Dissection of anterior mesendoderm segregation at single cell level in zebrafish

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, so far, the underlying mechanism of this segregation remains unclear. Here, taking zebrafish prechordal plate (PP) and endoderm (Endo) as research model, using single cell multi-omics and live imaging analyses, we show that anterior endodermal progenitors originate directly from prechordal plate. Deconvolution analysis for bulk RNA-seq datasets of Nodal-injected explants reveals that the specification of anterior endoderm from PP was determined by a relatively lower Nodal signaling. And a single-cell transcriptomic trajectory analysis of wild-type, ndr1 knockdown and lefty1 knockout Nodal explants confirms the diversification of Endo fate from PP progenitors. Genstoe Ontology (GO) enrichment analysis indicates that chromatin organization potentially underlies the segregation of endodermal cell fate from PP. A further single-cell ATAC and RNA sequencing analysis suggests a positive correlation between Nodal activity and chromatin openness. We then identify two transcriptional regulators, gsc and ripply1, which are differentially activated in PP and Endo, and manipulation of their expression levels tilts the cell fate decision between these two lineages. Collectively, our study suggests that different levels of Nodal activity promote transcriptional diversification between PP and Endo potentially through modulation of chromatin states, which eventually leads to diversification in cell fate decisions.

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