Date of Award

Spring 5-15-2016

Author's School

School of Engineering & Applied Science

Author's Department

Biomedical Engineering

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Mechanical forces play an essential role in morphogenesis, the shaping of embryonic structures. This research focuses mainly on eye development, a problem that has been studied for decades using a variety of approaches. However, the mechanics of the early stages of eye formation remain incompletely understood.

The embryonic eyes begin as bilateral protrusions called optic vesicles (OVs) that grow outward from the anterior end of the brain tube. The optic vesicles contact and adhere to the overlying surface ectoderm (SE) via extracellular matrix (ECM). Then, both layers thicken in the region of contact to form the retinal and lens placodes, which bend inward (invaginate) to form the roughly spherical optic cup (OC, primitive retina) and lens pit, respectively. These two structures then separate, and the lens pit continues to fold until it closes to create the lens vesicle (LV). The primary aim of this research is to determine the biophysical mechanisms that create the OC and LV in the chick embryo. Throughout, we used a combination of experiments and computational modeling.

First, we explored the hypothesis that the ECM locally constrains the OV as it grows, forcing it to thicken and invaginate. The integrity of the matrix was disrupted by removing its major source, the SE, and degrading the matrix chemically at different stages of development. At relatively early stages of invagination, 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 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. These results suggest that the ECM is required for the early stages but not the late stages of OV invagination. 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 changes in OV curvature, wall thickness, and invagination depth reasonably well. These results support the matrix constraint hypothesis for retinal placode formation and invagination.

Next, we studied the forces that create the LV. Studies have shown that, like the retinal placode, the lens placode is produced by growth of the SE constrained locally by ECM, while actomyosin contraction at the apical surface of the lens placode plays a major role in its subsequent invagination. Our experiments and modeling support this view but also suggest that apical contraction alone does not generate sufficient force to close the LV. We propose that programmed cell death, or apoptosis, which has been observed near the opening in the lens pit, supplies the required force for closure by causing tissue surrounding the opening to shorten circumferentially. Finite-element modeling and experiments involving apoptosis inhibitors support this idea.

Finally, we investigated the possible role of mechanical feedback in regulating the contractile behavior of neuroepithelia such as the OC and primitive brain tube. Using a novel loading technique, we subjected isolated brain tubes to compressive loads during culture. Normally, cell nuclei in the wall of the brain tube are elongated in the radial direction, and a previous study has shown that compression of the tube triggers a decrease in contractile force with the nuclei becoming rounder. We found that exposure to a gap junction blocker prevents this response, suggesting that compression without the blocker releases a molecular signal that diffuses through the wall to inhibit contraction. Our results also show that nuclear shape changes are likely caused by changes in wall stress. These results lay the groundwork for future studies of mechanotransduction in the embryonic brain.

Our experimental and computational findings expand our knowledge of early eye development and demonstrate key roles for extracellular matrix, actomyosin contraction, and apoptosis in morphogenesis. We also have shown that chemomechanical feedback can play a role in regulating contractility of neuroepithelial tissues such as the brain and retina. Future investigations of developmental mechanics may reveal similar mechanisms in the formation of other organs as well.

Language

English (en)

Chair

Larry A. Taber

Committee Members

Steven Bassnett, Philip V. Bayly, Spencer P. Lake, Ruth J. Okamoto,

Comments

Permanent URL: https://doi.org/10.7936/K7RN3641

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