A wave of WNT signaling balanced by secreted inhibitors controls primitive streak formation in micropattern colonies of human embryonic stem cells

Abstract

Long-range signaling by morphogens and their inhibitors define embryonic patterning yet quantitative data and models are rare, especially in humans. Here, we use a human embryonic stem cell micropattern system to model formation of the primitive streak (PS) by WNT. In the pluripotent state, E-cadherin (E-CAD) transduces boundary forces to focus WNT signaling to the colony border. Following application of WNT ligand, E-CAD mediates a front or wave of epithelial-to-mesenchymal (EMT) conversion analogous to PS extension in an embryo. By knocking out the secreted WNT inhibitors active in our system, we show that DKK1 alone controls the extent and duration of patterning. The NODAL inhibitor cerberus 1 acts downstream of WNT to refine the endoderm versus mesoderm fate choice. Our EMT wave is a generic property of a bistable system with diffusion and we present a single quantitative model that describes both the wave and our knockout data.

Mechanics regulate human embryonic stem cell self-organization to specify mesoderm

“…Our work extends prior findings, which illustrated that variations in BMP receptor accessibility and secreted morphogen concentrations lead to patterning of mesoderm specification within a concentric ring inwards from the hESC colony edge (Chhabra et al, 2019;Etoc et al, 2016;Manfrin et al, 2019;Martyn et al, 2019;Tewary et al, 2019Tewary et al, , 2017Warmflash et al, 2014), to implicate force as an additional regulator of self-organization, and mesoderm specification in particular. Our studies expand upon these findings to suggest that colonies of hESCs cultured on compliant substrates are particularly useful for studying the physical and molecular regulators of early embryogenesis, specifically because they recapitulate the morphogenesis and transcriptional reprogramming that regulates gastrulation in the embryo.…”

“…We identify a mechanism whereby regional high cell-adhesion tension modulates the conformation of β-catenin at cadherin-catenin complexes, permitting Src-mediated phosphorylation and release of β-catenin from cell junctions. The modified β-catenin is now free to translocate to the nucleus, where it exerts its transcriptional activity and induces the expression of multiple Wnt ligands that feed forward to promote and reinforce mesoderm specification and gastrulation (Bienz, 2005;Chhabra et al, 2019;Kemp et al, 2005;Lindsley et al, 2006;Martyn et al, 2019;Laralynne Przybyla et al, 2016). Notably, Wnt/β-catenin signaling is conserved among model organisms and plays multiple roles throughout the course of embryogenesis and in adult stem cells (Clevers, 2006;Eliazer et al, 2019;Petersen and Reddien, 2009;van Amerongen and Nusse, 2009), implying that the tension-regulated activation of the pathway that we illustrate here is likely also conserved and re-used throughout development.…”

“…To verify that cell tension specifies mesoderm by regulating Wnt/β-catenin signaling, we used FACS to isolate T-positive cells from patterned hESC colonies following BMP4 stimulation (36 hours; subsequent to β-catenin release from the E-cadherin junctions), and looked for evidence of Wnt signaling ( Figure 7A). Gene expression analysis revealed that the canonical Wnt ligands Wnt3a and Wnt8a, which are critical for mesoderm specification (Chhabra et al, 2019;Kemp et al, 2005;Lindsley et al, 2006;Martyn et al, 2019;Laralynne Przybyla et al, 2016), were expressed at significantly higher levels in the T-positive cells isolated from the regions of high cell-adheesion tension ( Figure 7B). By contrast, levels of the non-canonical ligand Wnt4 were higher in the isolated T-negative cells ( Figure 7B).…”

High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human embryonic stem cell gastruloid cultures

“…Corroborating previous studies (Warmflash et al, 2014;Martyn, Brivanlou and Siggia, 2019), transcriptomes of Mesoderm-1 and -2 contain an EMT signature. A hallmark of EMT during gastrulation is Cadherin switching, where CDH1 is downregulated and CDH2 is upregulated (Hatta and Takeichi, 1986;Radice et al, 1997).…”

N-cadherin stabilises neural identity by dampening anti-neural signals

“…This could help ensure that neural commitment proceeds robustly in the embryo. It has recently been proposed that cadherins propagate mesodermal differentiation from cell to cell in an in vitro model of the primitive streak, although in that case communication is propagated predominantly through changes in E-cadherin rather than N-cadherin (53). Differentiation is more variable and unpredictable in culture compared to the embryo, even though the extrinsic signalling environment in a culture dish can be tightly controlled.…”