Thesis

Immunofluorescence localization of dissociation supernatant and extracellular matrix components in Strongylocentrotus purpuratus sectioned embryos

A dissociation supernatant (DS), has been previously shown to promote re-aggregation of Strongylocentotus purpuratus blastula cells. The factor(s) was isolated from S. purpuratus blastula cells as they disaggregated from each other in calcium-magnesium-free sea water (CMF-SW). A further purification step was done to isolate a subset of molecule(s) that promote aggregation of fixed s. purpuratus blastula cells by absorbing DS with these fixed cells. This subset was termed (S2). Antibodies raised against S2 (A-S2) were shown, by an indirect immunofluorescence method, to label the extracellular martix of whole embryos and a fibrillar component present in the blastocoel of sectioned blastulae. The purpose of this study was to localize S2 component(s) on sectioned embryos from the unfertilized stage up to and including late gastrula. The localization pattern of fluorescence of S2 was compared to that of different extracellular components (fibronectin, laminin, and collagen types I and IV) that are normally found in the basement membrane of higher organisms. This was accomplished by using antibodies made against these different components and an indirect immunofluorescence method. Also included in this study is a comparison between the localization of S2 with that of hyalin, which is the major component of the hyaline layer. The results using embryo sections suggested that hyalin, fibronectin, and collagen type I were present in the embryos and may be components of DS; and that laminin and collagen type IV were not detected in the embryo sections. A-S2, however, did bind to sectioned agarose gels absorbed with fibronectin, laminin, or collagen types I and IV, suggesting that all of these molecules are present in DS and in blastula stage embryos, while laminin and collagen type IV may not be present in sufficient quantity or in a configuration available for staining on sections of embryos. All positive controls fluoresced. Negative controls did not fluoresce. The pattern of localization of hyalin was somewhat the same as S2 at the different stages of development. The results also suggested that resynthesis of hyalin occurs at the mesenchyme blastula stage. This study helps us to understand the nature of the molecules present during morphogenesis in the sea urchin embryo and lays the groundwork for future elucidation of their function.

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