Extended Data Fig. 4: Tissue organization in gastruloids.

a, Gastruloids formed from Sox1^GFP;Bra^mCherry (SBR) line and stained for Sox2 expression (Sox1^GFP and SOX2 signals are displayed in green and magenta, respectively). White arrowheads indicate tubular SOX2/Sox1-positive neural structures. Red arrowheads point to the presumptive digestive tube. b, WISH on 8-µm transverse cryosections of gastruloids at 144 h AA using Sox2 and Meox1 antisense probes, counter-stained with Nuclear Fast Red. Sox2-positive cells localized predominantly in a compact dorsal domain, whereas Meox1 signals were found in two bilateral domains. The domain of expression of each gene is outlined with white dashed lines. c, Haematoxylin and eosin staining of transverse paraffin sections of different gastruloids at 120 h AA, showing the diversity of cell types and several levels of tissue organization. d, Gastruloids formed from Sox1^GFP;Bra^mCherry ESCs were fixed and stained at 168 h AA for OLIG2 (top, white), PAX3 (middle, red) and PAX7 (bottom, red). Scale bars as indicated. c, d, Gastruloids formed from Sox1^GFP; Bra^mCherry ESCs collected at 168 h AA and stained for SOX17 (magenta, c) or CDX2 (magenta, d). Scale bars as indicated. All immunostaining experiments were repeated twice, with three biological replicates per experiment, with similar results.