Graeme Mardon, Ph.D.Professor, Department of Pathology
Professor and Co-Director, Program in Developmental Biology
Professor, Program in Cell and Molecular Biology
Professor and Co-Director of Graduate Studies, Department of Molecular and Human Genetics
Professor, Department of Ophthalmology
Professor, Department of Neuroscience
Program Director, NIH Training Grant T32 EY07102
Director, Darwin Transgenic Mouse Core Facility, Department of Molecular and Human Genetics
- Research Interests and Future Plans: A cross-species approach to understanding developmental neurobiology
- One of the most remarkable findings of the past decade has been the high degree of conservation of gene structure and function between insects and vertebrates, particularly during development.
- Our research takes advantage of this conservation of developmental mechanisms with the ultimate goal of understanding abnormalities that result in human disease. We use two animal model systems: the fruit fly Drosophila melanogaster and the house mouse Mus musculus. Employing molecular and genetic techniques, as well as nucleic acid database analysis, we identify and characterize conserved genes required for normal neural development and function. Drosophila provides the potential to decipher gene function with powerful genetic tools that are unsurpassed by any other higher eukaryote. We then apply insights from Drosophila to homologs of conserved genes in the mouse and investigate what role they may play in human disease. This two-pronged approach to understanding vertebrate development has proven to be informative and productive.
- A primary goal of our research is to understand molecular mechanisms of retinal development. In Drosophila, our main studies have focused on genes required for the earliest steps of retinal cell fate determination. A group of highly conserved retinal determination (RD) genes, including eyeless, dachshund, eyes absent, and sine oculis, function in a network to control eye development. Each RD gene is required for normal eye development: loss-of-function mutations in any one of these genes results in flies that have greatly reduced or no eyes. Moreover, targeted misexpression of each RD gene (except sine oculis) is sufficient to induce ectopic retinal tissue with the same structure and cellular composition found in the normal compound eye. These results indicate that this group of genes functions at the highest levels of the regulatory hierarchy controlling retinal cell fate determination and patterning in Drosophila. Finally, each RD gene is highly conserved in vertebrates and many are also required for mammalian retinal development.
- In addition to our work on the RD genes, we are studying senseless, which encodes a conserved zinc-finger transcription factor. senseless is both necessary and sufficient to direct R8 photoreceptor differentiation. Intriguingly, the R8 photoreceptor in Drosophila and the retinal ganglion cell in vertebrates may be developmentally equivalent. These two cells types are the first retinal cells to be specified, express orthologous genes with conserved function during their development, project axons directly to the brain, and play instructive roles in retinal patterning. We are investigating the mechanism by which senseless controls R8 differentiation in Drosophila.
- In Drosophila, we have two primary research goals in the area of retinal development. First, we are continuing to study the molecular mechanisms by which senseless acts during R8 differentiation. We have completed all the initial phases of a genetic screen to identify new genes that interact with a senseless overexpression phenotype and have identified 11 complementation groups and expect to soon know the identity of most (three are already known). These new genes will provide important tools to further pursue mechanisms of senseless function. In addition, we have identified distinct, small enhancer regions that are sufficient to drive reporter gene expression in the developing eye, antenna, and leg imaginal discs that closely match the normal pattern of senseless expression. Each enhancer depends upon normal function of the proneural gene atonal and yet separate enhancers are activated in only one tissue (i.e., the eye enhancer is not active in the leg and vice versa, even though both depend upon the same proneural gene, atonal). These reagents provide a unique opportunity to decipher tissue-specific mechanisms of gene regulation that are likely to provide broad insight into gene regulation in general.
- Our second Drosophila project is being conducted in collaboration with Dr. Rui Chen in the Department of Molecular and Human Genetics. This project aims to use a combinatorial approach of genetics, genomics, and computational biology to identify targets of the retinal determination gene eyeless. Although many transcription factors required for normal eye development are known, very few targets of such factors have been identified. This represents one of the largest gaps in our knowledge of development in general. Currently, we propose to use a combination of several different approaches to identify targets of Eyeless and then validate and characterize such targets in vivo. These approaches include microarray analysis of wild type and mutant eye discs (both loss- and gain-of-function and a combination of the two), chromatin immunoprecipitation using a tagged Eyeless protein, and computational biology to predict Eyeless binding sites in the genome. An R01 proposal to support this project has now been funded by the National Eye Institute. We have now shown direct binding of Eyeless protein to one or more binding site in each of the first five genes studied. Moreover, we have also shown that one novel predicted target is required for early steps of normal eye development, validating this approach. In the future, we plan to conduct similar analyses for each retinal determination gene.
- Finally, we are initiating a new genome-wide screening approach to identify novel genes whose expression is highly enriched in the developing mouse retina. Using a combinatorial approach of genetics, genomics, and computational biology we have identified a set of 40 genes that are highly and specifically expressed during early mouse retinal development. Since 16 of these 40 genes are already known to play an important role in mouse retinal development, the remaining novel genes are also highly likely to also be important players in this process. We have verified retinal enrichment by in situ hybridization for 11 new genes and have begun generating the first targeting constructs. We will generate conditional loss-of-function alleles for each of these genes and study their role during mammalian eye development.
- Chen, R., Amoui, M., Zhang, Z., and Mardon, G. (1997). Dachshund and Eyes Absent Proteins form a complex and function synergistically to induce ectopic eye development in Drosophila. Cell 91, 893-903
- Chen, R., Halder, G., Zhang, Z., and Mardon, G. (1999). Signaling by the TGFβ homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila. Development 126, 935-943
- Frankfort, B., Nolo, R., Zhang, Z., Bellen, H., and Mardon, G. (2001). senseless repression of rough is required for R8 photoreceptor differentiation in the developing Drosophila eye. Neuron 32, 403-414
- Frankfort, B., and Mardon, G. (2002). R8 Development in the Drosophila Eye: A Paradigm for Neural Selection and Differentiation. Development 129, 1295-1306
- Pappu, K., Chen, R., Middlebrooks, B.W., Woo, K., Heberlein, U., and Mardon, G. (2003). Mechanism of hedgehog signaling during early Drosophila eye development. Development 130, 3053-3062
- Frankfort, B., and Mardon, G. (2004). Senseless represses nuclear transduction of Egfr pathway activation. Development 131, 563-570
- Pappu, K., and Mardon, G. (2004). Genetic control of retinal specification and determination in Drosophila. International Journal of Developmental Biology 48, 913-924
- Chen, R., and Mardon, G. (2005). Keeping an Eye on the Genome. Developmental Biology 282, 285-93
- Pappu, K., Ostrin, E., Middlebrooks, B., Sili, B.T., Chen, R., Atkins, M., Gibbs, R., and Mardon, G. (2005). Dual regulation and redundant function of two eye-specific enhancers of the Drosophila retinal determination gene dachshund. Development 132, 2895-2905
- Ostrin*, E.J., Li*, Y., Hoffman, K., Liu, J., Zhang, L., and Mardon$, G., and Chen$, R. (2006). Genome-wide identification of direct targets of the Drosophila retinal determination protein Eyeless. *These authors contributed equally to this work. $These laboratories contributed equally to this work. Genome Research, in press
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