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Tissue growth constrained by extracellular matrix drives invagination during optic cup morphogenesis

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Abstract

In the early embryo, the eyes form initially as relatively spherical optic vesicles (OVs) that protrude from both sides of the brain tube. Each OV grows until it contacts and adheres to the overlying surface ectoderm (SE) via an extracellular matrix (ECM) that is secreted by the SE and OV. The OV and SE then thicken and bend inward (invaginate) to create the optic cup (OC) and lens vesicle, respectively. While constriction of cell apices likely plays a role in SE invagination, the mechanisms that drive OV invagination are poorly understood. Here, we used experiments and computational modeling to explore the hypothesis that the ECM locally constrains the growing OV, forcing it to invaginate. In chick embryos, we examined the need for the ECM by (1) removing SE at different developmental stages and (2) exposing the embryo to collagenase. At relatively early stages of invagination (Hamburger–Hamilton stage HH14\(-\)), removing the SE caused the curvature of the OV to reverse as it ‘popped out’ and became convex, but the OV remained concave at later stages (HH15) and invaginated further during subsequent culture. Disrupting the ECM had a similar effect, with the OV popping out at early to mid-stages of invagination (HH14\(-\) to HH14\(+\)). These results suggest that the ECM is required for the early stages but not the late stages of OV invagination. Microindentation tests indicate that the matrix is considerably stiffer than the cellular OV, and a finite-element model consisting of a growing spherical OV attached to a relatively stiff layer of ECM reproduced the observed behavior, as well as measured temporal changes in OV curvature, wall thickness, and invagination depth reasonably well. Results from our study also suggest that the OV grows relatively uniformly, while the ECM is stiffer toward the center of the optic vesicle. These results are consistent with our matrix-constraint hypothesis, providing new insight into the mechanics of OC (early retina) morphogenesis.

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Notes

  1. For simplicity, since our model does not include tissue remodeling, which occurs in vivo to partially maintain the relatively thicker OV after ECM degradation, the indentation tests were simulated for the OV in its initial state. As a check, we also ran these simulations for a partially invaginated OV with and without ECM. Although the computed stiffnesses were lower, the value of \(\bar{\mu }\) for the intact OV is approximately the same.

  2. Notably, the extensional stiffness of the ECM is most important for constraining cellular expansion in the OV, rather than the bending stiffness. The matrix does not need to be stiffer than the cells to cause a flat epithelium to bend. However, for a spherical shell the curvature stiffens the structure and complicates the mechanics. According to our model, a relatively large ECM stiffness is required to force a reversal of curvature (see Fig. S4).

  3. Natural variations in ECM and OV geometry or material properties could account for the different behaviors of some OVs at intermediate stages of invagination, especially near the critical invagination depth, which is especially sensitive to imperfections.

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Acknowledgments

We are grateful to Philip Bayly, Ruth Okamoto, Kristen Naegle, and members of the LAT laboratory for helpful discussions. We thank the Department of Mechanical Engineering and Materials Science for use of the confocal microscope. This work was supported by NIH Grant R01 NS070918 (LAT) and a fellowship for AO through the Imaging Sciences Pathway at Washington University (NIH T32 EB014855).

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Authors and Affiliations

  1. Department of Biomedical Engineering, Washington University, One Brookings Drive, Campus Box 1097, Saint Louis, MO, 63130-4899, USA

    Alina Oltean & Larry A. Taber

  2. Department of Ophthalmology and Visual Sciences, Washington University, Saint Louis, MO, 63130, USA

    Jie Huang & David C. Beebe

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  1. Alina Oltean
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Correspondence to Alina Oltean.

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D. C. Beebe: Deceased April 2015.

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Oltean, A., Huang, J., Beebe, D.C. et al. Tissue growth constrained by extracellular matrix drives invagination during optic cup morphogenesis. Biomech Model Mechanobiol 15, 1405–1421 (2016). https://doi.org/10.1007/s10237-016-0771-8

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