Fig. 4: Contractility and adhesion regulate surface epithelialization and goblet cell specification.
a Maximum intensity projection of F-actin stained aggregates at 5 hpa. Insets (boxes) in top row shown in lower row. Scale bar for all top row is 100 μm. b Epithelialization (Control, n = 39) is reduced after lowering contractility (Y27632, n = 9; Blebbistatin, n = 10; MBST695A; n = 12) and altering cell–cell adhesion (ΔE-C-cadherin, n = 15 and ΔC-C-cadherin, n = 22). Analysis for mosaic ΔC-C-cadherin expression (anti-Myc positive) are shown in a separate bar. Kruskal–Wallis test, two-sided, (P = <0.0001 for Y27632, P = 0.015 for Blebbistatin, P = 0.001 for MBST695A, P < 0.0001 for ΔE-C-cadherin, P = 0.147 for ΔC-C-cadherin, P = 0.001 for ΔC-C-cadherin (Myc positive)). c Epithelialization is increased after increasing contractility (control, n = 30; Calyculin A, n = 12; arhgef2C55R, n = 10). Red filled cells indicate epithelialized cells; surface areas are quantified in the graph. Significance of each treatment from the control is calculated using a Kruskal–Wallis H-test, two-sided; P = 0.046 for Calyculin A, P < 0.0001 for arhgef2. d Creep compliance at 30 and 60 s by micro-aspiration at 6 hpa after 4 h of small molecule inhibitor treatment. Data from four clutches were pooled with 22–25 aggregates per treatment. Statistical significance determined by Mann–Whitney U-test (*P < 0.05, **P < 0.01). (control, n = 25; Calyculin A, n = 23; Blebbistatin, n = 22). e Creep compliance for MBST695A expressing aggregates. Data from a single clutch with eight aggregates per treatment. Statistical significance determined by Mann–Whitney U-test (*P < 0.05, **P < 0.01). f Percentage of itln1 positive goblet cells quantified from two optical sections of 24 hpa in deep ectoderm aggregates (control, n = 8; Y27632, n = 10). Scale bar, 100 μm. Data from three clutches. Statistical analysis from unpaired t-test (two-tailed; P < 0.0001). g Schematic contrasting developmental sequence of native embryonic ectoderm with in vitro regeneration of surface goblet cells in deep ectoderm aggregates. Regenerated epithelium serves as a substrate for radial intercalation of multiple cell types including multiciliated cells, ionocytes, and small secretory cells. Regenerated epithelial cells differentiate into mucus secreting goblet cells. b–f Box plots show minimum, first quartile, median, third quartile, and maximum values.
