Glycosaminoglycans are a heavily sulfated component of the extracellular matrix (ECM) implicated in a variety of cell signaling events involved in patterning of embryos. Embryos of the sea urchin Strongylocentrotus purpuratus were exposed to several inhibitors that disrupt glycosaminoglycan function during development. Treatment with chlorate, a general inhibitor of sulfation that leads to undersulfated glycosaminoglycans, reduced sulfation of the urchin blastocoelar ECM. It also prevented correct specification of the oral-aboral axis and mouth formation, resulting in a radialized phenotype characterized by the lack of an oral field, incomplete gastrulation and formation of multiple skeletal spicule rudiments. Oral markers were initially expressed in most of the prospective ectoderm of chlorate-treated early blastulae, but then declined as aboral markers became expressed throughout most of the ectoderm. Nodal expression in the presumptive oral field is necessary and sufficient to specify the oral-aboral axis in urchins. Several lines of evidence suggest a deregulation of Nodal signaling is involved in the radialization caused by chlorate. Radial embryos resemble those in which Nodal expression was knocked down. Chlorate disrupted localized nodal expression in oral ectoderm, even when applied after the oral-aboral axis is specified and expression of other oral markers is resistant to treatment. Intriguingly, treatment of embryos with a sub-threshold dose of a Nodal receptor inhibitor rescued the radialization caused by low concentrations of chlorate.Glycosaminoglycans are typically found in association with core proteins as proteoglycans. Three cell-associated proteoglycans with possible roles in the regulation of cell signaling events leading to oral-aboral axis specification were identified in the urchin genome. These proteoglycans of the syndecan and glypican families are expressed in distinct but overlapping radially symmetrical patterns in the urchin embryo. Chlorate treatment upregulated proteoglycan expression, especially of glypicans. Our results indicate important roles for sulfated glycosaminoglycans (and possibly proteoglycans) in Nodal signaling and oral-aboral axial patterning, and in the cellular processes necessary for archenteron extension and mouth formation during gastrulation. We propose that interaction of the Nodal ligand with sulfated glycosaminoglycans limits its diffusion, and is required to specify an oral field in the urchin embryo and organize the oral-aboral axis.
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Thesis advisor: Brandhorst, Bruce P.
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