Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Harald J. Junge

Second Advisor

Leslie A. Leinwand

Third Advisor

Kenneth S. Krauter

Fourth Advisor

Kristi S. Anseth

Fifth Advisor

Natalie G. Ahn

Abstract

The Norrin ligand is a small (14 kDa) cystine-knot protein, required for the proper vascularization of retinal, cerebellar and inner ear neural tissue. Both inherited human disorders and murine genetic studies implicate Norrin mutations in major ocular and visual defects, including retinal hypovascularization. In this thesis, I describe the property of Norrin enrichment at specific layers in the retina. I address the question of whether proper Norrin distribution in retinal tissue is required for vascularization. To do so, I used ex vivo ligand in-situ staining to interrogate differences amongst vertebrate Norrin orthologues in retinal tissue retention. I then used sequence comparison amongst these proteins to identify protein regions required for tissue retention. In order to test the significance of the retinal tissue retention property of Norrin, I pursued a strategy to purify untagged versions of Norrin protein, and developed a protocol to intravitreally inject Norrin protein variants to rescue retinal hypovascularization defects in loss-of-function mice. I have identified a four-residue region required for retinal tissue retention of Norrin, making future in vivo investigations of a potential role for Norrin distribution in the retina possible. Using the strategy I have developed to pursue this question, future studies can also interrogate mutant versions of Norrin protein in vivo, using different rodent models of disease.

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