labs!

In sum, our findings highlight polyadenylation-mediated control of transcript stability as a critical mechanism in shaping the mammalian body plan and position gastruloid-based single-cell CRISPR screening as a powerful platform for systematic discovery of developmental regulators.

Cells adopting notochord identity in Cnot8 KO gastruloids closely matched embryonic notochord cell profiles. This was accompanied by increased Nodal signaling and a profound loss of Hox gene expression, with a shift toward dorso-ventral and anterior primitive streak fates.

Single-cell RNA sequencing of Cnot8-deficient gastruloids demonstrated severely altered mesodermal patterning, with paraxial mesoderm largely absent and a bias toward axial mesoderm and notochord differentiation.

Intriguingly, Cnot8 KO organoids exhibited indeed transcript stabilization of Nodal along with multiple components and modulators of the TGF-β and Wnt signaling pathways, consistent with profoundly altered patterning cues.

Given the established role of the CCR4-NOT complex in mRNA deadenylation and turnover, we profiled the poly(A) landscape in Cnot8 knockout organoids and observed marked poly(A) tail elongation of key signaling transcripts, including Nodal.

Interestingly, transcriptomic analysis of these Brachyury-positive cells in Cnot8 KO organoids revealed signatures of notochord cells, a cell type usually absent in mammalian gastruloids.

Our screen revealed several novel regulators of germ layer differentiation, including a strong paraxial mesoderm-specific role of the CCR4-NOT component Cnot8. Loss of Cnot8 resulted in gastruloid patterning defects such as the formation of multiple Brachyury poles and aberrant growth.

Validation of our screen revealed strong global depletion of general depletion controls, while germ layer controls were selectively depleted in their respective germ layers, accompanied by the loss of characteristic transcriptomic markers.

Led by the brilliant @dadita.bsky.social‬, we first established a single-cell CRISPR screening platform in gastruloids and designed a library targeting 200 RNA-binding proteins as well as known regulators of germ layer differentiation.