Dark-adapted albino zebrafish undergo loss of rods and cones when placed in constant intense light. The photoreceptors are regenerated from a population of adult stem cells in the inner nuclear layer. The Muller glia may also proliferate and either dedifferentiate into neuronal precursors or trans-differentiate into photoreceptors during regeneration. To examine the role of the Muller glia in regeneration, it is necessary to develop techniques to regulate the temporal-spatial expression of transgenes, such as the Cre-lox site- specific recombination and the Tet-On systems. While both of these systems are effectively used in mouse genetics, neither has been fully demonstrated to function in transgenic zebrafish. We will develop the Cre-lox system to express a reporter (EGFP) in a cell-specific manner to examine cell lineages. The Cre enzyme, which will be expressed in Muller glia using the GFAP or glutamine synthetase promoter, will catalyze the recombination between two lox sites in a second transgene. This will excise transcriptional and translational termination signals and express EGFP from a ubiquitous promoter. Thus, EGFP will only be expressed in Muller glia and any cells derived from these glia. Using immunohistochemical methods to examine retinas at different time points during regeneration, we will unambiguously identity the EGFP-positive cells and the lineage of the Muller glia during regeneration. We will also determine if the Tet-On system functions in zebrafish. We will express the Tet trans-activator (rtTA) using either an ubiquitous or cell-specific promoter. Addition of doxycycline will permit the rtTA to bind the TRE sequence and activate transcription of the EGFP reporter. Demonstrating that the Tet-On system is a tight inducible system will allow us to express a variety of transgenes to examine the roles of the inner nuclear layer stem cells and Muller glia in the regeneration response. ? Relevance: Regulating the temporal-spatial expression of transgenes in zebrafish will allow us to examine the function of either normal or mutant forms of a gene throughout development. We will initially use these techniques to examine the role of radial glia in the regeneration of photoreceptors. Elucidating the role of Muller glia in zebrafish photoreceptor regeneration may reveal inductive cues that could be used to induce Muller glia to regenerate photoreceptors in inherited human retinal degenerative diseases. ? ?
