Inhibition of the ROCK pathway prevents elongation but not cell type segregation.

A: Fixed gastruloids at 96 h treated with (left) or without (right) 10 μM of the ROCK inhibitor Y-27632 during the final 24 h of differentiation. Shown is a maximum z-projection. Cell nuclei were stained with DAPI. Scale bars: 200 μm. B: Wholemount immunostaining of Brachyury/T and Sox2 in fixed gastruloids at 96 h treated with (top) or without (bottom) 10 μM Y-27632. Cell nuclei were stained with DAPI. Scale bars: 200 μm. C: Wholemount immunostaining of Brachyury/T and Cer1 in fixed gastruloids at 96 h treated with (top) or without (bottom) 10 μM Y-27632. Cell nuclei were stained with DAPI. Scale bars: 200 μm. D: LOCO-EFA analysis of measured gastruloids at 96 h treated with (n = 110) or without (n = 106) 10 μM Y-27632. The results of two biological replicates were combined. Each data point is a gastruloid. The scatter plot shows the scaled LOCO-EFA coefficients L2/L1 and L3/L1.