Microplate assay for quantifying developmental morphologies: effects of exogenous hyalin on sea urchin gastrulation

摘要

It is often difficult to determine the effects of various substances on the development of the sea urchin embryo due to the lack of appropriate quantitative microassays. Here, a microplate assay has been developed for quantitatively evaluating the effects of substances, such as hyalin, on living sea urchin embryos. Hyalin (330 kDa) is a major constituent of the sea urchin hyaline layer, an extracellular matrix that develops 20 min postinsemination. Function of the hyaline layer and its major constituent, is the adhesion of cells during morphogenesis. Using wide-mouthed pipette tips, 25 pl of 24-h Strongylocentrotus purpuratus embryos were transferred to each well of a 96-well polystyrene flat-bottom microplate yielding about 12 embryos per well. Specific concentrations of purified hyalin diluted in low calcium seawater were added to the wells containing the embryos, which were then incubated for 24 h at 15 degrees C. The hyalin-treated and control samples were observed live and after fixation with 10% formaldehyde using a Zeiss Axiolab photomicroscope. The small number of embryos in each well allowed quantification of the developmental effects of the added media. Specific archenteron morphologies-attached, unattached, no invagination and exogastrula-were scored and a dose-dependent response curve was generated. Hyalin at high concentrations blocked invagination. At low concentrations, it inhibited archenteron elongation/ attachment to the blastocoel roof. While many studies have implicated hyalin in a variety of interactions during morphogenesis, we are not aware of any past studies that have quantitatively examined the effects of exogenous hyalin on specific gastrulation events in whole embryos.