Objectives: During development, synchronized morphogenetic events shape the adult body. A crucial step is the establishment of the antero-posterior axis, noticeable from 6.5 days post coitum (E6.5) in the mouse by the distinctive aspect of the primitive streak (PS). This site, where gastrulation will be initiated, is directed by Nodal signalling pathway. On the opposite side, cells of the visceral endoderm (VE) express antagonists like Lefty1 and Cer1 counteracting Nodal. The gradient thus establishes the body’s first axis, from the anterior visceral endoderm (AVE) to the posterior primitive streak (PPS). Studies from 2006 showed that progenitors of AVE (pro-AVE) cells could be traced back to the moment of implantation, in the late blastocyst at E4.5. At this stage the embryo is made of only three cell types: the epiblast at the origin of the individual; the trophectoderm which will lead to the placenta; and the primitive endoderm (PrE) which give rise to the yolk sac, an intermittent structure supporting the embryo development until the placenta becomes functional. Within the PrE emerges the pro-AVE cells displaying a complex expression pattern of Lefty1 and Lefty2. We aim to better understand how pro-AVE cells in the embryo decide on their fate early during development.

Methods: To this, we reinvestigated scRNA-seq data, performed HCR-FISH and Immunofluorescence on mouse embryos. To further study the pro-AVE cells molecular mechanism, and due to limited embryonic material, we improved embryoid bodies (EBs) technology (organoid-like) made from mouse embryonic stem cells (mESCs), to mimic the epiblast and PrE cell differentiation

Results: Data mining revealed that these cells have a distinctive molecular signature not previously described. Indeed, after integration of multiple scRNA-seq datasets, we showed that pro-AVE cells harbour an active Nodal signalling, as revealed by the high expression of its targets. Staining RNA in the embryos validated these findings. Aggregated mESCs are able to self-organize into an epiblast surrounded by an epithelium of PrE-like cells expressing Leftys, akin to the embryo. Activating Nodal pathway increased the number of pro-AVE, further reinforcing its role within the embryo.

Conclusions: By precisely manipulating the environment, we successfully directed mESCs towards the fate of a rare cell type, in the mouse embryo, allowing for further exploration of these cells, by genetic manipulation or single cell transcriptomic.