Extended Data Fig. 4: YAP signaling and YAP activation on posterior neuruloids.

a, Localization of total YAP during neuruloid formation. b, Treatment with TRULI promotes nuclear (total) YAP at 12 h post-induction and active YAP usually confined to the edge is now throughout the colony. c, Additional stains of posterior neuruloid at 12 h post-induction without and with TRULI showing a dampened pERK1/2 ring and no TBXT+ cells as well as the nuclear accumulation of YAP. Differences in phalloidin staining were also observed. M.I.P. is shown. d, Edge of neuruloid colony at 24 h with S5A-YAP-GFP expressing cells. e, Zoom of a neuruloid colony at 24 h showing ring of TBXT at the edge and YAP-S5A-GFP vs wtYAP-GFP expression. In contrast to wtYAP-GFP, YAP-S5A-GFP+ cells have low/no TBXT; they show high YAP levels both nuclear and cytoplasm and as expected from the mutated serine residues, show no signal for active, unphosphorylated YAP. Quantification of the colonies’ edge (30% radius, panel f) compares the proportion of TBXT+ cells in GFP+ cells versus all cells showing a significant reduction in YAP-S5A case (p-value = 0.00011, panel g). From the images analysed this is a conservative estimation, impaired by cell proximity or segmentation errors. Another way to quantify this effect is by using, colony-wise, the observed TBXT+ cells of all cells at the edge as expectation for the TBXT + GFP+ population. This shows ratios close to 1 for the wtYAP and closer to zero for the YAP-S5A (p-value = 0.00027; main Fig. 3k). Scale bars: 20μm (a,e), 50μm (d), 250 μm colony (b,c).