Probing photoreceptor cell biology using genetically modified amphibians.

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The vertebrate retina contains two principle types of photoreceptor: rods and cones. Within these cells, photodetection occurs in an elaborate structure called the outer segment (OS), a modified sensory cilium. This structure contains many proteins unique to photoreceptors that are involved in light detection or OS structure. For many years, my laboratory has used genetically modified transgenic amphibians (specifically X. laevis) to study these cells due to the ease with which transgenic animals can be generated, as well as other advantages related to the large size of amphibian photoreceptors. Recently, we have developed CRISPR/Cas9 based techniques for gene knockout, and we recently generated knockouts of several photoreceptor-specific genes. We propose to use these animals, and to generate novel genetically modified animals, to study uncharacterized aspects of photoreceptor cell biology including: 1. Alternate pathways for generation of chromophore. We have found that knockout animals lacking RPE65, the isomerohydrolase thought to be responsible for generation of chromophore, still have a measurable electroretinogram. We will further characterize the visual responses of these animals, and in collaboration with Dr. A. Sampath at the Jules Stein Eye Institute, determine which cone subtypes are responsible. We will conduct further genetic manipulations to determine the nature of the pathway involved, and attempt to identify alterations in visual responses in animals in which this pathway is eliminated. 2: Physiological consequences of arrestin multimerization. We have knocked out the arrestin (SAG) gene resulting in animals with delayed dark adaptation. Some arrestins multimerize, although he physiological consequences of this are unclear. Rod arrestin is known to migrate between different cellular compartments in response to light. We will transgenically restore fluorescent arrestins to these animals that are primarily monomeric, dimeric, or multimeric. In collaboration with Dr. P. Calvert at SUNY we will use time-resolved fluorescence anisotropy microscopy to determine their multimeric states in vivo in different compartments, and determine the consequences for arrestin migration kinetics and dark adaptation kinetics in vivo. 3. Dissection of peripherin-2 functions. Peripherin-2 is a structural protein found at photoreceptor disk rims, where it creates and maintains curvature, while participating in a large number of proposed interactions. We have identified six peripherin-2 homologues in X. laevis. The presence of a large number of genes suggests that they may have developed specialized roles. By knocking out each peripherin-2 sequence separately, we may be able to dissect different unique aspects of peripherin-2 function. The large number of peripherin-2 sequences will likely make the resulting phenotypes less severe, and therefore potentially more structurally informative, than mammalian knockouts.