We are interested in the mechanisms that direct development of the central nervous system (CNS) of vertebrates. We are focussing our studies on the vertebrate retina, a relatively simple and well-characterized area of the CNS. We have used genomics approaches (SAGE and microarrays) to systematically examine gene expression over time during murine retinal development. We have further investigated the cellular specificity of expression patterns by performing in situ hybridization for >2000 genes with dynamic temporal patterns, and by performing gene profiling on single retinal cells. The comprehensive analysis of gene expression in over 150 single retinal cells is allowing for a computational approach to discover the networks of genes involved in several basic developmental processes. These networks are being analyzed for their roles in cell cycle control, particularly with respect to the production of postmitotic neurons. In addition, the networks that potentially control the production of various types of retinal neurons are being investigated. Functional studies are being carried out using knock-out mice and RNAi approaches. Many of the 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 studies. 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.
Many of the genes identified in the genomic studies cited above are enriched or specific in their expression to photoreceptor cells. As many disease genes that affect vision have been found to exhibit photoreceptor-specific or enriched expression, this is an excellent set of candidate genes for these diseases. We are also examining gene expression changes that accompany photoreceptor death in murine models of the human diseases, retinitis pigmentosa, cone-rod dystrophy, and macular degeneration.
Genes that control the initial formation and pattern of the eye are being characterized. We have begun to define the network that controls the placement of the central rod-free zone, akin to the human fovea, in the proper location in the retina, as well as determine how the peripheral eye structures (iris and ciliary body) are determined. Several transcription factors, small molecules such as thyroid hormone and retinoic acid, and secreted proteins, such as Wnts and FGFs, are implicated in these processes and are being further investigated.
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References:
- Matsuda, T. and Cepko, C. Controlled expression of transgenes introduced by in vivo electroporation. PNAS 104(3): 1027-1032 (2007)
- Jadhav AP, Mason HA, Cepko CL. Notch 1 inhibits photoreceptor production in the developing mammalian retina.
Development. 2006 Mar;133(5):913-23. (2006).
- Corbo, J., Cepko, C. A hybrid photoreceptor expressing both rod and cone genes in a mouse model of enhanced S-Cone Syndrome. PloS Genetics 1(2):0140-0153. (2005)
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