Utilization of Forward and Reverse Genetic Approaches to Inform Ocular and Choroid Plexus Development

Utilization of Forward and Reverse Genetic Approaches to Inform Ocular and Choroid Plexus Development

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The zebrafish is one of the prominent animal models used to investigate questions in modern genetics and vertebrate development due to it ease of genetic manipulation, external fertilization, and the transparent embryo. Utilizing a morpholino-based forward genetic approach, we individually reduced the levels of over 250 bioinformatically defined secreted proteins with the intent of identifying genes involved in ocular development. We identified alpha-1-microglobulin bikunin precursor (ambp) as a novel regulator of retinal size. Further characterization of the retina suggested the decrease in size was related to a decrease in differentiated cell types in the retina and a predominance of undifferentiated cells. Further analysis of this morphant with a transgenic zebrafish, MnET16, created in an ongoing forward genetic enhancer trap screen prompted the subsequent identification and developmental characterization of the zebrafish choroid plexus. The knockdown of ambp causes disruption of both the vascular and epithelial components of the choroid plexus. In an attempt to isolate the mechanism by which ambp might function in the choroid plexus, we identified a requirement for Notch signaling in the development of the choroid plexus epithelial cells, but not the vasculature. Using a morpholino mediated reverse genetic approach, the phenotype was shown to require the Notch receptor, notch1b and the Notch ligands, deltaA and deltaD. The lack of a vascular phenotype when Notch signaling is inhibited contrasts from the ambp phenotype and may suggest that ambp is functioning independent from the Notch pathway. In conclusion, I discuss the use of morpholino screens to identify eye phenotypes, the possible mechanisms of ambp action, the role of Notch signaling in choroid plexus development, and uses of the zebrafish as a model for both normal choroid plexus development and associated pathologies.Also unsatisfied with the excel spreadsheet, I developed a Filemaker Pro database (Apple) to facilitate data entry and sorting. ... The interface was insufficient at online sharing and did not afford the ability to search by sequences; therefore a MySQL database was designed. ... With the addition of wireless internet in the screening room, we were able to make our screening data directly available to the consortium in a ... The pictures were taken, and printed at 8aquot; X11aquot; on a laser printer.


Title:Utilization of Forward and Reverse Genetic Approaches to Inform Ocular and Choroid Plexus Development
Author:Brent Roy Bill
Publisher:ProQuest - 2008
ISBN-13:

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