Erica M. Selva

Drosophila has many advantages: unbiased forward and reverse genetics, extensive genetic tools and a complete genome sequence. It can also serve as a model for human disease pathways because its well-studied signaling pathways are highly conserved. For example, mutations in patched, the Drosophila Hedgehog (Hh) receptor, have been implicated in the most common form of human cancer, basal cell carcinomas, while aberrant Wnt (a Drosophila Wingless (Wg) homolog) expression is linked to breast cancer. My research goals are to identify new components involved in the modification of extracellular signaling molecules as well as to elucidate the function of these molecular changes. Ultimately, I believe this work will cast light on mechanisms by which the extracellular environment might be modified to modulate aberrant signaling activities associated with human diseases.

The 1995 Nobel Prize winning work of Lewis, Nüsslein-Volhard and Wieschaus demonstrated that genetic screening of mutations in Drosophila embryos is an effective method to identify mutations that disrupt specific signaling pathways. Through similar genetic screens new classes of genes have been identified that modulate signaling through posttranslational alteration of the extracellular milieu. These genes can be roughly categorized firstly, as genes that control the modification of extracellular receptors and ligands to regulate their activity and secondly, as genes required for the biosynthesis of extracellular matrix molecules, which in turn regulate the activity of signaling pathways.

One group of genes identified in these embryonic screens are required for the biosynthesis of heparin sulfate (HS) and heparin sulfate proteoglycans (HSPGs). These include sugarless, a UDP-glucose dehydrogenase, sulfateless, an N-deacetylase/N-sulfotransferase (sfl), tout-velu, a glycosyltransferase (ttv) and fringe connection, a nucleotide-sugar-transporter (frc). Despite the fact that these genes are required the biosynthesis of sugar polymers, remarkable signaling pathway selectivity can be ascribed to each. For example, sgl and sfl modulate the activity of the Wg pathway while ttv specifically supports Hh signaling. Indeed, HS and HSPGs have been shown to play important roles in mammalian Wg/Wnt and Fibroblast Growth Factor signaling pathways.

In addition to the HS biosynthetic function of these genes, more specific roles in modifying components of signaling pathways has also been demonstrated. For example, further analysis of Frc has revealed a role for this enzyme in the modification of Notch signaling. The activity of the Notch receptor is modulated by Fng, a glycosyltransferase that catalyzes the elongation of a novel O-linked sugar chain on Notch. This modification enhances Notch responsiveness to the ligand Delta while inhibiting signaling with the alternative ligand Serrate, an exquisite example of a seemly minor posttranslational modification having a profound effect on signaling. Whether Frc supplies the substrate directly for Notch glycosylation or is important of the modification of a factor required for Notch ligand selectivity is question my laboratory will pursue in the future.

Yet another gene identified in this embryonic screen illustrates the importance of posttranslational modification of the signaling activity of secreted ligands. The rasp gene product has been shown to be absolutely required for the signaling activity of Hh. Rasp represents an entirely new class of modifying enzymes, membrane bound acyltransferases, catalyzing the palmitoylation of Hh. Clearly the posttranslational changes necessary to regulate signaling are dynamic, yet the mechanisms by which these alterations modulate signaling remain poorly understood.

Given that these genes play important roles in development and more specifically in regulating signaling, the further elucidation of additional gene products involved in this process in combination with more mechanistic understanding of the roles of the known players will allow us to better understand how the interplay between different cues are regulated developmentally.

• Identification of new genes that influence signaling through posttranslational events - A key to understanding the function of a gene is to identify other genes that play a role in the same process. Performing modifier screens using a sensitized genetic background to identify novel genes involved in a process is a powerful tool of Drosophila genetics. For this project novel genetic screens will be developed using known components like Frc and Ttv as well as the genes associated with the novel mutations 7E4, 7H24 or 8J16. A starting point for this project will take advantage of the readily scorable wing phenotype associated with the misexpression of porcupine, an acteytransferase involved in Wg signaling.
• Biochemical characterization of frc and brainiac (brn) in Notch signaling - This project will involve a combined biochemical and genetic approach to elucidate the role of frc and a fringe related glycosytransferase, brainiac (brn), in Notch signaling. This will also entail the development of novel strategies and techniques necessary to dissect the function of these and other gene products with posttranslational phenotypes. These techniques will include the development of a tissue culture model and RNA interference assays.