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. ? ? Functional analysis of the three syndecans and ten glypicans that we cloned from zebrafish 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. Using this specific, quantifiable assay, we anticipate that we will be able to thoroughly dissect core protein epistasis.? ? We have also take a focussed approach to examine how one core protein, syndecan 3, functions in neural crest development. Morpholino directed RNA knockdown of endogenous syndecan 3 and over-expression of syndecan 3 leads to similar heart and head defects, supporting previous research demonstrating that having too many HSPG molecules has similar detrimental effects as not enough HSPG. Analysis of syndecan 3-disrupted embryos using probes for genes known to regulate neural crest development demonstrated that syndecan 3 is specifically involved in a cranial neural crest development at a time after the neural crest is initially determined but before it migrates to its target tissue. We are examining the timing of syndecan 3 function using time-lapse imaging of zebrafish reporter lines. Current research is also aimed at determining what neural crest signalling pathways are being regulated by syndecan 3. Further experiments are also planned using transgenesis and mutagenesis to look for genetic modifiers of syndecan 3 function in development.? ? To complement these genetic approaches, we are developing tools to metabolically engineer the HS of zebrafish so as to visualize the molecular interactions of HS during early development. The difficulty of metabolically labeling a whole organism lies in that the saccharide may be incorporated into glycoproteins not of immediate interest. However, we have accounted for this by choosing a sugar that should be specifically incorporated into HS. Using this method in combination with mutant fish or knock-downs defective in HS metabolism potentially will allow studying HS function in much greater detail than presently allowed.? ? 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 have developed a pharmacologically-regulated subcellular trafficig tool that is allowing us to address important questions regarding the cell biology of HSPGs. Control experiments to test the system have finally been completed; we will soon begin selecting, screening, and analyzing binding of the peptides to specific HS disaccharides using Surface Plasmon Resonance. This technique will allow us to determine how strongly and specifically these peptides bind to different HS disaccharides. ? ? 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.
