Cell fate specification is imposed by transcriptional regulators that activate cell type specific gene expression patterns. Many of these master regulators are conserved from invertebrates to vertebrates, making the Drosophila eye an excellent model system in which to address this process. This proposal will investigate the Drosophila zinc finger transcription factor Glass (Gl), which appears to act as a key determinant in controlling the developmental switch from neuronal precursors to photoreceptor cell types. In gl mutants, eye progenitor cells initiate neuronal differentiation as detected by the presence of neuronal markers, but fail to express photoreceptor-specific genes, extend axons aberrantly into the brain, and die before reaching adulthood. We hypothesize that characterizing the role of Gl in photoreceptor differentiation will reveal novel molecular features underlying eye development. These findings will have translational implications in developing stem cell-based therapies for patients suffering from retinal degenerative diseases.
The first aim i s to identify the specific stages of photoreceptor development that require Gl function. Premature cell death has prevented a careful analysis of this requirement and it is presently not known if Gl is required at later stages for the differentiation and maintenance of photoreceptors or if its only role is to ac as an early determinant that activates the photoreceptor transcriptional network. To address this question we will inactivate gl expression at late stages, and examine the fate of cells incapable of inducing cell death in a gl mutant background.
The second aim will determine if Gl is sufficient to induce photoreceptor development in Drosophila or mammalian cells. Using genetic tools we will misexpress Gl in fly tissues and determine by phenotypic analysis, immunohistochemistry and gene expression profiling if it can induce photoreceptor-specific genes or traits. Gl was shown to bind to vertebrate rhodopsin promoters making it a potentially powerful tool with which to induce gene expression changes in mammalian cells. Gl shares homology with an uncharacterized human zinc finger protein ZNF500, which is also highly expressed in the retina. We propose to misexpress Gl or ZNF500 together with neuronal inducers in mouse embryonic stem cells to determine whether it can activate the expression of photoreceptor-specific genes. In the third aim we will use an RNAi strategy to investigate the role of each of the identified direct targets of Gl activation in photoreceptor development. As many of these genes remain uncharacterized, we expect to identify novel factors that contribute to photoreceptor recruitment, differentiation, survival or axon targeting.
Retinal degeneration is a disease for which there is no known cure, affecting millions of people worldwide resulting in the loss of photoreceptors and vision. Understanding the molecular program that controls the developmental switch from neuronal precursors to photoreceptors will be important for the development of stem cell-based therapies for patients suffering from retinal degenerative diseases. We aim to elucidate the activity and effects of a master gene regulator that defines this transition and to determine whether this knowledge could be used to generate photoreceptors and restore vision to patients.
