Guiding mouse embryoid self-organization with Nodal signaling inhibtion promotes neuromesodermal-driven axial elongation

Abstract

The formation of the primitive streak is the earliest patterning event during gastrulation that marks the anterior-posterior axis. This event promotes morphogenetic changes that lead to the stereotypical axially elongated shape of the embryo. These changes are coordinated by the combination of different signaling gradients during the process. The Anterior Visceral Endoderm (AVE) regulates this process through the secretion of signaling pathways inhibitors. The goal of this thesis is to understand how the intrinsic self-organizing potential of the epiblast can be guided to promote axial elongation by mimicking specifically the Nodal signaling inhibition role of the AVE. In order to do that, we used three-dimensional stem cell aggregates called embryoid bodies (EBs). This model can reproduce the formation of the primitive streak in-vitro, showing that anterior-posterior patterning is a self-organizing process. However, unlike embryos, they do not undergo axial elongation. To address our hypothesis we used a combination of experimental work on embryoid bodies and computer modeling. First, it was found that inhibition of Nodal signaling at a specific developmental stage triggers convergent extension movements that lead to axial elongation in a way significantly different to other well-established in-vitro models. Secondly, we wanted to characterize the genetic network that controls this process. For that, a single-cell RNA-seq was performed. It was found that Nodal inhibition stimulates differentiation of neuromesodermal progenitors (NMPs) into neuroectodermal fates, while NMPs from control EBs differentiate to mesodermal progenitors. To understand the spatial distribution of these fates in the EBs, some specific markers for neural and mesodermal fates were selected and a whole-mount immunofluorescence combined with Light-sheet microscopy was carried out. The preliminary results showed that upon Nodal inhibition cells with neuroectodermal and mesodermal fates were spatially segregated in the EB for a short period of time. After that, mesodermal progenitors changed to neuroectodermal fates creating highly polarized structures in coordination with axial elongation. Because it became evident that a cross-talk between these two fates can exist, we performed a merging of aggregates made of neuroectodermal fates with aggregates made of mesodermal fates. It was found that mesodermal aggregates elongate more when combined with neuroectodermal ones than when they are subjected to Nodal inhibition in absence of neuroectodermal fates. Finally, to explore more broadly the elongation process observed experimentally, a theoretically 3D agent-based model was developed. The results showed that to achieve an elongation like the one observed in our experimental work a coordinated polarization between neighbor cells is more important than a localized proliferation gradient, although both are needed. Taken together, the data collected in this thesis show how the inhibition of Nodal signaling in EBs guides NMPs towards neuroectodermal fates that promote elongation through the formation of highly polarized neural structures.