Project Summary The long-term goal of our research is to understand fundamental principles of cell communications mediated by heparan sulfate proteoglycans (HSPGs). HSPGs are a special type of carbohydrate-modified proteins that serve as co-receptors for various growth factors, including bone morphogenetic proteins, Wnt/Wingless, and Hedgehog. These HSPG co-receptors play critical roles in quantitative and robust control of signaling output. We study in vivo functions of HSPGs using the Drosophila model. Our earlier research has established critical roles of HSPGs in development, namely in morphogen signaling and gradient formation. In addition, we have demonstrated that HSPGs regulate the stem cell niche as a universal factor. Although proteoglycan biology has made significant progress, several major questions still remain to be elucidated. For example, the molecular mechanisms of co-receptor activities of HSPGs are poorly understood. It is also unknown how distinct HS structures regulate specific signaling and patterning events. It is now critical to develop an interdisciplinary approach, which can directly link detailed HS structural motifs, ligand binding, quantitative signaling output, and developmental/physiological phenotypes. Our previous studies suggested that HSPGs cooperate with other factors to exert co-receptor activity on the cell surface. To reveal the molecular basis for co-receptor function, we use proteomic and genetic approaches to identify these missing players. We recently found that Windpipe (Wdp), a transmembrane protein containing four leucine-rich repeats, acts as a novel negative co-receptor for Hh signaling. We also showed that Wdp is a chondroitin sulfate proteoglycan (CSPG). Thus, the Hh pathway is controlled by two classes of proteoglycan co-receptors: HSPGs (positive regulators) and Wdp (CSPG, a negative regulator). We will elucidate how such a dual PG co-receptor system achieves quantitatively controlled signaling output and precise pattern formation. To understand how a change in HS structure affects signaling and morphogenesis, we will establish an in vitro model using Drosophila cells. Despite the many strengths of the Drosophila model for in vivo studies, information on Drosophila HS structure is limited. This is mainly due to the difficulty of metabolic radio-labeling of HS in vivo using Drosophila animals. To fill this gap, we have recently generated novel Drosophila cell lines mutant for five HS modifying enzymes. Our studies using these cell lines will provide a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic information obtained over the last two decades using this animal model. Together, this application will advance our field by: (1) defining fundamental molecular mechanisms of co-receptor function and (2) comprehensive understanding of the structure-function relationship of HS.