Fig. 4: Nodal and Lefty spread via a relay mechanism in 2D-gastruloids.

a smFISH results showing mRNA for Nodal and Lefty1/2 at 12 h, 24 h, 30 h, 36 h post-BMP4 treatment, scale bar, 100 µm. b Quantitative analysis of the smFISH mean intensity as a function of distance from colony center at the indicated times, individual colonies are shown as colored line. Black line shows mean values + /− SEM, n = 10 biological replicates. c Representative images of gastruloids at 42 h, showing distribution of RNA and protein of Nodal and pSmad2/3, scale bar, 100 µm. The rectangular box indicates the enlarged area in the right panel, scale bar, 50 µm. d Simultaneous quantification of Nodal RNA and protein and pSmad2/3. Normalized mean intensity is quantified over time as function of distance from colony center. Data are presented as mean values + /− SEM, n = 6 biological replicates, quantification of individual colonies is shown in Supplementary Fig. 8c. e Representative images of gastruloids at 42 h, showing Lefty mRNA and protein, scale bar, 100 µm. f Quantification of Lefty RNA and protein over time, mean intensity as function of distance from colony center. Data are presented as mean values + /− SEM, n = 6 biological replicates, quantification of individual colonies is shown in Supplementary Fig. 8d.