At the late pluteus stage, sea urchin larvae form an adult rudiment left–right (LR) asymmetrically, on only the left side. Little is known about how the LR asymmetry of the adult rudiment is established in earlier stages during which the larval body is basically LR symmetric. To investigate how the different regions of the embryo function to establish LR asymmetry, we removed different regions at the gastrula stage and assessed the effects of the operation on the establishment of LR asymmetry of the adult rudiment.
Surgery was performed on mid- to late-gastrula embryos of two indirect developing species: Scaphechinus mirabilis and Hemicentrotus pulcherrimus. The embryos were placed under a dissecting microscope (SMZ8, Leica) and ectodermal epithelium together with underlying mesenchyme cells was dissected out with a glass needle. The region along the midline of the embryo at the width of the archenteron was designated the ‘midline region’, and the region lateral to the ‘midline region’ was designated the ‘lateral region’. When the left and/or the right side was excised, the whole lateral region was precisely removed on the animal side, but the plane of incision deviated more laterally on the vegetal side to avoid the ventro-lateral cluster of primary mesenchyme cells, so that a part of the defined ‘lateral region’ was left on the vegetal side. The vertical excision was made by cutting mid-gastrula embryos vertically to the archenteron at a level just superior to the tip of the archenteron. In the sham operation, embryos were pressed with the needle as in the vertical excision, but the incision was stopped slightly before the animal and vegetal halves of the embryos were completely divided from each other. The operated embryos were examined through an optical microscope (Optiphoto, Nikon) to confirm that the excision was correct, and they were then cultured with a food supply (diatoms, Chaetoceros gracilis). The handedness of the adult rudiment was examined at the six-armed or eight-armed pluteus stage through an optical microscope (Table 1).
