Abstract
It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock‐in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis.
Highlights
The recent single-cell genomics revolution has generated transcriptional and epigenetic roadmaps for the formation of the three principal germ layers during gastrulation[1,2,3]
Epithelial-to-mesenchymal transition (EMT) and mesenchymalto-epithelial transition (MET) are evolutionarily conserved processes that occur during development and are essential for gastrulation and embryonic morphogenesis, but if they are dysregulated in adulthood they lead to cancer metastasis[4,5,6]
This short timespan seems insufficient for a cell to down- and upregulate molecular machineries on the messenger RNA and protein level to undergo a complete EMT–MET cycle
Summary
Definitive endoderm is formed in the absence of an EMT–MET cycle. To test this hypothesis and study endoderm differentiation and morphogenesis in more detail, we performed time-resolved and simultaneous lineage labelling and tracking using Foxa2–Venus fusion (FVF) and Sox17–mCherry fusion (SCF) double knock‐in reporter mouse embryonic stem cells (mESCs) and mouse embryos[29,30]. In line with our Foxa[2] knockout results from in vitro differentiations (Fig. 4b), we observed a lack of Cer[1] expression in Venus+ lineage-labelled cells of Foxa2Venus/Venus aggregation chimeras (Fig. 5g) and concomitant upregulation of the Wnt target gene Lef[1] and the EMT transcription factor Snail[1] (Fig. 6e–g). These results suggest that Foxa[2] directly induces the expression of Wnt inhibitors and thereby indirectly inhibits Wnt/β-catenin signalling and target genes, such as Lef[1] and Snail[1] (Fig. 6h)[37,44,45,46,47].
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