The many roles of 2-0-sulfotransferase in early zebrafish development

Heparan sulfate proteoglycans (HSPG) are a family of cell surface and extracellular matrix proteins with glycosaminoglycan (GAG) chains covalently attached to a protein core. O-sulfotransferases modify these GAG chains by catalyzing the transfer of a sulfate to a specific position on HSPG GAG chains. Although the role of specific HSPG modifications have been described in cell culture and invertebrates, little is known about their functions or abilities to modulate specific cell signaling pathways in vertebrate development. My thesis research focused on the many roles a particular GAG chain modifying enzyme, 2-O-sulfotransferase (2-OST), plays in early development. To characterize the function of 2-OST in early zebrafish development we knocked down this gene via morpholino and found that 2-O-sulfation controls epiboly. Epiboly is the process by which the cells of the blastodisc move towards the vegetal pole from the animal pole to envelop the yolk cell. Knocking down 2-OST results in a failure to initiate and progress through epiboly due to alterations in assembly of filamentous actin, in microtubule organization, and in yolk cell endocytosis. Syndecans 2 and 4, HSPG core proteins, appear to be targets of the 2-OST activity which contribute to this phenotype. 2-OST activity modulates the activity of multiple signaling cascades. We found that 2-OST is an essential component of canonical Wnt signaling in zebrafish development. 2-OST deficient embryos have decreased β-catenin and E-cadherin protein levels, reduced cell adhesion, and altered cell cycle regulation. The cell cycle and iv adhesion defects in 2-OST-deficient embryos can be rescued by reactivation of intracellular Wnt pathway components, but not by overexpression of Wnt8 ligand. Together these results indicate that 2-OST functions within the Wnt pathway downstream of ligand signaling and upstream of intracellular localization and function. Knocking down 2-OST also affects FGF signaling. While Wnt epistasis experiments show loss of 2-O-sulfation impairs some signaling interactions at the cell surface, epistasis experiments with components of the FGF signaling cascade suggest loss of 2-O-sulfation in some contexts can promote signaling which occurs at lower levels in regular developmental contexts. These results suggest HSPG function is essential for early development.