The goal of this proposal is to use zebrafish as a model system to identify novel regulators of ocular lens development and examine how loss of single and combined ?-crystallins, small heat shock proteins important for lens function, affect lens aging and the regulation of lens crystallin expression. These experiments will produce a better understanding of lens development and maintenance of lens transparency important for developing treatments to prevent lens opacities, the leading cause of blindness worldwide. Work in our laboratory has helped make the zebrafish a powerful model system for studying lens development, function and disease. Undergraduate students supported by past funding have cloned and characterized three zebrafish ?-crystallins and used CRISPR/Cas9 gene editing to disable each to examine the impact of their loss on early lens development. Here, we propose in Aim I to use our already produced knockout lines to model the impact of ?-crystallin loss on the maintenance of lens transparency and the gene regulatory networks that control lens protein expression.
This aim will include an integration of transcriptomic and proteomic analyses, along with anatomical examination of resulting lens phenotypes. We hypothesize that the resulting lens phenotypes will depend on the specific ?-crystallin lost, providing a novel examination of ?B-crystallin function due to the presence of two orthologs in zebrafish for the single-copy mammalian gene.
In Aim II we will use transcriptomic data sets generated by our collaborators from mouse and zebrafish tissues to identify possible novel transcription factors (TFs) regulating lens development. We will then efficiently target these TFs using CRISPR/Cas9 methods that will allow us to screen for lens phenotypes directly in injected embryos. Mutant TF alleles that disrupt normal lens development will be carried into stable lines to detail the role they play in establishing and maintaining lens transparency. We expect that these two aims will efficiently identify novel regulators of lens development and clarify the role of ?- crystallins in maintaining lens transparency. The experiments proposed will provide excellent training opportunities for undergraduates in a research setting with a strong track record of student success.

Public Health Relevance

This project will use the zebrafish as a cost-efficient model system to examine how proteins called alpha crystallins contribute to the ocular lens' ability to stay transparent as it ages, preventing the formation of cataracts, the leading cause of human blindness worldwide. We will also identify novel genes regulating lens development, providing a better understanding of lens disease and possible treatments. Undergraduate college students at Ashland University will gain valuable experience using innovative genome manipulation and other molecular biology techniques while conducting these studies.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15EY013535-05A1
Application #
10046498
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Araj, Houmam H
Project Start
2001-08-01
Project End
2023-06-30
Budget Start
2020-08-01
Budget End
2023-06-30
Support Year
5
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Ashland University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
076751825
City
Ashland
State
OH
Country
United States
Zip Code
44805
Posner, Mason; Murray, Kelly L; McDonald, Matthew S et al. (2017) The zebrafish as a model system for analyzing mammalian and native ?-crystallin promoter function. PeerJ 5:e4093
Wages, Phillip; Horwitz, Joseph; Ding, Linlin et al. (2013) Changes in zebrafish (Danio rerio) lens crystallin content during development. Mol Vis 19:408-17
Posner, Mason; Skiba, Jackie; Brown, Mary et al. (2013) Loss of the small heat shock protein ?A-crystallin does not lead to detectable defects in early zebrafish lens development. Exp Eye Res 116:227-33
Posner, Mason; Kiss, Andor J; Skiba, Jackie et al. (2012) Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein ?A-crystallin. PLoS One 7:e34438
Posner, Mason; Hawke, Molly; Lacava, Carrie et al. (2008) A proteome map of the zebrafish (Danio rerio) lens reveals similarities between zebrafish and mammalian crystallin expression. Mol Vis 14:806-14
Smith, Amber A; Wyatt, Keith; Vacha, Jennifer et al. (2006) Gene duplication and separation of functions in alphaB-crystallin from zebrafish (Danio rerio). FEBS J 273:481-90
Dahlman, Jason M; Margot, Kelli L; Ding, Linlin et al. (2005) Zebrafish alpha-crystallins: protein structure and chaperone-like activity compared to their mammalian orthologs. Mol Vis 11:88-96
Runkle, Stephanie; Hill, Julie; Kantorow, Marc et al. (2002) Sequence and spatial expression of zebrafish (Danio rerio) alphaA-crystallin. Mol Vis 8:45-50