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. 2021 May 21;12:3020. doi: 10.1038/s41467-021-23294-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a Two SOX10-expressing neural crest cell (NCC) populations are distinguished by TFAP2A expression (purple) and non-expression (red) in day 16 EMLOs by scRNAseq. NCC cluster 14 is expanded. b TFAP2α (magenta) and SOX10 (cyan) double-positive cells in day 6 H3.3.1 EMLO (top) and diverging NCC lineages by distinct SOX10/TFAP2α immunostaining in day 20 EMLO (middle). High magnification representative day 20 EMLO is shown (bottom). White dotted arrows depict potential NCC migration about the gut tube. c Two TFAP2A-expressing NCC populations are distinguished by TUBB3 expression (blue) and non-expression (orange) (top). The NCC cluster 14 is expanded. The subsets of putative neuronal (TFAP2α+ in green, TUJ1+ in red) and non-neuronal (TFAP2α+, TUJ1−) NCCs are visualized as well by IF that is best seen at the ganglion in the white dotted box (bottom). TFAP2α signal is higher outside of the ganglion. d EMLOs lacking the spinal cord (SC) compartment does not contain NCCs, supporting an origin in SC. e N-cadherin (cyan) expression restricted to SC and the gut tube anterior pole, counterstained with ZO-1 (magenta) and shown at days 13 and 16. High magnification images provided. f ISL1 (green) and BMP2/4 (ref) IF in day 22 H3.3.1 EMLOs. ISL1+ cellular pools in proximity to the gut tube are observed in both lines, and increased expression in budding regions of the tube versus non-budding regions. High magnification ISL1 colocalization with TUJ1 is shown (right). NCC biomarkers were stained in H3.3.1 EMLOs in N = 8 of 11 formation experiments, and N = 3 formation experiments for the other representative lines. g Histogram of TFAP2α (purple) and SOX10 (yellow) positive nuclei in mesoderm–endoderm (ME) over time at day 13 (n = 5 EMLOs TFAP2α, n = 6 EMLOs SOX10) versus day 22 (n = 7 EMLOs TFAP2α, n = 8 EMLOs SOX10) in H3.3.1 EMLOs. (unpaired two-tailed t test **p = 0.0033, t = 3.814, df = 10 TFAP2α; ***p = 0.0004, t = 4.873, df = 12). h Plot of cell number (DAPI/TUJ1) per ganglion over time at day 13 (n = 10 EMLOs) versus day 22 (n = 9 EMLOs) in H3.3.1 EMLOs (****p < 0.0001, t = 5.854, df = 17). Data reported as (mean ±s.e.m.). i Histogram quantification of EMLOs with neuronal ganglionic structures in ME in F3.5.2 (n = 25), H3.3.1 (n = 31), and A2.1.1 (n = 19) ED-hiPSC lines at day 22 (median). Colors denote N = 3 separate formation experiments. Individual scale bars provided.