Research ArticleCELL BIOLOGY

Strain-triggered mechanical feedback in self-organizing optic-cup morphogenesis

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Science Advances  21 Nov 2018:
Vol. 4, no. 11, eaau1354
DOI: 10.1126/sciadv.aau1354
  • Fig. 1 Quantitative simulations of 3D multicellular dynamics during optic-cup morphogenesis.

    (A) Time-lapse imaging of in vitro optic-cup formation. (B) Immunostaining assay of the in vitro optic cup. (C and D) Spatial pattern of Mitf intensity and the curvature and thickness of epithelium along the epithelial sheet in (B). (E) Shape of in vitro single cell at NR and the NR-RPE boundary in day 9 (also shown in movie S1). (F) Quantities of cell behaviors obtained from experiments [morphologies were measured as shown in fig. S2; epithelial thickness and surface stiffness were partly obtained from our previous study (11)]. (G) Geometric structures of OV and cells on the 3D vertex model (described in fig. S3). The model involves several physical parameters, a part of which are given by experiments in (F), and the other unknown parameters are entirely varied in computational simulations. (H) Phase diagram resulting from computational simulations for all of the unknown physical parameters (enlarged in fig. S4). Several phenotypes in the phase diagram are verified to correspond to those resulting from pharmacological assays in experiments (fig. S5; in silico phenotypes also shown in movie S3). (I to K) Outer and cross-sectional views of the recapitulated optic-cup formation steps (movies S2 and S3). For this recapitulation, two key factors are required: the autonomous bending of NR in the apically convex direction and the constriction of the NR-RPE boundary along the apicobasal (lateral) direction (K). (L) Shape of in silico single cell at NR and the NR-RPE boundary, where apical surfaces are colored red. Tissue morphologies are shown in 3D coordinates with the distal-proximal (Di-P), dorsal-ventral (Do-V), and anterior-posterior axis in (G) and (I) and are projected on the plane normal to the anterior-posterior plane in (F), (J), and (K).

  • Fig. 2 Autonomous epithelial bending in apically convex direction due to apical actomyosin reduction.

    (A) The NR autonomous bending in the apically convex direction driven by the inversion of NR spontaneous curvature. (B) In silico screening assay of optic-cup formation with respect to the NR spontaneous curvature (fig. S6A and movie S5). Tissue morphologies are shown in the 3D coordinates with the distal-proximal, dorsal-ventral, and anterior-posterior axis. (C to F) Mechanical assays of spherical OV at day 6 (D) and spherical NR at day 9 (E). The angle displacements by incising (F). (G) Normalized bending rigidities of OV, NR, and RPE estimated from their apical elasticities and cell heights. (H) Immunostaining assay of pMLC in vitro. (I) pMLC distributions along the apicobasal (lateral) axis in (H). (J) Correlation between local pMLC intensity and curvature in epithelium shown in (H). (K) pMLC intensity on the apical side in the distal area, normalized by those in the proximal area, in vitro and in vivo. (L to N) Inhibitor assays of in vitro OV. (M) Inhibitor assay of in vitro OV with ROCK inhibitor, Y27632, at late phase 1 (movie S6). (N) Inhibitor assay of in vitro OV with calyculin. (O) Invagination probability in in vitro and ex vivo cultures. (P and Q) Inhibitor assay of in vitro OV at early phase 1, where Y27632 was locally applied to the distal portion of OV. The apical length, basal length, and height of the epithelium were measured (Q). Bars in (F), (J), (K), and (O) indicate SEs.

  • Fig. 3 Calcium-dependent lateral cell constriction in epithelial hinge structure formation.

