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Fig. 1.
Folding of epithelial patches. (A) SEM picture of ectoderm with epithelial (e) and inner ectoderm (ie) layer; ap, apical surface. (A′�A′′′) Epithelial patch immediately after isolation. (A′) apical surface; (A′′) lateral view; (A′′′) basal surface with loosely connected cells and protrusions (arrowheads). (B) The apical surface (pigmented) of two cells (outlined in yellow; 1a, 2a) expands during rounding up. Cells were followed frame by frame. Boundaries between basal parts of same cells (1b, 2b) are barely discernible. (C) Fractured epithelial spherule, with wedge-shaped cells (white arrow), bottle cell (red arrow), and central cells (yellow arrow). (D) Fractured cylindrical wrinkle, arrows as in C. (E) Sectioned ectoderm double stained with antibody against aPKC (red) and actin (green). (F) Epithelial spherule stained as in E. (G) Spherule stained with C-cadherin antibody. (H) Explants above 66 μm radius wrinkle into connected spheres. (I) Even larger explants wrinkled into bent cylinders. (J) Explant structure and explant size. To determine critical sizes, patches were cultured for 4 h and scored for being completely or incompletely covered or wrinkled. Transition between incompletely (n = 32) and fully covered (n = 33) at a radius of 37 μm is shown. Onset of wrinkling, n = 14; radii of cylinders, n = 57. Horizontal lines are at multiples of epithelial height. (K) Cells of a folding sheet, with apical (brown) and lateral and basal (white) surfaces, subapical junctional complex (red), and actin cortex (green). Arrows and magenta/blue dots indicate movement of basal surface, and double arrows show apical expansion. (L) Patch size and spherule size. The apical side of the single-layered patch is shaded. Extreme cell elongation (at r = 3h) is avoided by wrinkling.
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Fig. 2.
Elasticity of epithelium. (A) Spherical epithelial explant treated with EDTA after 3 h. Apical (pigmented) cell surface (yellow outline) shrinks. (B�E) Flattening of spherules by compression. Spherules (3 h after explantation) before (B) and 30 min after compression (D) are shown. (C) Flattened explant 120 min after start of compression. (E) Recoil after compression; τ, time constant. (F) Recoil for flattened explants treated with cytochalasin D and explants treated with cytochalasin D followed by washing with MBS for 1 h. (G�I) Recoil of epithelial patch in situ. Fixed and fractured embryos, ectodermal region, are shown. The epithelium (e) was cut at three sides and peeled off the inner ectoderm (ie). The patch, attached at the fourth side (arrow), curls back, shortening its apical side (ap) (G), and then straightens within <1 min (H). No straightening occurs in EDTA buffer even after 5 min (I). Patches were oriented randomly; no indication of in-plane anisotropy was noted. (J) Two stage 10+ epithelial patches attached with their basal sides (arrowheads) form a double layer: fixed and fractured 1 h after explantation. (K) Height of epithelium at different stages in intact embryo (dark gray bars) and after straightening of patch, as in H, or in double layer (DL), as in J (light gray bars). Error bars, SDs; significant differences at same stage and between stages are marked with brackets. Numbers inside bars indicate numbers of measurements and embryos.
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Fig. 3.
Spreading of epithelial patches. (A) Surface tensions and elastic force during spreading. Gray, epithelium; yellow, mesenchyme. γe, epithelial; γm, mesenchymal surface tension; γem, interfacial tension. (B) Mesenchymal surface tension and epithelial spreading (Eq. 1), with interfacial tension estimated according to Eq. 2 (blue) or Eq. 3 (red). (C) Regions of embryo: mesend, leading edge mesendoderm; ect, ectoderm; prechord, prechordal mesoderm; end, endoderm; chord, chordamesoderm; dbp, dorsal blastopore. (D and E) Patches spread on inner ectoderm (D) or retract (E) (arrowhead) or detach (arrow) on mesendoderm. (F�H) Epithelial patches (between red arrows; yellow bar, thickness) on mesenchymal explants, fixed after 3 h, and transected. (I�K) Surface tension of coated explants. Transected inner ectoderm explant after 5 h of flattening is shown without (I) or with (J) excess epithelial (dark; epi) coating. Red, Laplacian contour fit to explant. (K) Time course of flattening of epithelium-coated inner ectoderm, lateral view.
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Fig. S1. Immunostaining of sectioned ectoderm and epithelial explants. (A�C) Ectoderm or epithelial explants stained with aPKC antibody (red) to indicate apical surface (ap), tubulin antibody (green) showing microtubules, and DAPI to reveal nuclei or chromosomes (blue); viewed in fluorescence microscope. (D�F) Epithelial explants stained for aPKC (red) and tubulin (green), viewed in a confocal microscope. (E and F) Same explant as in B in red and green channels, respectively. Orientation of mitotic spindles (yellow arrowhead): A, B, and F, parallel to apical surface; C and D, perpendicular or oblique to surface. (G and H) Staining of ectoderm (G) or epithelial explant (H) with an integrin antibody shows lack of integrinβ1 on apical (ap) surfaces and expression on all other membranes.
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Fig. S3. Effect of 0.5 μM cytochalasin D on elasticity. In preliminary experiments that explored a range of concentrations (0.2�10 μM), 0.5 μM was found to be effective in our experiments without strongly affecting explant integrity. (A�D) Continuous incubation in cytochalasin D: rounded epithelial explant at the onset of cytochalasin treatment, before compression (A), after 1 h of compression before (B) and directly after (C) lifting of coverslip, and 1 h after removing coverslip (D). Four of five explants behaved as shown. (E�H) Pulse treatment with cytochalasin D: rounded explant at onset of cytochalasin incubation, before compression (E); after 1 h of cytochalasin D treatment followed by 1 h of washing in MBS, directly before (F) and after (G) lifting of coverslip; and 1 h later (H). Five of five explants showed this behavior. A second independent experiment gave similar results.
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Fig. S4. An explanted epithelial patch can be cultured in Ca2+- and Mg2+-free MBS for prolonged times without dissociation into single cells (in contrast to EDTA-containing buffer). Explants are uneven and cells tend to move slowly in and out of focus. Nevertheless, the areas of 10 cells were followed from t = 20 min to either t = 40 min or t = 60 min. Apical areas were fit to straight lines in time; the average of the fitted slopes was not significantly different from zero (P = 0.20); i.e., there was no significant overall change in cell area.
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Fig. S5. Spreading of ectodermal epithelial patches on inner ectoderm layer. (A) Frames from time-lapse recordings of epithelial patch (center, brown) on inner ectoderm (white) at beginning and end of spreading and after subsequent EDTA treatment. (B) Time course of spreading; τ, time constant. (C) Spreading occurred by intercalation of inner cells; inner cells ahead of the advancing front of epithelial cells moved with the epithelium rather than being overtaken by it. (D) Exponential spreading. The logarithm of the equivalent radius as a function of time is shown for four explants (blue). The curves do not deviate sys- tematically from the straight lines fitted to them (red). Differences in slopes reflect natural variation (Table S1).
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