Date of Award

12-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Neuroscience

Research Advisor

Michael R. Taylor, Ph.D.

Committee

Alessandra D’Azzo, Ph.D. Kristin Hamre, Ph.D. Charles A. Lessman, Ph.D. John D. Schuetz, Ph.D.

Keywords

blood-brain barrier, zebrafish, development, genetic screen, chemical screen

Abstract

The blood-brain barrier (BBB) maintains a homeostatic environment as well as prohibits the entrance of xenobiotics into the brain. Because of these qualities, drug delivery is a fundamental challenge for the treatment of many diseases of the central nervous system (CNS). Along with limiting the access of therapeutics into the brain, the BBB is also impaired in CNS pathologies. Understanding the molecular cues that are essential for healthy BBB development and integrity may reveal targets for drug delivery leading to decreased progression or possible treatment of many detrimental CNS diseases. While the central features of the BBB have been accepted, an innovative model has yet to creatively utilize this established knowledge to decipher how the unique signals controlling BBB properties can be optimally targeted. To overcome these scientific barriers, we used zebrafish as a model organism to study BBB development in vivo. We hypothesized that zebrafish could be used to genetically dissect the molecular mechanisms important for the development, function, and maintenance of the BBB. The ultimate goal of this project was to use the zebrafish model for unbiased genetic and small molecule screens. To fulfill these goals, we produced a BBB reporter line, Tg(glut1b:mCherry)sj1, to characterize the in vivo development of the BBB. We were able to show through live imaging and proof-of-principle experiments, that the processes of CNS angiogenesis and barriergenesis occur concurrently, but do so via independent mechanisms. We then initiated a forward genetic and small molecule screen with our BBB reporter line. From these unbiased, high-throughput approaches, we uncovered several BBB mutants and chemical hits that modulate CNS angiogenesis and the glut1b promoter. We also cloned one of these genetic mutants whereby the mutation was in gpr124, an established gene important for BBB development in mammals. The gpr124 mutant validates that we were able to find genetic modifiers of BBB development in our forward genetic screen. The execution of these screens will not only advance our understanding of BBB development but potentially reveal novel genes and molecules that could be targeted for CNS drug delivery.

DOI

10.21007/etd.cghs.2014.0329

Comments

One year embargo expired December 2015

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