Fractionation of Micromeres, Mesomeres, and Macromeres of 16-cell Stage Sea Urchin Embryos by Elutriation*. (sea urchin embryo/blastomere/elutriation/micromere/mesomere/macromere)

A protocol was developed to fractionate micromeres, mesomeres and macromeres of 16-cell stage sea urchin embryos by elutriation. The purities of these fractions were 99%, 93%, and 90%, and their recoveries were 75%, 31%, and 42%, respectively. Using this method, several hundred milligrams of each blastomere type were obtainable from a single-pair mating. On culture, micromeres formed spicules in the presence of horse serum, mesomeres developed into ciliated ectodermal vesicles and macromeres formed gastrula-like or exogastrula-like embryoids with spicules. To analyze the different structures characteristic of the blastomere lineage, we examined the expressions of marker genes. Cells of the micromere lineage expressed the primary mesenchyme-specific SM50 gene exclusively, those of the mesomere lineage expressed the ectoderm-specific arylsulfatase gene strongly, and SM50 and the endoderm-specific Endo 16 genes weakly, whereas those of the macromere lineage expressed all three marker genes. These results indicate that blastomeres fractionated by elutriation were equivalent to those isolated by hand under a microscope with respect to development and gene expression.