The Role of Heparan Sulfate Proteoglycans as Co-Receptors of Wnt-signaling during Neural Development.
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Cell migration is a hallmark of many biological processes, including development, immunity, and malignancies. In particular, structures part of the nervous system, including synapses and circuitries, are formed and maintained through the process of cell migration. The extracellular space plays a significant role in this process, as it facilitates exposure between the traveling cell and molecules within the extracellular environment. However, neurodevelopmental and physiological diseases, as well as metastatic cancer, can be caused by aberrant migrations. My thesis project aims to understand the role of heparan sulfate proteoglycans (HSPGs), extracellular glycoproteins present in the majority of cells, in cell migration and neural development. The polysaccharide chains attached to HSPGs contain a vast number of complex modification patterns in the form of acetylation, sulfation, and epimerization. Moreover, HSPGs have been shown to act as essential co-factors on multiple signaling pathways. To determine the significance of HSPGs in cellular migration, I created or obtained fluorescently labeled reporters that allowed me to visualize the positioning of single-cell(s) in live Caenorhabditis elegans. Using these reporters, I analyzed the migration and final positioning of five different cells in the worm. I found that membrane-bound HSPGs sdn-1/Syndecan and lon-2/Glypican are required for all the migration events tested; in contrast, some HSPGs are only needed in particular migrations. I also determined that a given HSPG is necessary to promote migration, while others are primarily required to establish the exact position of each moving cell(s). In sum, my genetic analyses suggest a unique combination of HSPGs modulate each migration event. While revising the literature, I found that a similar genetic study was conducted by surveying the role of different Wnt ligands and Frizzled receptors genes in cell migration. Previous research performed on loss-of-function mutants in Wnt-signaling genes demonstrated strikingly similar phenotypes to the ones observed in HSPG mutant animals. Based on these observations, I hypothesized HSPGs act together with Wnt ligands and frizzled receptors to control cellular migrations and positioning. Indeed, I found that the Wnt ligand EGL-20 requires the presence of SDN-1/Syndecan to promote cell migration. A second Wnt ligand, lin-44, functionally interacts with sdn-1 to promote axon guidance of a separate neuron. These findings suggest SDN-1 is capable of interacting with different Wnts in a context-dependent manner. To further determine the role of HSPGs with Wnt-signaling, I performed coimmunoprecipitation experiments between SDN-1/Syndecan, EGL-20/Wnt, and MIG1/Frizzled. These experiments suggested that SDN-1/Syndecan, EGL-20/Wnt, and MIG1/Frizzled form a biochemical complex when they are transfected in a heterologous system. These findings suggested SDN-1 is a co-receptor of the Wnt-signaling pathway. Next, I established the function of HS glycans during cellular and axonal migrations and demonstrated that SDN-1/Syndecan contains unique HS epitopes that may confer selectivity to the Wnt-Frizzled complexes acting in each migration event. Taken together, my thesis findings suggest HSPGs genetically and biochemically interact with Wnt-signaling genes to coordinate cell and axonal migrations, as well as positioning. To my knowledge, this is the first instance HSPGs and Wnt-signaling have been implicated in conjunction with neural development. I expect the research conducted during my thesis work will provide knowledge necessary to comprehend the process of cell migration in normal and diseased states.