The monoclonal antibody Sp12 recognizes an epitope shared by a diverse group of developmentally regulated cell surface and extracellular matrix antigens which are expressed during Strongylocentrotus purpuratus development. Immunofluorescent localization reveals antigen in the cortical granules of eggs and in the hyaline layer from fertilization through to gastrula. Primary (skeletogenic) mesenchyme and two secondary mesenchyme derivatives (blastocoelar cells and pigment cells) express antigen after their release into the blastocoel and maintain it throughout the remainder of larval development. The antibody allows detailed descriptions of blastocoelar cells, a prominent yet poorly described fibroblast-like mesenchyme lineage. In adults antigen is localized to the organic matrix which invests the calcified stereom of the test and spines. Immunogold electron microscopy shows antigen in the cortical granules of eggs, and on cell surfaces, within membrane bound vesicles, and within the Golgi apparatus of mesenchyme cells. On western blots a confluent smear of antigen (Mr primarily >$180K) is present in eggs, but is resolved into seven antigen bands (Mr from 35K to >$200K) after fertilization. A prominent antigen at 140K is newly expressed coincident with mesenchyme immunoreactivity. Antigens at 140K and 120K fade as prisms develop into plutei, while an antigen at 105K appears in older larvae. In adult test six antigens are shared with larvae, and there are two novel antigens at 75K and 110K. Immunoreactivity is eliminated by digestion of samples with endoglycosidase F, is reduced by periodate oxidation, but is unaffected by boiling. Calcium-magnesium-free-seawater or 1M glycine extract a subset of antigens from dissociating embryos, leaving a complementary subset of antigens associated with cell membranes. Membrane antigens are not extracted, at 4 C, with high or low ionic strength, organic solvents, or non-ionic detergents but are solubilized by ionic detergents. Whole antibody has no effect on development of embryos cultured in it from fertilization on, nor is there a specific effect from injection of antibody into the blastocoel of developing embryos. The shape of dissociated cells cultured in Sp12 whole antibody is markedly constrained compared to that of controls, however, this difference is not seen in cells incubated in Sp12 Fab fragments. Sp12 appears to recognize a carbohydrate moiety shared by ten glycoproteins of widely variable Mr and cell membrane affinity which are differentially expressed on mesenchyme throughout S. purpuratus development. The antibody does not disrupt development in vivo, but does affect cell shape in vitro, probably by crosslinking cell surface antigens. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9478 |
| Date | 19 June 2018 |
| Creators | Tamboline, Colin Richard |
| Contributors | Burke, Robert D. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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