    (A) Concept of apical and lateral cell constrictions at the NR-RPE boundary. (B to E) In silico screening assay of optic-cup formation with respect to the apical and lateral cell contractility (movie S7). External view of the entire tissue and the sections around the NR-RPE boundary obtained under conditions with no constriction (B), lateral constriction (C), and apical constriction (D). Dependence of NR curvature on apical and lateral constrictions (E), where apical and lateral constrictions are expressed as the length strains of the spontaneous perimeter and height of cells (nondimensions). Tissue morphologies are shown in the 3D coordinates with the distal-proximal, dorsal-ventral, and anterior-posterior axis in (B) to (D). (F to H) Theoretical analyses of cell mechanics; a simple mathematical model of cell mechanics based on assuming a conical frustum as an average cell shape (F). Epithelial bending rigidity as a function of apical/lateral strain (G). Sensitivity of epithelial curvature to apical/lateral strain as a function of the reference curvature of epithelium (H). (I and J) Time-lapse imaging of the in vitro optic cup at the NR-RPE boundary (movie S8). Time displacement of epithelial height (J). (K and L) Calcium observation in the in vitro optic cup; time variance of local GCaMP intensity at NR, RPE, and the NR-RPE boundary (L). (M and N) Kymographs of GCaMP intensity and cell shape along the apicobasal axis, respectively. (O) Time-lapse imaging of calcium transients within a single cell. (P and Q) Actin accumulation within individual cells (movie S9). Actin aligning along the apicobasal axis in the NR-RPE boundary (P) and actin accumulated around the apical surface in OV (Q). DIC, differential interference contrast.

  • Fig. 4 Strain-triggered cell constriction in epithelial folding.

    (A to I) Mechanical assay in the in vitro OV using a micropipette (movie S10); the distal portion of the vesicle forcedly invaginated, and the surrounding portion is forcedly folded (A and B). The pipette was gently removed after sustained pushing for 30 min (C). Enlarged frames in (B) and (C) show the time-lapse images of the folded region of the epithelium. (D) Difference in the epithelial angles before and after pushing for 30 min in (B) and (C). (E and F) Calcium distribution and local dynamics in (B). Arrows in (F) indicate the peaks of GCaMP intensity corresponding to calcium transients. (G) Time-lapse images of calcium transients in a single cell around the folded region under pushing. (H and I) Spatial distribution of the calcium transient events under pushing (H), from which the average frequencies are counted in each region (I). (J and K) Time-lapse imaging of calcium responses to shear stress on the basal surface (movie S11). (L to P) Time-lapse imaging of single-cell dynamics under the assay of focusing two-photon laser locally on the basal cell surfaces (movie S12) (41): single-cell shape (L), epithelial height (M and N), and calcium concentration (O and P). (Q to T) Proof of concept by computational simulations under the condition with the strain-triggered lateral constriction using the 3D vertex model; in silico optic cup obtained by the simulation at t = 48 (R) (movie S13), probability density of strain-triggered lateral constriction along the proximal-distal axis (S), and dependence of NR curvature on lateral contractility (T). Tissue morphology is represented in the 3D coordinates with the distal-proximal, dorsal-ventral, and anterior-posterior axis in (R). In (R) and (T), lateral constriction is expressed as the length strain of the spontaneous height of cells (nondimensions). (U) Proposed model for the stepwise optic-cup morphogenesis with the strain-triggered mechanical feedback. Bars in (D), (I), (N), and (P) indicate SEs, and bars in (T) indicate SDs.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/11/eaau1354/DC1

    Supplementary Text

    Fig. S1. Concept models of optic-cup formation and epithelial bending.

    Fig. S2. Versatile 3D vertex model describing multicellular dynamics in 3D space.

    Fig. S3. Quantification of optic-cup morphogenesis.

    Fig. S4. Quantitative simulations of optic-cup morphogenesis for all of the unknown parameters.

    Fig. S5. Comparison between in silico and ex vivo optic-cup formation under perturbations.

    Fig. S6. Actomyosin activities in in vivo and ex vivo optic-cup morphogenesis.

    Table S1. Main symbols used in the 3D vertex model of optic-cup formation.

    Table S2. Standard physical parameter values of cell states in computational simulations of optic-cup formation.

    Table S3. Standard physical parameter values of boundary regions in computational simulations of optic-cup formation.

