Ripply1 and Gsc collectively suppress anterior endoderm differentiation from prechordal plate progenitors

During gastrulation, the mesendoderm is firstly specified by morphogens such as Nodal, and then segregates into endoderm and mesoderm in a Nodal concentration-dependent manner. However, the mechanism underlying the segregation and crosstalk of different sub-groups within the meso- and endoderm lineages remains unclear. Here, taking zebrafish prechordal plate (PP) and anterior endoderm (Endo) as research model, using single-cell multi-omics and live imaging analyses, we show that anterior Endo progenitors originate directly from PP progenitors. A single-cell transcriptomic trajectory analysis of wild-type,ndr1knockdown andlft1knockout Nodal explants confirms the diversification of anterior Endo fate from PP progenitors. Gene Ontology (GO) enrichment analysis indentifies that the change of chromatin organization potentiates the segregation of endodermal cell fate from PP progenitors. Single-cell ATAC & RNA sequencing further reveals that two transcriptional regulators,gscandripply1, exhibit varied activation patterns in PP and Endo lineages at both the chromatin and RNA expression levels. We further demonstrate that Ripply1 functions coordinately with Gsc to repress endodermal cell fate by directly binding to thecis-elements ofsox32andsox17. Modulating the expression levels of these regulators tilts the cell fate decision between the PP and Endo lineages.