SUMMARY
The mechanisms that cells use when they are choose their fate during the development of the central nervous system is the main problem under study in our lab. We have focussed our studies on the retina, a tractable model for the rest of the central nervous system. In addition, we are interested in why photoreceptor cells die in many forms of human retinal degeneration.
RETINAL CELL FATE DETERMINATION
Over 15 years ago, we used a lineage marking technique to discover that the mitotic progenitor cells that generate the various neuronal and glial cell types of the retina are multipotent. Products of a final division can be as different as a specialized sensory neuron, such as a rod photoreceptor, and a non-neuronal cell type, a Muller glial cell. We have been addressing several questions concerning the mechanisms that lead each retinal cell to choose its fate in the retina. One question concerns the properties of the progenitor cells themselves. We have found that they change over time in terms of their ability to divide, and in terms of their ability to make different types of retinal cells. We are now using genomics methods to discover the genes that are differentially expressed among retinal progenitor cells. This cataloguing method should help us determine how many types of progenitor cells there are, as well as lead us to the genes that are responsible for the differences. The level of expression of such genes is being manipulated to provide information about the function of a gene. Effects on cell fate, proliferation, differentiation, and survival will be assessed. To date, homeobox genes and basic helix-loop-helix genes have been analyzed in this way. Another approach is to recapitulate some aspects of retinal development in vitro. The environment of retinal cultures can be varied to determine how the environment affects the cell fate decisions. For example, the formation of rod photoreceptors can be influenced positively and negatively by several factors. These factors include small molecules that can act as neurotransmitters as well as growth factors. In addition, production of all retinal neurons is influenced by the signals produced by a large transmembrane receptor, notch, first identified in fruit flies.
PATTERNING OF THE RETINAThe retina is not a uniform sheet of cells, but exhibits various types of patterns. For example, in humans and chicks, there is a central region, the fovea, devoid of rod photoreceptors. We have identified several genes that control some aspects of retinal pattern. They are expressed asymmetrically across the retina of mice and chicks early in development when the patterns are set up. For example. The homenpx. Vax, is expressed in a ventral pattern early in development. Overexpression of Vax ventralizes gene expression patterns of the retina. It causes a gross pertubation in the ability of dorsal retinal ganglion cells to find their appropriate target cells in the tectum. It can also disturb the formation of the central rod-free zone. In addition to transcription factors, we have begun to explore the role of two small molecules, retinoic acid and thyroid hormone, in the formation of retinal pattern as the genes that regulate or respond to them are patterned during critical stages of retinal development.
As many of our studies rely on knowing the gene expression patterns in retinal cells, we have used genomics methods to comprehensively catalogue the genes that are expressed in retinal cells in development and disease. These methods include microarrays and serial analysis of gene expression (SAGE). We have cDNA microarrays of genes expressed in the retina at several stages of development, in the adult, and in the degenerating retina from the rd1 mouse. We use these arrays to characterize gene expression in single cells and in retinal tissue from a variety of conditions. Mature retinal neurons, as well as retinal cells that are in the process of choosing their fates and differentiating, are being characterized. SAGE is a different genomics method that we have used to describe gene expression during development, and in a mouse mutant for the Crx gene, a photoreceptor-specific transcription factor. Genes that were found to be differentially expressed using these genomics methods were further investigated using in situ hybridization. A catalogue of the gene expression patterns and the associated in situ hybridization images can be found here.
We are using retinal microarrays for a application aimed towards elucidating the mechanism of retinal disease. In many forms of human retinal degeneration, rod photoreceptors express a mutant gene, while cone photoreceptors do not. Interestingly, the cones still die. This implies a non-autonomous process in the death of cones. Humans rely most heavily on cones, and thus we would like to understand why the cones die so that gene therapy or a pharmaceutical intervention can be developed. To this end, we are using microarrays to examine several mouse models of human disease to discover the non-autonomous process leading to cone death.
We are grateful to the following institutions for support of our studies: The National Eye Institute, The Foundation for Retinal Research, The Macular Disease Research Foundation, and Merck.