Johns Hopkins School of Medicine

Molecular Approaches to the Study of Retinal Development

My laboratory uses molecular biology approaches in an effort to better understand retinal function and pathology, and attempts to use this increased understanding to develop new approaches for the diagnosis and treatment of retinal disease. We are particularly interested in defining the mechanisms regulating photoreceptor and ganglion cell gene expression, determining how gene expression changes in disease, and identifying and characterizing novel retinal genes that are important for retinal function and disease. Amongst the ongoing research projects are the following:

In order to better understand photoreceptor gene expression, we have for a number of years studied the regulation of rhodopsin gene expression as a model system. Using a combination of transgenic mouse, cell culture, and in vitro transcription approaches, we have defined a number of the cis-acting elements important in rhodopsin regulation. More recently, using the yeast one-hybrid and a number of related approaches, we have cloned several of the transcription factors that bind to these elements. One of these, cone rod homeobox (CRX), turns out to regulate not only rhodopsin, but also a number of other photoreceptor-specific genes. After mapping the CRX gene to the site of a known photoreceptor degeneration, we set-up collaborations with a number of colleagues and found that CRX mutations are associated with cone rod dystrophy and the childhood degeneration Leber congenital amaurosis. In a parallel set of studies with Anand Swaroop, we are using mutation analysis to dissect the functional domains of CRX. We are particularly interested in defining the transactivation domain and the domain that binds to the bZIP protein neural retinal leucine zipper (NRL), a protein that we had previously shown to be important in rhodopsin expression. The binding between CRX and NRL is interesting because interaction between a homeobox protein and a leucine zipper protein have not been previously reported. Since "downregulation" of mutant gene products in the retina may slow down photoreceptor death, we are hoping to "translate" these advances in our understanding of retinal gene expression into practical approaches for modifying photoreceptor gene expression in vivo.

We are also using photoreceptor promoters to ask biological questions about the retina and to develop new animal models of retinal disease. In collaboration with Peter Campochiaro's lab, we generated transgenic mice expressing a dominant-negative form of the FGF receptor to demonstrate that adult photoreceptors are dependent on FGF signal transduction for maintenance. As a model system for the abnormal neovascularization that is important in the pathogenesis of both AMD and diabetic retinopathy, we generated transgenic mice whose photoreceptors overexpress vascular endothelial cell growth factor (VEGF). To refine these approaches, we have successfully adapted the tetracycline system for inducible retinal gene expression and are also developing retinal pigment epithelial (RPE)-specific promoters. In an additional collaboration with Ruben Adler, we are exploring the mechanisms by which neurotrophic factors retard retinal degeneration.

In collaboration with Harry Quigley's lab, we are exploring the mechanisms by which retinal ganglion cells (RGCs) die in glaucoma. We have shown that in this disease RGC death involves apoptosis, and possibly activation of caspase 3. Based on these results, we have generated transgenic mice and rats that express anti-apoptosis genes such as P35 and CrmA in their RGCs to determine if inhibition of apoptosis might provide a new "neroprotective" strategy for the treatment of glaucoma.