Transcriptional and epigenetic regulation of gene expression; retinal stem cell biology; molecular genetics of retinal development and differentiation.

During development, distinct and often dynamic patterns of gene expression are critical for cell fate specification and differentiation as well as regulating the patterns of the synatpic connections of retinal neurons. The long term goal of my research is to understand the transcriptional mechanisms that establish and maintain spatial and cell-specific patterns of gene expression in retinal neurons during development, differentiation and synaptogenesis. We are pursuing two main lines of research. First, we are studying the cellular mechanisms regulating neuronal differentiation by glial-derived retinal stem cells. Second, we are characterizing the molecular mechanisms controlling expression of a family of signaling molecules, the Ephrin receptors (Eph), that are known to play a critical role in patterning the connections of retinal ganglion cells in the brain.

Retinal Stem Cell Biology Some species, notably teleost fish and amphibians, are able to regenerate retinal neurons following injury. In contrast, in mammals there is no spontaneous retinal regeneration. In fish, the stem cells present in the inner retina arise from the Muller glia therefore, there is considerable interest in the neurogenic capacity of Muller glia from the mammalian retina. We have isolated a novel cell line of Muller glia from post-natal mice that have stem cell characteristics in vitro. We have found that culturing cells in three-dimensional hydrogel cultures increases expression of specific genes that expressed in differentiated retinal neurons in the mature retina. Using combinatorial treatment with specific growth factors, over-expression of developmentally important transcription factors and targeted gene knockdown, we are identifying mechanisms that will suppress glial identity and enhance the neuronal potential of these cells. The long term goal is to generate sheets of stem cells that are biased to generate specific cell types that can be used for transplantation into the retina to replace neurons that are lost as a result of degenerative retinal disease.

Transcriptional Regulation of Eph Receptor Expression Eph receptors are tyrosine kinase receptors involved in diverse patterning events during embryogenesis. In the developing retina, gradients of Eph receptors along the nasal/temporal and dorsal/ventral axes specifies the position of retinal ganglion cells within the retina. Eph receptors on the growth cones of the retinal ganglion cells respond to counter gradients of ligands in visual centers in the brain and patterns the spatial organization of retinal ganglion cell terminals. In order to regenerate the optic nerve in degenerative diseases such as glaucoma, we need to first understand how to generate new ganglion cells, and then how to direct them to re-establish the appropriate patterns of connectivity within the brain. To address this, we have cloned the promoters of four Eph receptors that are expressed in the retina are studying the role of specific transcriptional factors and changes in chromatin structure including DNA methylation on promoter activity. A new and related initiative we are undertaking is to characterize the global patterns of DNA methylation and gene expression in retinal ganglion cells and how these change during the disease progression in a mouse model of inherited glaucoma.

Some of the techniques used in these studies include tissue culture, in vitro promoter activation assays, histology, immunostaining and in situ hybridization, light and fluorescence microscopy; confocal microscopy and molecular biological methods such as Northerns, Southerns, Westerns, gel mobility shift assays, cloning, quantitative RT-PCR, DNAseI footprinting, chromatin immunoprecipitation, bisulfite sequencing, transgenic animals, in vitro transcription and translation, site directed mutagenesis.