WNT signaling memory is required for ACTIVIN to function as a morphogen in human gastruloids

Abstract

Self-organization of discrete fates in human gastruloids is mediated by a hierarchy of signaling pathways. How these pathways are integrated in time, and whether cells maintain a memory of their signaling history remains obscure. Here, we dissect the temporal integration of two key pathways, WNT and ACTIVIN, which along with BMP control gastrulation. CRISPR/Cas9-engineered live reporters of SMAD1, 2 and 4 demonstrate that in contrast to the stable signaling by SMAD1, signaling and transcriptional response by SMAD2 is transient, and while necessary for pluripotency, it is insufficient for differentiation. Pre-exposure to WNT, however, endows cells with the competence to respond to graded levels of ACTIVIN, which induces differentiation without changing SMAD2 dynamics. This cellular memory of WNT signaling is necessary for ACTIVIN morphogen activity. A re-evaluation of the evidence gathered over decades in model systems, re-enforces our conclusions and points to an evolutionarily conserved mechanism.

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

“…Some hiPSCs located on the colony's edge could translocate interiorly while others remained at the edge during colony expansion ( Figure 4G). Tracking spatial locations of a cell within a colony can yield new information regarding geometric cues, as recent work identified a subpopulation of PSCs on colony edges that share hallmark properties with primitive endoderm, with distinct cell states compared to those in the interior (Yoney et al, 2018;Nakanishi et al, 2019). To understand the kinetics of PSC translocation from the perimeter to the interior, PSCs at the edge of colonies were tracked over a period of 12 hours and analyzed for position changes from the edge to the interior ( Figure 4H).…”

“…We found this reporter of single cell lineage and clonal expansion had broader utility and enabled longitudinal quantification of cell morphology and migration. Studies have recently identified a subset population of PSCs that reside on the perimeter of PSC colonies (Li et al, 2010;Warmflash et al, 2014;Rosowski et al, 2015;Yoney et al, 2018;Nakanishi et al, 2019).…”

“…These edge PSCs share hallmark properties with primitive endoderm and display distinct non-canonical Wnt signaling and gene expression that enhance their colony initiation capacity compared to PSCs localized in the colony interior (Yoney et al, 2018;Nakanishi et al, 2019).…”

Communication codes in developmental signaling pathways

“…Lastly, it is interesting to contrast TGF-β with BMP4. Both ligands belong to the same superfamily of signaling ligands, but BMP4 induces sustained signaling activity whereas temporal adaptation appears to be built into the TGF-β pathway (Nemashkalo et al, 2017;Yoney et al, 2018). How and why the pathways exhibit such distinct dynamic behaviors remains unclear.…”

“…Similar reconstitution approaches have been applied to embryonic stem cells to enable spatially organized differentiation in 2D and 3D. Some of the 2D models are especially compatible with quantitative time-lapse imaging and spatio-temporally controlled perturbations, and hence are particularly suitable for studying signaling activities in space and time at the single cell level Morgani et al, 2018;Thorne et al, 2018;Warmflash et al, 2014;Yoney et al, 2018). Together, these systems are poised to address fundamental questions about how developmental signaling information is encoded in space and time during early embryonic development.…”