We are interested in the mechanisms that direct development and degeneration of the central nervous system (CNS) of vertebrates. We are focusing our studies on the vertebrate retina, a relatively simple and well-characterized area of the CNS. We have used genomics approaches and in situ hybridization to characterize genes with dynamic temporal patterns to better determine which genes are candidates for playing a role in cell fate determination. These data include profiles of >200 single retinal cells. We are now trying to determine how the retina uses this large repertoire of genes to form this complex tissue of >60 neuronal cell types. We are particularly interested in the diversification of the different types of interneurons as these cells form critical elements in retinal circuitry. To aid in these studies, we also carry out lineage studies wherein we mark individual progenitor cells in vivo, and analyze the types of neurons produced. Of interest is whether progenitor cells produce cells that are connected in various types of retinal circuits. To this end, and more generally to discover the circuitry of CNS neurons, we have recently developed novel viral vectors which move transsynaptically in vivo. These vectors can be modified to transmit either retrogradely or anterogradely, and can be used to transduce a variety of genes, encoding fluorescent proteins, ion channels, or calcium indicators. We are interested in their applications in neuroscience, as well as what they can tell us about how viruses transmit among neurons. We are also using them to learn more about retinal circuitry.

Many of our studies are facilitated by the electroporation of multiple plasmids in vivo. We have developed an electroporation method and a series of plasmids that promote the regulated expression of short hairpin RNAs, cDNA, or multiple genes and shRNA species. This method allows for relatively rapid assessment of gene function, including genetic epistasis. We are also investigating the regulatory sequences that control selected genes using electroporation of reporter plasmids. These data are then analyzed with respect to the function of suspected trans-acting factors, which can be identified through computational approaches, as well as inspection of the gene catalogues created by the single cell microarray data.

We are also interested in the mechanisms that lead to the death of photoreceptors in the many inherited forms of human blindness. Through examination of gene expression changes that accompany photoreceptor death in murine models of the human diseases, retinitis pigmentosa, we have discovered that the metabolism of cone photoreceptors appears to be stressed to the point that the cells undergo autophagy. We have found that we can slow down the death of these cells through administration of insulin, whereas the death is accelerated if animals are depleted for insulin. In addition, we have found that delivery of the histone deacetylase 4 gene can slow down the autonomous death of mutant rod photoreceptors via stabilization of the hypoxia inducible factor 1 alpha. We are now investigating whether gene therapy approaches that follow from these findings might extend vision in animal models, with the goal of developing a therapy for humans.