Extended Data Fig. 15: Morphological, molecular and functional characterization of patient-like iPSC-derived axioloids with mutations in HES7 and MESP2.
Panels a–e, show data for HES7 KO2, panels f–j, show data for HES7R25W Mt2 and panels k–o, show data for MESP2 KO2; all control and patient-like axioloids were grown under +MG +RAL conditions. a,f, and k, Serial brightfield images of a forming axioloid at 72 h, 96 h and 120 h. b,g, and l, Immunofluorescence staining and signal quantification of +MG +RAL embedded axioloids at 120 h. Top, merged channel images of axioloids stained for F-actin (Phalloidin) in gray, TBXT (BRA) in cyan, FN1 in yellow, and MEOX1 in red. Bottom, corresponding quantification along the posterior to anterior axis of TBXT (blue line) and MEOX1 (in red) signal intensity for HES7 KO2 (3 independent experiments, n = 9), HES7R25W MT2 (3 independent experiments, n = 9) and MESP2 KO2 (3 independent experiments, n = 12). c,h, and m, HCR staining and signal quantification of +MG +RAL embedded axioloids at 120 h, UNCX in cyan and TBX18 in magenta, MESP2 in yellow, and corresponding signal intensity measurements along the posterior to anterior axis normalized to the position of the MESP2 signal peak for HES7 KO2 (3 independent experiments, n = 8), HES7R25W Mt2 (3 independent experiments, n = 8) and MESP2 KO2 (3 independent experiments, n = 9). d,i, and n, Kymograph of HES7 oscillatory activity for d, HES7 KO1 and KO2, i, HES7R25W Mt1 and Mt2, and n, MESP2 KO1 and KO2 patient-like axioloids embedded in +MG +RAL. All experiments were performed under +MG +RAL condition. e,j,o, Average measured HES7:Luciferase signal over time for HES7 KO1 (5 independent experiments, n = 8), HES7 KO2 (6 independent experiments, n = 9), HES7R25W Mt1 (5 independent experiments, n = 10), HES7R25W Mt2 (5 independent experiments, n = 9), MESP2 KO1 (3 independent experiments, n = 6), MESP2 KO2 (3 independent experiments, n = 7) and 201B7 Luc +MG +RAL (5 independent experiments, n = 9 data identical to the one shown in Fig. 14j). Lines in b,c,e,g,h,j,l,m and o correspond to mean values, error bands represent the 95% confidence interval, of which the top and bottom show the 2.5 and 97.5 percentiles for each data point. Scale bar is 200 μm. a.u., arbitrary units; n = number of axioloids.
