Photoreceptor development and survival requires the efficient transport of proteins from the inner segment to the outer segment. The central hypothesis of this proposal states that intraflagellar transport (IFT) proteins are essential for outer segment formation and protein transport through the connecting cilium and mutations in IFT genes cause retinal degeneration. The IFT proteins form a multisubunit complex that is transported along microtubules in the connecting cilium via the action of kinesin motors. We will use zebrafish as an experimental system because zebrafish photoreceptors are well-characterized, established techniques exist in zebrafish to manipulate gene expression and create genetic mosaic embryos, and we have zebrafish mutants for three IFT genes. These mutants exhibit mislocalized rhodopsin, disrupted photoreceptor outer segment formation, and photoreceptor degeneration. Our objective is to understand the function and regulation of the IFT particle in ciliary protein transport within the photoreceptor. Specifically, we propose to: 1) To determine the function and effects of mutations of three IFT proteins on zebrafish photoreceptor development by examining outer segment structure and protein localization using electron microscopy and light microscopy combined with immunohistochemistry. 2) To examine the regulation of IFT particle assembly and function by the Par-aPKC polarity complex. The IFT particle and Par-aPKC complex co-localize within the cilium and both interact with kinesin-ll. We will investigate the interactions between these complexes using immunoprecipitation and genetic mosaic analysis. 3) To measure IFT complex stability and movement in IFT57 mutants by analyzing the localization and movement of IFT-GFP fusion proteins assembled in the IFT particle using fluorescence microscopy and time-lapse confocal microscopy. 4) To determine the requirement for each IFT protein in photoreceptor morphogenesis by using morpholino oligonucleotides to generate Bardet-Biedl Syndrome phenotypes in zebrafish and assess photoreceptor development by light and fluorescence microscopy. As the mechanisms of protein transport required for photoreceptor development also function in the maintenance of adult photoreceptors, our studies can provide insights in the processes that lead to retinal pathologies.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Mariani, Andrew P
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Texas A&M University
Schools of Arts and Sciences
College Station
United States
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Song, Ping; Perkins, Brian D (2018) Developmental expression of the zebrafish Arf-like small GTPase paralogs arl13a and arl13b. Gene Expr Patterns 29:82-87
Miller, Andrew H; Howe, Hollis B; Krause, Bryan M et al. (2018) Pregnancy-Associated Plasma Protein-aa Regulates Photoreceptor Synaptic Development to Mediate Visually Guided Behavior. J Neurosci 38:5220-5236
Lessieur, Emma M; Fogerty, Joseph; Gaivin, Robert J et al. (2017) The Ciliopathy Gene ahi1 Is Required for Zebrafish Cone Photoreceptor Outer Segment Morphogenesis and Survival. Invest Ophthalmol Vis Sci 58:448-460
Bell, Brent A; Yuan, Alex; Dicicco, Rose M et al. (2016) The adult zebrafish retina: In vivo optical sectioning with Confocal Scanning Laser Ophthalmoscopy and Spectral-Domain Optical Coherence Tomography. Exp Eye Res 153:65-78
Fogerty, Joseph; Denton, Kristin; Perkins, Brian D (2016) Mutations in the Dynein1 Complex are Permissible for Basal Body Migration in Photoreceptors but Alter Rab6 Localization. Adv Exp Med Biol 854:209-15
Song, Ping; Dudinsky, Lynn; Fogerty, Joseph et al. (2016) Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish. Invest Ophthalmol Vis Sci 57:4517-26
Daniele, Lauren L; Emran, Farida; Lobo, Glenn P et al. (2016) Mutation of wrb, a Component of the Guided Entry of Tail-Anchored Protein Pathway, Disrupts Photoreceptor Synapse Structure and Function. Invest Ophthalmol Vis Sci 57:2942-54
Babino, Darwin; Perkins, Brian D; Kindermann, Aljoscha et al. (2015) The role of 11-cis-retinyl esters in vertebrate cone vision. FASEB J 29:216-26
DiCicco, Rose M; Bell, Brent A; Kaul, Charles et al. (2014) Retinal regeneration following OCT-guided laser injury in zebrafish. Invest Ophthalmol Vis Sci 55:6281-8
Krock, Bryan L; Perkins, Brian D (2014) The Par-PrkC polarity complex is required for cilia growth in zebrafish photoreceptors. PLoS One 9:e104661

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