Extended Data Fig. 5: Roles of CDX2 and TBXT in R-C patterning of µNTLS.
From: A patterned human neural tube model using microfluidic gradients
a. Representative stitched confocal micrographs showing R-C patterned µNTLS on day 4, generated from wild type (WT) or CDX2-knockout (CDX2-KO) H9 human ES cell lines as indicated, stained for SOX2, CDX2, and BRACHYURY (BRA). Zoom-in views of boxed regions are shown on the right. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. White arrowheads mark SOX2+BRA+ NMPs. n ≥ 2 experiments for each condition. For WT, ncolony = 20 and nexperiment = 3; for CDX2-KO, ncolony = 14 and nexperiment = 2. b. Representative stitched confocal micrographs showing R-C patterned µNTLS on day 7, generated from WT or CDX2-KO H9 human ES cell lines as indicated, stained for HOXB1, HOXB4, and HOXC9. Zoom-in views of boxed regions are shown on the right. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. n ≥ 2 experiments for each condition. For WT, nHOXB1 = 22, nHOXB4 = 22, nHOXC9 = 12, and nexperiment = 3; for CDX2-KO, nHOXB1 = nHOXB4 = nHOXC9 = 13, and nexperiment = 2. c. Plot showing relative R-C positions of BRA+ domains in day 4 R-C patterned µNTLS and HOXB1+, HOXB4+, and HOXC9+ domains in day 7 R-C patterned µNTLS, generated from either WT or CDX2-KO human ES cells as indicated. Rostral and caudal ends of µNTLS are designated as 0 and 1, respectively. Error bars represent mean ± s.e.m. n values are provided in a & b. Two-sided Student’s t-tests were performed to calculate p values. d. (Left) Cartoon illustrating definition of normalized contour length, calculated as the ratio between contour length and region length. (Right) Box-and-whisker plot showing normalized contour length of µNTLS generated from WT and CDX2-KO human ES cells in indicated R-C regions (box: 25–75%; bar-in-box: median; whiskers: 1.5 × interquartile range). For WT, ncolony = 10 and nexperiment = 2; For CDX2-KO, ncolony = 11 and nexperiment = 2; Two-sided Student’s t-tests were performed to calculate p values. e. (Left) Protocol for clonal growth assay. Single H2B-GFP human ES cells are mixed with non-fluorescent human ES cells during cell seeding at a ratio of 1:200. Length of H2B-GFP cell colonies is recorded daily. (Right) Representative brightfield and fluorescence images showing clonal growth of single WT and CDX2-KO H2B-GFP human ES cells in µNTLS from day 1 to day 6. Experiments were repeated three times with similar results. f. Box-and-whisker plot showing length of H2B-GFP cell colonies in different R-C regions of day 6 µNTLS generated from WT and CDX2-KO human ES cells (box: 25–75%; bar-in-box: median; whiskers: 1.5 × interquartile range). Only clonal growth from a single H2B-GFP human ES cell is included for quantification. For WT, ncolony = 10, 11, 10, and 10 for Region 1, 2, 3, and 4, respectively, and nexperiment = 2; For CDX2-KO, ncolony = 12, 11, 14, and 14 for Region 1, 2, 3, and 4, respectively, and nexperiment = 2. Two-sided Student’s t-tests were performed to calculate p values. g. Representative stitched confocal micrographs showing day 4 R-C patterned µNTLS generated from WT or TBXT-KO WIBR3 human ES cells as indicated, stained for SOX2, CDX2, and BRA. Zoom-in views of boxed regions are shown on the right. Arrowheads mark SOX2+BRA+ NMPs. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. For WT, ncolony = 20 and nexperiment = 3; For TBXT-KO, ncolony = 18 and nexperiment = 3. h. Representative stitched confocal micrographs showing day 7 R-C patterned µNTLS generated from WT or TBXT-KO WIBR3 human ES cells as indicated, stained for HOXB1, HOXB4, and HOXC9. Zoom-in views of boxed regions are shown on the right. Intensity maps depict relative mean values of indicated markers as a function of relative R-C position in µNTLS. For WT, ncolony = 14 and nexperiment = 2; for TBXT-KO, ncolony = 12 and nexperiment = 2. i. Plot showing relative R-C positions of CDX2+ ___domain in day 4 R-C patterned µNTLS and HOXB1+, HOXB4+, and HOXC9+ domains in day 7 R-C patterned µNTLS, generated from either WT or TBXT-KO human ES cells. Rostral and caudal ends of µNTLS are designated as 0 and 1, respectively. Error bars represent mean ± s.e.m. n values are provided in g & h. Two-sided Student’s t-tests were performed to calculate p values. In a, b, g and h, nuclei were counterstained with DAPI. Scale bars, 400 µm (a, b, g and h) and 200 µm (e).
