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.

Spreading of epithelial patches. (A) Surface tensions and elastic force during spreading. Gray, epithelium; yellow, mesenchyme. γ_e, epithelial; γ_m, mesenchymal surface tension; γ_em, 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.

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.