Extended Data Fig. 9: Ventral neural patterning in trunk-organoids/ notoroids.

a, Cryosection of H9 notoroid treated with vismodegib showing ventral neural patterning associated with notochord is abolished. b, Optical confocal section of H9 notoroid stained with the ventral neural markers NKX6.1 and OLIG2. OLIG2 expression appears polarized toward the TBXT+ notochord streak. c, Cryosections of MShef4 notoroids from three independent experiments (per row, n = 2) stained for TBXT, PAX6 and OLIG2, the ventral motor neuron progenitor maker, showing association of notochord and ventral patterning. d-f, Analyses of the proximity of notochord and neural cell types. d, Density plot showing distances of FOXA2 + SOX2+ are significantly closer to notochord cells (n = 663, n organoids=9) than PAX6 + SOX2+ cells. The thinner, overlayed density curves for each individual trunk-organoid are scaled by the number of pairwise distances contributing to the overall density plot. e, Cryo-sections of notoroids stained for neural markers SOX2, PAX6, the notochord marker TBXT and FOXA2 (both neural floor plate and notochord). Example of cryo-section segmentation and notochord cells selection (TBXT + SOX2-). Example of the 30 closest PAX6 + SOX2+ and FOXA2 + SOX2+ cells from an example notochord cell picked. f, zoomed inset of cryosection in panel e showing different marker combinations. Scale bars: 50 μm (a,c,e,f).