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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. 2 — a Single-cell RNA-Seq (scRNAseq) cluster annotation of H3.3.1 day 16 EMLOs (n = 15,576 cells, N = 1 biological sample) plotted with UMAP. b Canonical gut tube epithelial genes by scRNAseq with violin plots for FOXA2 (1,707/15,576 cells) and GATA6 (1,244/15,576 cells) (log2Exp). Violin plot statistics are as follows: FOXA2 (cluster 7 max = 5.36, min = 0, median = 1.59, q1 = 1, q3 = 2.32; cluster 8 max = 5.25, min = 0, median = 2, q1 = 1, q3 = 2.59), GATA6 (cluster 6 max = 3.32, min = 0, median = 0.66, q3 = 1; cluster 8 max = 3.91, min = 0, median = 1.06, q3 = 1.58; cluster 17 max = 4.09, min = 0, median = 1, q3 = 1.58). c Phase contrast of EMLO shaking cultures at day 15. Asterisk indicates gut tube (*). d Multi-dimensional visualization of SOX2 (magenta) and GATA6 (yellow) primitive gut tube in day 13 EMLO. Sagittal and transverse planes are shown. High magnification Z-slice depicts mitotic figures in apical (Ap) but not basal (Bs) epithelium. e SOX2 and GATA6 distribution as percent biomarker calculated from maximally projected Z-stacks of whole EMLOs over time. n = 60 total EMLOs measured (H3.3.1). f Length of gut tube major and minor axes over the elongation period in the three representative lines (n = 47 EMLOs measured per line). g Z-slices depict robust FOXA2 (cyan) expression in gut tube and associated TUJ1 (red) neuronal fibers at day 20. Inset is phase contrast. h Z-slice of CDH1/E-cadherin (cyan) and TUJ1 (red) IF at day 20. Maximally projected TUJ1 Z-volume is overlaid onto E-cad Z-slice to show 3D spatial relationship (right). i Increased gut tube morphological complexity at day 40 by FOXA2 and TUJ1 IF and phase contrast. Asterisk highlights meandering gut tube at this time point (*). j Identification of specified gastrointestinal cell types in day 16 EMLOs by scRNAseq that are intestinal stem cell (LGR5/SOX), esophagus–trachea junction (NKX2-1/SOX2)¹³, Paneth cell-like (LYZ/SOX2), and enterocyte-like (VIL1/SOX2). Individual scale bars provided. H3.3.1 EMLOs were formed in N = 11 separate biological repeat experiments over the course of this study with similar results. Phase and IF data were acquired each time (c, d, g–i). Data reported as (mean ± s.e.m.).