Extended Data Fig. 3: Additional posterior neuruloid characterization.

a, Single-z plane images and maximum intensity projection (M.I.P.) of immunofluorescence staining of SOX2, TBXT and phalloidin of day 3 posterior neuruloid. b, SOX1 immunostaining overview illustrating the robustness of the platform. c, SOX2, TBXT and FOXA2 nuclear levels in day3 posterior neuruloids were discretized into low, mid and high-levels. Optical section showing high SOX2 cells located at the centre of the colony whereas high TBXT cells occupy a position at the periphery. Mid TBXT + SOX2+ cells localize to an intermediate ring in the colony. d, UMAP plot of neuruloid day3 single-cell RNA-seq showing additional gene markers for mesoderm population and SHH expression in the notochord progenitors. e, UMAP plot highlighting 7 NotoP single cells co-expressing NOTO + CHRD + FOXA2 + . The presence of these cells was both rare and variable across replicates which formed the basis for our exploration of conditions to further increase them. f, M.I.P. of mesodermal populations (TBX6 and TBXT) in the day3 neuruloid with various levels of FGF. Images in a-b are representative of at least three independent experiments and images in f representative of two independent experiments.