Zvi MurryFollow

Document Type

Dissertation/Thesis

Date Created

5-6-2020

Embargo Date

11-6-2020

Abstract

During vertebrate eye development the optic vesicles protrude from either side of the brain and form the optic cups. As an optic cup starts to surround the lens a groove on the ventral side of the eye forms, known as the choroid fissure (CF). Normally, the CF will close around the optic nerve and hyaloid vasculature. If this process does not occur properly it results in a keyhole opening in the eye known as coloboma. This results in blindness and affects nearly 1 in 4-5,000 births. Zebrafish were utilized as a model for eye development to study CF closure (CFC) as they utilize similar gene expression and cellular signaling. Previously, a transient β-catenin/actin fusion seam within the fusing CF was observed indicating the formation of cell-to-cell contacts. Rac, a small G-protein, regulates actin cytoskeleton reorganization and formation of lamellipodia required for cell-to-cell adhesion. These lamellipodia increase interactions between cells increasing contacts that could form adherens junctions. I hypothesized Rac would be expressed prior to CFC and dissipate upon CFC completion, similar to adhesion proteins. To determine Rac’s localization, embryos were cryosectioned at 47 and 49-hours post-fertilization (hpf) and Rac immunofluorescence was observed. These data demonstrated Rac is present within the CF edges at 47 hpf and dissipates the CF fusion seam as CFC progresses in wildtypes embryos around 49 hpf. Quantification of these data further demonstrated a progressive fusion event that initiates in the central section of the CF and moves bidirectionally towards the proximal and distal edges, emulating a zipper-like fashion. in vivo analysis of Rx3:GFP embryos (neuroretina labeled) identified a subpopulation of cells that are present within the CF at 24 hpf. This population of cells appear highly protrusive and “reach” in multiple directions. Further analysis of Rac embryos identified these “reaching cells” as Rac positive. in vivo analysis of this cell population revealed that seven identified categories of reaching cells can be divided into three stages of CFC. Rac is also required for reaching cells. When observing the Rac-DN embryo no reaching cells were ever observed, regardless of heat-shocking time. The Rac-DN embryos showed an abnormal optic cup angle and unusual cuboidal cells shapes (early heat-shock). In later heat-shocked times the abnormal angle and unusual cell shapes were resolved, however, there were unusual division patterns that were observed. Further investigation is ongoing to identify the role of Rac in this cell population and the role of “reaching cells” during zebrafish eye development.

Keywords

Coloboma, Zebrafish, Rac, Eye Development

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