Extended Data Fig. 8: Molecular characterization of human axioloids exposed to agonists or inhibitors of retinoic acid (RA) signaling.
a, Brightfield images of 201B7 Luc-derived axioloids at 96 h (top) and 120 h (bottom) embedded in MG alone and supplemented with (from left to right) DMSO (2 independent experiments, n = 12 for 96 h and n = 13 for 120 h), BMS493 (a pan-RAR inverse agonist) (3 independent experiments, n = 18 for 96 h and n = 17 for 120 h), or +MG +RAL supplemented with DMSO (3 independent experiments, n = 9 for both 96 h & 120 h), BMS493 (3 independent experiments, n = 9 for both 96 h & 120 h), AGN193109 (a pan-RAR inhibitor) (3 independent experiments, n = 11 for somite number and n = 18 for length measurements for 96 h & 120 h) or ER50891 (a RARα-specific inhibitor) (3 independent experiments, n = 12 for somite number and n = 18 for length measurements for 96 h & 120 h). b, Corresponding axioloid length measurements and somite numbers for axioloids shown in a (axioloids embedded in MG only are not plotted). Boxes represent the first and last 4th percentile intervals, whiskers show the minimum and maximum values within that range, middle line the median, and dots represent individual data points. Somite numbers for +MG +RAL (3 independent experiments, n = 9 for both 96 h & 120 h) are shown in upper panel. Length measurements for +MG +BMS (3 independent experiments, n = 18 for 96 h and n = 17 for 120 h) and +MG + DMSO (3 independent experiments, n = 9 for 96 h and n = 9 for 120 h) are shown in the lower panel. c, Serial images of forming +MG +RAL axioloids at 5 h intervals, from 74 h to 119 h in the absence or presence of RA signaling inhibitors (extracted from Supplementary Video 6). Red arrowheads pinpointing to areas where segmentation is ongoing whereas blue arrowheads highlight the areas where segmentation is completed. d–m, Immunofluorescence and in situ hybridization-based characterization of axioloids treated with different RA signaling inhibitors. d,j, and l, Immunofluorescence staining of axioloids embedded in +MG +RAL supplemented with d, BMS493, j, AGN193109 and l, ER50891 at 120 h with F-actin (Phalloidin) in white, TBXT in cyan, FN1 in yellow and MEOX1 in red. e, Signal intensity quantification of d, along the posterior to anterior axis (3 independent experiments, n = 11). f,h,k, and m, HCR staining of UNCX in cyan, TBX18 in magenta and MESP2 in yellow of axioloids embedded in +MG +RAL supplemented with BMS493 at f, 96 h and h, 120 h of culture or supplemented with k, AGN193109 or m, ER50891 at 120 h of culture. g, and i Corresponding quantification of signal intensities along the posterior to anterior axis for +MG +RAL axioloids treated with BMS493 at g, 96 h (2 independent experiments, n = 9) and i, 120 h (4 independent experiments, n = 8) of culture. Lines in e,g and i 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.
