Biomechanics of dorsal closure studied using holographic laser microsurgery

Abstract

Dorsal closure is an important morphogenetic event in the embryogenesis of Drosophila melanogaster and serves as a useful model system for studying wound healing, palatogenesis, and neural tube closure in vertebrates. During this stage of development, cells in the amnioserosa – a tissue that fills a gap left in the epithelium of the embryo as a result of germband retraction – undergo periodic contractions in their apical surfaces. These contractions play an important part in reshaping the amnioserosa tissue. To study the physical forces driving this apical constriction cycle, I built a multi-point (holographic) laser microsurgical system. This system utilizes a spatial light modulator to diffract a single 5-ns pulse from a UV laser, creating a user-defined pattern in the focal plane of a confocal fluorescent microscope.

This system was then used to investigate cell-autonomous behavior in amnioserosa cells in vivo. A model of the tissue was constructed to simulate the behavior seen in the cell-isolation experiments. To verify the model, further experiments were performed on embryos anesthetized using CO2 and Argon gases, both of which pause the apical contraction cycle. The experiments and model suggest that internally generated contractile forces are largely responsible for the behavior seen in individual cells of the amnioserosa tissue. Passive elastic strain plays a much smaller role.

To investigate possible secondary effects of multi-point ablation, we studied the dynamics of laser-induced cavitation bubbles using a bright-field, high-speed imaging system. The cavitation bubbles formed in embryos are much larger than the laser-disrupted region of tissue, raising the possibility that these bubbles are expanding in the uncompartmentalized space between the tissue and the surrounding vitelline membrane. Furthermore, shockwaves radiating from certain, highly-symmetric patterns of ablation sites can both cause secondary cavitation in un-ablated material, and enhance the growth of existing cavitation bubbles. Therefore, the possibility of such interactions should be accounted for when simultaneously ablating multiple closely-spaced sites.