    Table S4. Standard physical parameter values of cell behaviors used in computational simulations of optic-cup formation.

    Table S5. Varied physical parameter values obtained from computational simulations of optic-cup formation.

    Movie S1. Hinged cell shape at the NR-RPE boundary in the in vitro optic cup.

    Movie S2. In silico recapitulation of optic-cup morphogenesis using the versatile 3D vertex model.

    Movie S3. Cell proliferation, constriction, and apoptosis in in silico optic-cup formation.

    Movie S4. Dependence of in silico optic-cup morphogenesis on cell heightening, proliferation, apoptosis, and differentiation.

    Movie S5. Dependence of in silico optic-cup morphogenesis on formation of spontaneous curvature of NR.

    Movie S6. Pharmacological assays of actomyosin activities in vitro.

    Movie S7. Dependence of in silico optic-cup morphogenesis on apical and lateral cell constrictions.

    Movie S8. Lateral cell constrictions in vitro.

    Movie S9. Characteristic alignment of intracellular actin fibers along the apicobasal axis in vitro.

    Movie S10. Elastic and plastic responses of in vitro neuroepithelium to mechanical stimuli.

    Movie S11. Calcium response to shear stress on the basal surface in vitro.

    Movie S12. Lateral constrictions triggered by local up-regulation of intracellular calcium concentration in vitro.

    Movie S13. In silico recapitulation of optic-cup morphogenesis with strain-triggered lateral constriction.

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Text
    • Fig. S1. Concept models of optic-cup formation and epithelial bending.
    • Fig. S2. Versatile 3D vertex model describing multicellular dynamics in 3D space.
    • Fig. S3. Quantification of optic-cup morphogenesis.
    • Fig. S4. Quantitative simulations of optic-cup morphogenesis for all of the unknown parameters.
    • Fig. S5. Comparison between in silico and ex vivo optic-cup formation under perturbations.
    • Fig. S6. Actomyosin activities in in vivo and ex vivo optic-cup morphogenesis.
    • Table S1. Main symbols used in the 3D vertex model of optic-cup formation.
    • Table S2. Standard physical parameter values of cell states in computational simulations of optic-cup formation.
    • Table S3. Standard physical parameter values of boundary regions in computational simulations of optic-cup formation.
    • Table S4. Standard physical parameter values of cell behaviors used in computational simulations of optic-cup formation.
    • Table S5. Varied physical parameter values obtained from computational simulations of optic-cup formation.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Hinged cell shape at the NR-RPE boundary in the in vitro optic cup.
    • Movie S2 (.mp4 format). In silico recapitulation of optic-cup morphogenesis using the versatile 3D vertex model.
    • Movie S3 (.mp4 format). Cell proliferation, constriction, and apoptosis in in silico optic-cup formation.
    • Movie S4 (.mp4 format). Dependence of in silico optic-cup morphogenesis on cell heightening, proliferation, apoptosis, and differentiation.
    • Movie S5 (.mp4 format). Dependence of in silico optic-cup morphogenesis on formation of spontaneous curvature of NR.
    • Movie S6 (.mp4 format). Pharmacological assays of actomyosin activities in vitro.
    • Movie S7 (.mp4 format). Dependence of in silico optic-cup morphogenesis on apical and lateral cell constrictions.
    • Movie S8 (.mp4 format). Lateral cell constrictions in vitro.
    • Movie S9 (.mp4 format). Characteristic alignment of intracellular actin fibers along the apicobasal axis in vitro.
    • Movie S10 (.mp4 format). Elastic and plastic responses of in vitro neuroepithelium to mechanical stimuli.
    • Movie S11 (.mp4 format). Calcium response to shear stress on the basal surface in vitro.
    • Movie S12 (.mp4 format). Lateral constrictions triggered by local up-regulation of intracellular calcium concentration in vitro.
    • Movie S13 (.mp4 format). In silico recapitulation of optic-cup morphogenesis with strain-triggered lateral constriction.

    Files in this Data Supplement:

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