Overview
My lab is interested in understanding how spatially and temporally coordinated inputs from multiple signaling pathways mediate the implementation of complex developmental decisions. To address such issues we are studying the RTK pathway which is one of the major routes through which extracellular signals are transduced to the nucleus. We use Drosophila as our model system both because of the ease with which one can combine genetic, molecular, biochemical and cellular approaches, and because it is increasingly clear that the signaling mechanisms controlling basic developmental processes have been highly conserved in evolution. Thus knowledge of the molecular circuitry of cell-cell communication used in Drosophila is relevant to the study of mammalian development and its associated defects and diseases.

Research Summary
Receptor Tyrosine Kinase Signaling: We are studying the evolutionarily conserved signal transduction pathway mediated by receptor tyrosine kinases (RTK) using Drosophila as a model system. RTK-mediated signaling is critical for mitogenesis, cell fate specification and differentiation during normal development of all multicellular organisms. In mammals, uncontrolled activity of the pathway has been implicated in tumorigenesis, and several components of the pathway, most notably the GTPase Ras, have been identified as oncogenes. While the basic RTK/Ras/MAPK signaling cassette is well understood, very little is known about the nature of the downstream targets of the pathway and how these effectors regulate the specificity of response to RTK-initiated signals. In addition, complex feedback loops and as cross-talk with other signaling networks appear likely to contribute to signaling specificity.
Understanding how YAN activity is regulated in response to RTK signaling: We have been studying the function and regulation of Drosophila YAN, an ETS family transcriptional repressor that acts as an antagonist to the RTK/Ras/MAPK signal transduction pathway. During development, YAN functions to prevent inappropriate differentiation of both neuronal and non-neuronal cells types in the eye and embryo. Interestingly, just as improper regulation of the YAN homolog TEL leads to uncontrolled proliferation and carcinogenesis in humans, loss of yan function results in overproliferation of Drosophila epithelial tissues. Thus proper downregulation of YAN is essential to normal differentiation and development. We are taking a cell biological and biochemical approach in Drosophila cultured cells to investigate the molecular mechanisms controlling YAN function and regulation. Specifically we are seeking to understand how a complex interplay between DNA binding, phosphorylation, alterations in subcellular localization, dimerization and other protein-protein interactions regulates YAN activity.
A genetic approach to identifying new RTK pathway components: Because YAN functions as a transcriptional repressor downstream of multiple RTK pathways, it offers an ideal tool with which to address the issue of how specificity of response to RTK-mediated signaling events is generated in different developmental contexts. Toward this goal, we performed a genetic modifier screen designed to investigate how extracellular signals converge on nuclear transcription factor activity during Drosophila eye development. The underlying assumption of our screen was that cross-talk between the RTK and other signaling networks was likely to occur, at least in part, at the level of coordinate regulation of downstream nuclear transcription factor activity. We are currently using a combination of genetic, biochemical and microarray approaches to investigate how the genes identified in this screen may mediate RTK signaling specificity and crosstalk.
RTK signaling during retinal determination: Among the modifiers we isolated were 10 alleles of eyes absent. Eyes absent is a member of an evolutionarily conserved network of nuclear transcription factors and cofactors that have been shown to mediate retinal determination. In both flies and mammals, loss of function mutations in these genes are generally associated with loss of or severe defects in the eye; conversely, overexpression can induce formation of eye tissue outside of the normal eye field. Because the genes in this network all encode nuclear factors, their activity must ultimately be responsive to input from one or more signal transduction pathways. We have found that RTK signaling appears to influence retinal determination gene activity using EYA as a point of cross-talk.
Identification of split ends, a novel RRM motif protein involved in RTK signaling: In the course of our yan-based genetic screen we isolated a gene called split ends (spen). Drosophila SPEN and its orthologs in C. elegans, M. musculus and H. sapiens are characterized by a distinct set of RNA recognition motifs (RRMs) and a highly conserved C-terminal domain of unknown function, termed the SPOC domain. We have demonstrated that spen functions as an RTK signaling pathway regulator or effector in several developmental contexts. In particular, SPEN serves as a positively acting component of the RTK pathway that regulates migration and survival of the midline glial cells in the developing embryonic central nervous system.
Modeling neurodegenerative disease in Drosophila: We are using both a genetic and a candidate gene approach to modeling human neurodegenerative diseases in Drosophila. By recapitulating neurodegenerative phenotypes in the fly, we can then initiate genetic modifier screens designed to identify genes that suppress or enhance the phenotype. This approach will help to elucidate the signaling pathways that normally regulate, or are regulated by, these disease genes, and by extension, will shed light on the molecular mechanisms underlying neurodegenerative disorders. The developing fly eye is a particularly informative tissue to study neurodegeneration. First, the eye is dispensable for viability of the adult thereby allowing us to induce neurodegenerative phenotypes specifically in the eye without compromising survival of the animal. Second, the stereotyped regularity of structure of the Drosophila compound eye, together with a large array of cell type specific markers, allows us to identify each developing cell type and analyze changes in cell fate at a single cell resolution at multiple stages in development.

Selected Publications
Rebay, I. Keeping the Receptor Tyrosine Kinase signaling pathway in check: lessons from Drosophila. Developmental Biology, submitted (2002).

Tootle, T., Lee, P. and Rebay, I. Down-regulation of the RTK inhibitor YAN involves CRM1-mediated nuclear export. Genes and Development, submitted (2002).

Hsiao, F. C., Williams, A., Davies, E. L., and Rebay, I. Eyes absent mediates cross-talk between retinal determination genes and the receptor tyrosine kinase signaling pathway. Developmental Cell, 1: 51-61. (2001).

Chen, F. and Rebay, I. Split ends is a new component of the RTK/Ras pathway that regulates development of the midline glial cells. Current Biology 10: 943-946. (2000).

Rebay, I., Chen, F., Hsiao, F., Kolodziej, P.A., Kuang, B.H., Laverty, T., Suh, C., Voas, M., Williams, A., and Rubin, G.M. A genetic screen for novel components of the Ras/MAPK signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RRM motif containing protein. Genetics 154: 695-712. (2000).