Emergence and patterning dynamics of mouse-definitive endoderm

Abstract

The segregation of definitive endoderm (DE) from bipotent mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE commitment and onset has been challenging as it occurs within a narrow spatiotemporal window in the embryo. Here, we employ a dual Bra/Sox17 reporter cell line to study DE onset dynamics. We find Sox17 expression initiates in vivo in isolated cells within a temporally restricted window. In 2D and 3D in vitro models, DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17 + cells. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Self-sorting coincides with upregulation of E-cadherin but is not necessary for DE differentiation or proliferation. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development

“…From E7.25 onward, some DE cells intersperse in vivo with the VE ( 55 ), whereas the noninterspersed DE cells will translocate to the anterior side of the embryo along with the migrating axial mesoderm. DE cells at the anterior side of the embryo will subsequently also intercalate in the VE ( 57 , 58 ). Because of their large size, the imaging of immunostained ETX embryoids beyond 5 days was difficult; nonetheless, live imaging displayed enhanced GFP (EGFP) ESC-derived cells that were found intercalated in the DSRED-positive VE (for those ETX embryoids that retained DsRed expression somewhat longer) ( Fig.…”

Coordination between endoderm progression and gastruloid elongation controls endodermal morphotype choice

“…Gastruloids start elongating towards the end of Day 4. As Sox17+ cells sort out of the Bra+ population, and then upregulate E-cad to create epithelial domains (24), we hypothesized that in GB morphology this epithelialization occurs before the elongation process, thus leading to formation of tight epithelia that fail to migrate later along the elongating axis. Thus, the stage of endoderm progression at the start of gastruloid elongation may be critical to the resulting morphotype.…”

“…To study the drivers of endoderm morphology in gastruloids, we employed a dual reporter mESC line, Sox17-RFP/Brachyury-GFP, in order to track in real time the onset, expansion and morphology of mesoderm and endoderm in developing gastruloids (24).…”

“…To test this hypothesis, we generated a short delay in endoderm progression. As the Nodal/Activin pathway promotes the differentiation of the DE from the mesendoderm layer (24)(25)(26)(27), we hypothesized that a temporary inhibition of this pathway could cause a short delay in the progression of endodermal differentiation, so that the majority of DE cells do not undergo MET until after the gastruloid substantially elongates, thus shifting DE morphology towards other narrow/tube containing patterns. Using the small molecule SB-431542 ('SB'), a nodal-pathway inhibitor via activin receptor, we partially inhibited endoderm onset and/or expansion.…”

“…Here we use gastruloids and the progression of definitive endoderm (DE) in them to study divergence of morphogenetic programs, employing a dual Bra-GFP and Sox17-RFP reporter cell line (24). We characterize and quantify the possible endoderm morphologies in gastruloids.…”