Date of Award

8-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Michael R. Taylor, Ph.D.

Committee

Charles A. Lessman, Ph.D. Paul E. Mead, Ph.D. Leta K. Nutt, Ph.D. Radhakrishna Rao, Ph.D. Clinton F. Stewart, Pharm.D.

Keywords

choroid plexus, forward genetic screen, Sart1, zebrafish

Abstract

The choroid plexus (CP) is an epithelial based structure localized within the brain ventricles and functions as the blood-cerebrospinal fluid barrier (BCSFB). Under normal conditions, the CP is responsible for generating the cerebrospinal fluid (CSF) and regulating its components. Abnormal CP function has been associated with neurodegenerative diseases, tumor formation in CP epithelia (CPe), and hydrocephalus. Despite the significant role of the CP in these disorders, little research has been done to characterize its functional properties and genetically dissect the pathways involved in normal CP development and disease. For this study, we have utilized zebrafish, (Danio rerio), as a model system to better understand the genetic components of CP development. Their transparent nature and rapid ex utero development provide mechanisms to visualize CP formation that is not possible in other model systems. By utilizing an enhancer trap line, Et(cp:EGFP) sj2 that expresses the enhanced green fluorescent protein (EGFP) in CPe, we have demonstrated that the zebrafish CP possesses barrier properties such as tight junctions, transporter activity, and regulating central nervous system (CNS) homeostasis. After validating that the zebrafish CP is similar to higher vertebrates, we have initiated a genetic screen to answer questions such as: 1) what genes are needed for CP development, function, and maintenance, 2) what are the signaling pathways involved, and 3) how do these pathways interact with one another to form the BCSFB? This study is the first to employ an unbiased approach using a forward genetic screen to genetically dissect the CP and identify genes essential for its formation and function. As a result of this genetic analysis, we have confirmed 10 mutant lines with CP abnormalities. The mutants generated in this study will be used in future investigations to elucidate specific genes and signaling pathways essential for CP development, function, and maintenance and will provide a better understanding of how genetic mutations contribute to CP- related diseases. We have also mapped the gene for one mutant line, cp27.5, to Chromosome 21. Using a combination of classical positional cloning and whole-exome sequencing (WES), we identified the mutated gene as squamous cell carcinoma antigen recognized by T cells (sart1). sart1 plays a role in assembling the spliceosome, a multi-ribonucleoprotein complex essential for processing pre-mRNA. By characterizing cp27.5 mutants, we identified a number of proteins with altered expression levels and patterning primarily within the brain and eye. Using RNA-Seq analysis, we also identified multiple genes up-regulated or down-regulated due to the sart1 mutation. While sart1 itself was up-regulated, we also identified increased expression of genes involved in apoptosis such as tp53 and mdm2. A loss of photoreceptors and lack of retinal lamination in mutants is related to a down-regulation of vision-related genes such as phosphodiesterase 6H, (pde6h) and opsin-1, short-wave-sensitive 1 (opn1sw1), both of which are expressed in photoreceptors. Future studies will investigate the role of sart1 in splicing and the mechanisms involved in producing the phenotypes observed in cp27.5 mutants.

DOI

10.21007/etd.cghs.2014.0134

Comments

One year embargo expired August 2015

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