The focus of this lab is to characterize both the developmental and cell biological function of heparan sulfate proteoglycans (HSPGs) during early zebrafish development. We use zebrafish because the early embryos are transparent, easy to manipulate, and a large number of mutants exist that aid in our analysis. Studies in a number of model systems have demonstrated that most of the cell-cell signaling pathways that regulate early development are regulated by heparan sulfate (HS). HS is covalently attached to core proteins in the extracellular matrix and at the cell surface, and proteins to which heparan sulfate attaches are referred to as HSPGs. At the cell surface, the predominant HSPGs belong to two families of core proteins: transmembrane syndecans and glycophosphatidylinositol-linked glypicans (GPCs). Initial cloning and analysis of the full zebrafish GPC family suggested that two zebrafish GPCs, GPC7a and GPC7b, are paralogs of a GPC not previously identified in higher vertebrates. We further investigated the evolutionary origin of GPCs by cloning the entire family from organisms that diverged at key evolutionary branch points. We assessed the orthology of vertebrate and invertebrate sequences by alignment with ClustalW, constructed maximum likelihood phylogenetic trees, and compared the predictions to conserved syntenic regions. Our analysis demonstrated that all GPCs emerged as a result of whole genome duplications, with the exception of GPC2. While all bony fish retain a full compliment of 10 GPCs (including 3 paralogs), we observed that a single GPC was stochastically lost in all organisms that evolved after teleosts. We are currently examining the specificity of gene expression across multiple organisms to assess whether structurally close or divergent GPCs compensate for the loss of a family member. We expect that these results will help us better understand how the GPC gene family functions. Our functional analysis of each zebrafish GPC demonstrated that each core protein has a distinct function, despite the fact that each core protein has the same early, ubiquitous expression. To address how the each core protein specifically functions, we are focussing on how the core proteins differ in their ability to regulate dorsal-ventral patterning, a very early decision that preceeds other HS-dependent roles in the early embryo. Knockdown of GPC2, GPC6a, or GPC6b resulted in strong dorsoventral phenotypes between gastrulation and 24hpf. To better understand the molecular pathways in which GPCs are involved, we used a candidate gene approach to study changes in transcription of BMP, Wnt and FGF target genes because these cell-cell signaling pathways are known to regulate dorsoventral patterning. Using QPCR, we identified seven genes that are significantly up-regulated in at least one of the three GPC knockdowns. The gene expression patterns were different between each GPC knockdown, with GPC2 having a distinct affect on BMP2b expression while GPC6a and 6b differentially increased expression of Ved/Vox/Vent. These results were confirmed by in situ hybridization, demonstrating that BMP2b expression is significantly expanded to the dorsal side in GPC2 morphant embryos. Our preliminary results suggest that GPC2, GPC6a, and GPC6b regulate distinct steps in dorsoventral patterning, an observation that would add a new dimension to our understanding of how proteoglycans might work together to mediate early development. We have also taken a focussed approach to examine how two core proteins, GPC7a and SDC3. GPC7a is very specifically and uniquely expressed in the earliest vascular endothelial cells, including those destined to form the endocardium of the heart. While expression appears transient such that it disappears before heart morphogenesis begins, knockdown of GPC7a results in zebrafish that have a severely deformed ventricle. We are currently using in situ analysis of the knockdown fish coupled with transgenesis to elucidate how GPC7a is functioning. We have also demonstrated that SDC3 mediates zebrafish melanogenesis and have further detailed the molecular mechnisms by demonstrating that SDC3 mediates an interplan between the extracellular signaling protein AGRP and attractin. To complement these genetic approaches, we are developing tools to examine how the HS chains contribute to the proteoglycan function. HS is an unbranched sugar chain consisting of repeated disaccharides that can be modified at up to six positions, leading to an extraordinary level of sequence diversity. Commonly referred to as its fine structure, the pattern of HS modification over 2-7 disaccharides creates a specific ligand binding site. In many cases, a cell can only respond to a cell-cell signaling molecule if it has an appropriate HS fine structure at its cell surface. To characterize the HS fine structure, we are using directed evolution to bioengineer a set of novel, more efficient tools by designing peptide modules that can engage in high affinity, targeted binding to specific disaccharide units in the HS chain. A major hurdle of this project has been the stability of the peptide module, a problem that was recently overcome through mutational analysis. We developed a pharmacologically-regulated subcellular trafficking tool that is allowing us to address important questions regarding the cell biology of HSPGs. These peptides have virtually the same interval length as a disaccharide, so one expectation is that we will be able to bring the peptides together in multiple combinations for characterizing HS tetrasaccharides. Furthermore, by attaching a defined oligonucleotide to the modules and then ligating oligonucleotides on modules bound to adjacent disaccharides, we intend to create a nucleotide sequence that we can amplify, sequence, and ultimately read to obtain the HS sugar sequence from entire chains. The hope is that this approach will help us define the 'HS code', a widely hypothesized model in which distinct HS modification patterns may provide information that is required for metazoan development. Because the HS fines structure and many steps in zebrafish embryogenesis are similar to those in humans, the tools, mechanisms and modifiers that we identify may be applicable to better understanding a wide range of cell-cell signaling events in development and disease.

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National Heart, Lung, and Blood Institute
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