Kim HY , Jackson TR , Stuckenholz C .

R01 HD044750 NICHD NIH HHS , T32 HL076124 NHLBI NIH HHS , R56 HL134195 NHLBI NIH HHS

             cell aggregation

	Fig. 1: Surface cells of deep ectoderm aggregates undergo epithelial-like phenotypic transition. a) Schematic of the assembly of deep ectoderm cell aggregates from early Xenopus embryo (Stage 10). b) Surface F-actin and fibronectin (FN) from maximum intensity projections at 1.5, 5, and 24 h post aggregation (hpa). Three panels on the right are higher resolution views of the inset region (white box) in the third column. Arrows indicate margin of FN where dense circumapical F-actin suggests epithelial cell phenotype. Scale bar for aggregate images is 100 µm. c) Transverse sectional view through the ectoderm of NHS-Rhodamine surface-labelled embryos. Scale bar, 50 µm. Rhodamine is restricted to the apical surface of outer epithelial cells. d) Deep ectoderm aggregates generated from NHS-Rhodamine surface-labelled embryos. Scale bar, 100 µm. Lack of rhodamine indicates absence of contaminating epithelia. e) Percent of epithelial cell phenotype found on the surface of different-sized deep ectoderm aggregates at 24 hpa. Aggregates assembled with varying amounts of embryo-ectoderm explants (1/2 explant, n = 6; 1 explant, n = 5; 2 explants, n = 5; 4 explants, n = 8). Box plot shows minimum, first quartile, median, third quartile, and maximum values. f) Labeled nuclei shown in deep ectoderm aggregates from 1/2- and 4-embryo-ectoderm explant containing aggregates. Scale bar, 100 µm.
	Fig. 2: Epithelialization precedes differentiation of mucus-secreting goblet cells. a) Apical polarity protein aPKC localizes on the apical surface of aggregates by 5 hpa. Scale bar, 10 µm. b) Apical localization of epithelial cytoskeleton keratin is restricted to the outer surface of deep ectoderm aggregates. Cross sectional view of aggregates at 5 hpa (Scale bar, 100 µm) and 24 hpa (Scale bar, 50 µm). c) Maximum intensity projection of epithelial tight junctional protein ZO-1 expression in aggregates at 5 hpa (top; immunofluorescence staining) and 24 hpa (bottom; GFP-ZO-1 expression). Epithelialized cells are marked by arrows (inset). Scale bar, 100 µm. d) Representative frames from a time-lapse sequence of an aggregate expressing GFP-ZO-1. Top panel: maximum intensity projection of GFP-ZO-1 shows cells undergo epithelialization (outlined with red on lookup-table-inverted images in lower panel) on the surface of aggregates from 2 to 6 hpa. Scale bar, 100 µm. e) Percentage of cells having undergone epithelialization increases over time (9 aggregates tracked from three clutches). Box plot shows minimum, first quartile, median, third quartile, and maximum values. f) qPCR expression profiling in aggregates made of ectoderm, deep layer, or superficial layer cells. Expression of epithelial (Cdh1, ZO-1, Krt12, and Itln1) and mesenchymal (FN, VimA, and Snai1) genes are analyzed for CT-based fold changes from 3 hpa to 24 hpa. g) At 24 hpa the deep ectoderm aggregate is covered by epithelial cells including differentiated mucus-secreting goblet cells (itln1) and radially intercalated multiciliated cells (acetylated tubulin). Scale bar, 100 μm.
	Fig. 3: YAP nuclear translocation and tissue stiffening both coincide with epithelialization. a) Maximum intensity projection of deep ectoderm aggregate expressing YAP1-GFP and ZO-1-RFP and stained for nuclei. Scale bar, 100 µm. b) Color coded nuclear YAP ratios for control and blebbistatin treated deep ectoderm aggregates at 5 hpa. Scale bar, 100 µm. c) Nuclear YAP ratios of 5 hpa aggregates (n = 2040 nuclei from 7 aggregates) are higher than blebbistatin-treated aggregates (3471 nuclei from 7 aggregates) and 2 hpa aggregates (2167 nuclei from 7 aggregates). Statistical analysis from one-way ANOVA followed by Tukey multiple comparison test (***P < 0.001). d) Schematic of micro-aspirator used to measure tissue compliance of aggregates by adjusting pressure at the opening of the microchannel (see Methods). e) Representative kymographs of tissue displacement over the length of representative micro-aspiration experiments at 3, 6, and 12 hpa. Red arrows indicate the time suction pressure is applied and then released. f) Aspirated distances of an aggregates in (e) at 3 (black), 6 (dark gray), 12 hpa (light gray) relative to the pressure applied (red). The power-law model of creep compliance (blue dots) fit to this data. g, h) Creep compliance at 30 and 60 s indicating steady-state from micro-aspiration at 3 (black), 6 (gray) and 12 hpa (white). 12 to 16 aggregates were measured for each time point and repeated over three clutches. i, j) Creep compliance at 30 and 60 s from micro-aspiration of aggregates consisting of only deep mesenchymal cells (gray) or both deep mesenchymal and superficial epithelial cells (white) 3, 6, and 12 h post aggregation (one clutch, n = 5–8 aggregates each). g–j) Box plots show minimum, first quartile, median, third quartile, and maximum values. Statistical analysis by Mann–Whitney U-test is shown as either not significant (n.s.) or by asterisk (*P < 0.05; **P < 0.01; ***P < 0.001).
	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.
	Supplementary Figure 1: Epithelialization and goblet cell specification is independent from Notch signaling. a-b, Activation of Notch signaling by over-expression of the intracellular domain of NotchICD inhibits the differentiation of multiciliated cells but does not inhibit regeneration of goblet cells in deep ectoderm aggregates (24 hpa). Inset regions of third columns (white boxes) are shown in fourth column. Scale bars are 100 μm for all image panels except insets.
	Supplementary Figure 2: F-actin and ZO-1 co-localize on the boundary of epithelial cells. Aggregates expressing ZO-1 RFP (red) and stained for F-actin (green) reveal co-localization of tight junctions and circumapical actin in epithelialized cells (white) in both control and Calyculin A treated aggregates. Inset regions from first and third rows (white boxes) are shown in second and fourth rows, respectively. Scale bars are 100 µm for all panels except insets.
	Supplementary Figure 3: ΔC-C-cadherin expressing cells are not able to transition to epithelial on the surface of deep ectoderm aggregates. F-actin and myc stained deep ectoderm aggregates at 5 hpa. Cells expressing myc-tagged ΔC-C-cadherin are detected by Anti-myc staining. Non-expressing cells epithelialize (a) while ΔC-C-cadherin expressing cells (yellow) do not (b) within same aggregate. Scale bar for whole aggregate (top row) is 100 µm.
	Supplementary Figure 4: ROCK inhibition blocks epithelialization driven by constitutive active Rho-GEF. a. Representative maximum projection confocal images of F-actin labeled aggregates. Epithelialization proceeds in control 5 hpa aggregates is enhanced in arhgef2C55R expressing aggregates. Incubation of arhgef2C55R expressing aggregates in ROCK inhibitor Y27362 inhibits epithelialization. Inset regions in first column (white boxes) are shown in second column. Scale bar for all panels except the insets are 100 µm. b. Percent of epithelialization from treatments shown in (a; controls, arhgef2C55R , arhgef2C55R + Y27362, n=6, 5, 6, respectively). (See Statistical Analysis in Methods for detailed description of statistical methods.)

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