Genetic Study of Heparan Sulfate Function in Development

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DESCRIPTION (provided by applicant): Heparan sulfate proteoglycans (HSPGs) are involved in a variety of biological processes such as growth factor signaling. Many human diseases have been found to be associated with defects in the HSPG biosynthesis. The heparan sulfate (HS) chains have markedly heterogeneous structures that are mainly produced by the regulated introduction of N-, 2-O-, 6-O-, and 3-O-sulfate groups. Evidence suggests that these "fine structures" of HS control discrete signaling events at the cell surface. The molecular basis for this control, however, is largely unknown. Our goal is to understand the roles of specific HS fine structures in morphogenesis using a genetically tractable model organism, Drosophila melanogaster. We recently identified Drosophila HS 6-O-sutfotransferase (dHS6ST) as a positive regulator of FGF signaling during tracheal system development. This is consistent with recent biochemical data, which suggested that 6-O-sulfation is responsible for the activation of FGF signaling in vertebrate systems. However, the molecular mechanism by which growth factor signaling is controlled by this specific sulfation of HS remains to be elucidated. We have also investigated sulfation at the 3-O-position of HS, which is known to be critical for blood coagulation and viral infection in mammals. However, the role of 3-O-sulfation in development is poorly understood. In our preliminary studies, Drosophila HS 3-O-sulfotransferase-b (dHS3ST-b) is involved in Notch signaling and the assembly of many tissues, implicating 3-O-sulfation in development. In the proposed research, we will study the in vivo functions of three genes for Drosophila HSSTs, dHS6ST, dHS3ST-a, and -b, during development. Specific aims are: Aim 1. Explore the molecular functions of dHS6ST in growth factor signaling during Drosophila development. Aim 2. Determine the molecular functions of the dHS3ST-b gene in the Notch signaling pathway. Aim 3. Localize the ligand-specific HS in Drosophila tissues. Two recently developed techniques, transgenic RNA interference (Aim 2) and an in situ ligand binding assay (Aim 3), will be used in this study.