Extended Data Fig. 11: Characterization of neural crest (NC) development in µNTLS.

a. Representative confocal micrographs showing NC cells from R-C and D-V patterned µNTLS on day 11 stained for PAX7, SNAI2, and SOX10. Dashed lines mark dorsal boundaries of µNTLS. Experiments were repeated twice with similar results. b. Representative confocal micrographs showing NC cells and their derivatives in rostral and caudal regions of R-C and D-V patterned µNTLS on day 11, stained for SOX10, TWIST1, PHOX2B, ISL1, and S100B. Dashed lines mark dorsal boundaries of µNTLS. Experiments were repeated three times with similar results. c. Plots showing percentages of TWIST1⁺, PHOX2B⁺, ISL1⁺, and S100B⁺ cells among all disseminated cells in rostral and caudal halves of µNTLS. Error bar represents mean ± s.e.m. n_TWIST1 = n_PHOX2B = n_ISL1 = n_S100B = 8 and n_experiment = 2. Two-sided Student’s t-tests were performed to calculate p values. d. Protocol for generating control R-C and D-V patterned µNTLS and ectopic caudalization of µNTLS. From day 2 to day 5, CHIR (3 µM), FGF8 (200 ng mL⁻¹), and RA (500 nM) are supplemented into the right reservoir of the central channel in addition to NIM to induce caudalization and R-C patterning of µNTLS. From day 5 to day 9, BMP4 (25 ng mL⁻¹) and RA (500 nM) / smoothened agonist (SAG, 500 nM) are supplemented into the top and bottom channels, respectively, to induce D-V patterning of µNTLS. For ectopic caudalization of µNTLS, caudalizing factors CHIR (3 µM), FGF8 (200 ng mL⁻¹), and RA (500 nM) are added together into the left reservoir of the center channel from day 5 to day 9. Culture medium in all reservoirs is then switched back to fresh basal medium (BM) from day 9 to day 11 to allow for further development of NC cells. e. Representative stitched confocal micrographs showing day 5 µNTLS cultured following the protocol in d, stained for OTX2, HOXB1, and SOX10. Zoom-in views of boxed regions are shown on the right. Experiments were repeated three times with similar results. f. Live imaging with the TBXT::T2A-Cre lineage tracer to track progenies of NMPs during the development of R-C and D-V patterned µNTLS from day 5 to day 11 (see Supplementary Video 6). Only caudal ends of µNTLS are monitored as indicated. Dashed lines mark dorsal boundaries of µNTLS. Zoom-in views of boxed regions are shown. Experiments were repeated three times with similar results. g. Representative confocal micrographs showing caudal ends of R-C and D-V patterned µNTLS generated from the TBXT::T2A-Cre lineage tracer, stained on day 11 for SOX10, HOXC9, ISL1, and PHOX2B. Dashed lines mark dorsal boundaries of µNTLS. White arrowheads mark ZsGreen⁺ NC cells, and yellow arrowheads mark ZsGreen⁻ NC cells. Experiments were repeated twice with similar results. h. Plot showing percentages of ZsGreen⁺ cells among all HOXC9⁺ trunk NC cells, ISL1⁺ sensory neurons, or PHOX2B⁺ sympathetic neurons. n_HOXC9 = 11, n_ISL1 = 9, n_PHOX2B = 11, and n_experiment = 2. Error bars represent mean ± s.e.m. i. Representative confocal micrographs showing NC cells in caudal regions of R-C and D-V patterned µNTLS on day 11 generated from wild type (WT) and CDX2-KO human ES cell lines, stained for indicated markers. Dashed lines mark dorsal boundaries of µNTLS. Zoom-in views of boxed regions are shown. Experiments were repeated twice with similar results. In a, b, e, g and i, nuclei were counterstained with DAPI. Scale bars, 100 µm (a, b, and i), 200 µm (e and f), and 50 µm (g).

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