DESCRIPTION (provided by applicant): Understanding the transcriptional networks that progressively specify cell fates in normal development will improve our ability to regenerate damaged cells for therapeutic purposes. The Glass transcription factor was identified as a critical determinant of photoreceptor cell fate based on the phenotype of glass mutants in Drosophila. In these mutants, cells in the eye primordium differentiate as neurons, but they fail to express photoreceptor-specific genes, develop abnormal axonal projections, and die before the adult stage. Further analysis of Glass and its target genes, using methods and concepts that have been developed in the years since its initial discovery, has the potential to reveal how the photoreceptor fate decision is implemented. The first aim of this proposal will probe the function and regulation of Glass. The glass mutant phenotype will be analyzed to determine how Glass affects the progressive recruitment of distinct photoreceptor cell types early in development. To study later events in photoreceptor differentiation, an initiator caspase mutation will be used to prevent cell death in glass mutants, or glass will be depleted after differentiation has begun using RNA interference. Investigation of glass regulation will test the hypothesis that it integrates input from Pax6 and other retinal determination transcription factors with input from signaling pathways that provide temporal and spatial control of photoreceptor differentiation. The second aim will test the effects of Glass misexpression to determine whether it is sufficient to drive any aspects of photoreceptor development. The ability of two different isoforms of Glass to transform neuronal or non-neuronal cells towards a photoreceptor identity will be evaluated by phenotypic and gene expression analysis. A collaborative project to test the ability of Glass and its mammalian homologues to transform mouse embryonic stem cells from a neuronal to a photoreceptor identity will also be initiated. In the third aim, the role of individual Glass targe genes will be investigated. Transgenic RNA interference will be used to determine which of the genes likely to be directly activated by Glass mediate its effects on photoreceptor recruitment, differentiation, survival and axon targeting. In addition to depleting single target genes, related genes that might act redundantly, including two homologous transcription factors of the Scratch family will be removed simultaneously. Together, these experiments will improve our understanding of the transcriptional regulation of photoreceptor identity, and may provide useful tools for regenerative medicine.

PUBLIC HEALTH RELEVANCE: New stem cell technologies hold great promise for restoring vision to patients suffering from retinal damage and degeneration. However, converting stem cells into replacement photoreceptors requires us to understand how this cell fate is specified. We propose to bridge this gap by characterizing the function of a transcription factor that is required for neurons in the developing eye to differentiate as photoreceptors.