We have re-observed in detail the development of the sea urchin species Temnopleurus toreumaticus, which is considered to be a typical indirect-developing species with a feeding larval stage. In this re-observation, we discovered two new morphological traits in the early embryonic stages of T. toreumaticus. The first trait is that, immediately after fertilization, the egg enters a stage in which wrinkles form on its surface as a result of actin polymerization. The second new trait is that the blastulae form wrinkles; in sea urchins, this has previously been known only in direct-developing species that have a nonfeeding larval stage and form wrinkles during the blastula stage, before hatching. These phenomena indicate that after fertilization, the egg of T. toreumaticus undergoes a surface transformation that is unprecedented in echinoderms, and that an indirect-developing sea urchin can form a wrinkled blastula.

Many sea urchins, including the strongylocentroid Hemicentrotus pulcherrimus, produce an amniotic cavity on the left for adult rudiment formation at the late larval stage. In contrast, temnopleurids form a cell mass at the early larval stage instead of an amniotic cavity. Although the mechanisms establishing left-right polarity of the amniotic cavity involve cell-cell interactions and signaling pathways, corresponding pathways for the cell mass are unknown. We analyzed the effects of blastomere isolation on the specification of larval axes in the temnopleurid Temnopleurus toreumaticus and compared them to those in H. pulcherrimus. Blastomere isolation at the two- or four-cell stages in T. toreumaticus disturbed the location of the cell mass and adult rudiment in approximately 10-20% of specimens. In contrast, isolation at the two-cell stage in H. pulcherrimus caused the left-right polarity to become random. When blastomeres isolated at the two-cell stage were cultured as pairs, approximately 20% of pairs had atypical polarity in both species. Following isolation at the four-cell stage, 71.4% of quartets produced larvae with atypical polarity in T. toreumaticus. Thus, cell-cell interaction between two daughter blastomeres after the second cleavage may be involved in the mechanism determining left-right polarity. Dye injection into a blastomere and subsequent observations indicated that the location of the boundary of the first cleavage showed similar patterns in both species. These observations suggest that species-specific mechanisms establish the larval axes and blastomeres at the two- and four-cell stages redistribute their cytoplasm, forming gradients that establish left-right polarity.