Item Details

Rx Gene Regulation and Early Vertebrate Eye Development

Wang, Hui
Format
Thesis/Dissertation; Online
Author
Wang, Hui
Advisor
Grainger, Robert
Abstract
Study of vertebrate eye development is important for understanding not only the eye-related genetic diseases, but also the biological mechanisms in general. Here, I present detailed studies on an enhancer of Rx, the vertebrate retina homeobox gene, and the identification of an up-stream regulatory factor, the processed form of Gli3 protein, which binds to this enhancer and activates Rx gene expression in anterior neural plate of Xenopus. Deletion analysis on the previously identified -1.7kb promoter region identified a 259bp minimal enhancer element which is both necessary and sufficient for regulating reporter expression. Comparative genomic studies of the Rx loci from multiple vertebrate species agree with the result from traditional deletion analysis. Site-directed mutagenesis of the predicted transcription factor-binding motifs in the highly-conserved sequence stretches within the 259bp enhancer element indicates that a putative Gli-binding motif and YY1-binding motif are critical for activating Rx enhancer. At neural plate stage, the expression pattern of Gli3 overlaps with the Rx expression pattern. Mis-expression of the C-terminal truncated form of Gli3 protein activates Rx expression ectopically. Morpholino antisense oligo-based loss-of-function study indicates that loss of Gli3 protein function leads not only to the disturbance of Rx gene expression, but also the failure of eye formation in Xenopus. The electrophoretic mobility shift and competition assay shows that the C-terminal truncated form of Gli3 protein binds to the Gli-binding motif found in the Rx enhancer in vitro. Mis-expressed full length Gli3 protein is shown to be processed into a short form of the same size as that of the predicted C-terminal truncated form in the anterior neural plate where Rx is expressed, which is evidence of the co-localization of the processed form of Gli3 protein with Rx mRNA. In collaboration with another group, we find Yin Yang 1 (YY1) protein binds to the predicted YY1 motif found in Rx enhancer in vitro and activates the enhancer activity in yeast-one-hybrid system. These results suggest that YY1 protein and the processed form of Gli3 protein bind to adjacent motifs in Rx enhancer and may cooperate in regulating Rx expression in Xenopus. Note: Abstract extracted from PDF text
Language
English
Date Received
20140123
Published
University of Virginia, Department of Biology, PHD (Doctor of Philosophy), 2008
Published Date
2008-01-01
Degree
PHD (Doctor of Philosophy)
Collection
Libra ETD Repository
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