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Microplate Assay for Quantifying Developmental Morphologies: Effects of Hyalin on Sea Urchin Gastrulation
Author(s) -
Razinia Ziba,
Carroll Edward J.,
Oppenheimer Steven B.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1155
Subject(s) - blastocoel , sea urchin , strongylocentrotus purpuratus , embryo , pipette , gastrulation , chemistry , biophysics , seawater , microbiology and biotechnology , blastula , anatomy , biology , embryogenesis , ecology
A microassay was developed for quantitatively evaluating the effects of various substances on the development of living embryos. Here, this assay is used to examine the effects of purified hyalin, a large glycoprotein constituent of the sea urchin hyaline layer. Using wide‐mouthed pipette tips, 25 microliters of 24‐hour 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 (0–0.225 mg per ml) diluted in low calcium seawater, or control solutions including low calcium seawater without hyalin, were added to the wells containing the embryos that were then incubated for 24 hours at 15 degrees C. The small number of embryos in each well allowed quantification of the developmental effects induced by the added media. Results were plotted as mean percentages plus/minus standard error of the mean of 10 replicate experiments. The specific archenteron morphologies scored in dose dependent response curves were: attached, unattached, no invagination, and exogastrula. High hyalin concentrations blocked invagination, while low concentrations inhibited archenteron elongation and/or attachment to the blastocoel roof. Previous work indicated that exogenously added large molecules are able to enter the blastocoel of sea urchin embryos (Latham, et al., Acta Histochemica 100:–200, 1998; Itza and Mozingo, Zygote 13: –264, 2005). Added hyalin, therefore, may exert its effects from the inside as well as the outside of the embryo. This simple system may improve our understanding of mechanisms of adhesive interactions in higher organisms that are not as exquisitely accessible (Supported by NIH NIGMS SCORE, RISE, MARC and the Joseph Drown Foundation).