First evidence of autofluorescence of a cell mass in developing temnopleurid sea urchins

The pigment cells of some sea urchin embryos emanate autofluorescence in response to light irradiation. However, it is unclear if this feature is maintained throughout larval development. In the present study we observed embryos and larvae of the temnopleurid sea urchins Temnopleurus hardwickii, T. reevesii, T. toreumaticus and Mespilia globulus exposed to light irradiation, and compared our findings with those of a strongylocentroid, Hemicentrotus pulcherrimus. After exposure to ultraviolet irradiation for a few minutes, there was a strong signal from the temnopleurid sea urchins. The signal was detected from a cell mass that is part of the adult (juvenile) rudiment, formed during development of the prism to the two-armed larval stage and not from pigment cells. This signal was observed in both live and formalin-fixed specimens. Fluorescence was also detected from the digestive organs, coelomic pouches, the ciliary band on the oral hood, from some yellowish-green cells and ectodermal cells, although there were some differences among species. In live H. pulcherrimus larvae, the amniotic cavity that is part of the adult rudiment emanated autofluorescence in response to ultraviolet irradiation. These results indicate that the autofluorescence observed in the cell mass of temnopleurid sea urchins is caused by a different mechanism than previously described. This feature may be a useful marker to trace development of the cell mass.

Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side

Indirect-developing sea urchins eventually form an adult rudiment on the left side through differential left-right development in the late larval stages. Components of the adult rudiment, such as the hydropore canal, the hydrocoel and the primary vestibule, all develop on the left side alone, and are the initial morphological traits that exhibit left-right differences. Although it has previously been shown that partial embryos dissected in cleavage stages correctly determine the normal left-right placement of the adult rudiment, the timing and the mechanism that determine left-right polarity during normal development remain unknown. In order to determine these, we have carried out a series of regional operations in two indirect-developing sea urchin species. We excised all or a part of tissue on the left or right side of the embryos during the early gastrula stage and the two-armed pluteus stage, and examined the left-right position of the adult rudiment, and of its components. Excisions of tissues on the left side of the embryos, regardless of stage, resulted in formation of a left adult rudiment, as in normal development. By contrast, excisions on the right side of the embryos resulted in three different types of impairment in the left-right placement of the adult rudiment in a stage-dependent manner. Generally, when the adult rudiment was definitively formed only on the right side of the larvae, no trace of basic development of the components of the adult rudiment was found on the left side, indicating that a right adult rudiment results from reversal of the initial left-right polarity but not from a later inhibitory effect on the development of an adult rudiment. Thus, we suggest that determination of the left-right placement of the adult rudiment depends on a process, which is directed by the right side, of polarity establishment during the gastrula and the prism stages; however, but commitment of the cell fate to initiate formation of the adult rudiment occurs later than the two-armed pluteus stage.

Regulative potential to form an amniotic cavity in mesomeres of a direct developing echinoid, Peronella japonica

Peronella japonica, a direct developer, exhibits certain peculiar features during development, particularly heterochrony, a change in the relative timing of expression among tissues and organs. One of the important heterochronical changes in the species was found in the development of the amniotic cavity, a component of an adult rudiment. In indirect developers the amniotic cavity is formed on the left side of the larval body in the late pluteus stage. In P. japonica the organ is formed at the gastrula stage in the region located on the midline of the larval body.In the present study, the ability of partial embryos isolated from 8- or 16-cell stage embryos of P. japonica to differentiate an amniotic cavity was investigated to assess the regulative potential of a direct developer.The embryos were dissected at 8-cell stage with a glass needle to obtain half embryos. Some of the half embryos were further divided into four blastomeres to obtain mesomere pairs. Each half embryo and blastomere that did not form micromeres but divided equally during the next cleavage was identified as an animal cap and presumptive mesomere pair. Isolated animal caps and mesomere pairs were cultured, and differentiation of the amniotic cavity was examined at 24 and 48 h after fertilisation, when the organ in the normal embryos had already completed differentiation.

An analysis of sea urchin metamorphosis

Metamorphosis of sea urchin larvae is initiated by one or more cues from the environment. The cues can be from bacterial films (Cameron & Hinegardner, 1974), algae (Kitamura et al., 1993) or sand and seawater from adult habitats (Highsmith, 1982). The substances from sand are peptides (Burke, 1984), and those from red algae are free fatty acids (Kitamura et al., 1993) and dibromomethane (Taniguchi et al., 1994). Burke (1983a) suggested that chemical and physical stimuli were received by sensory receptors, probably podia of the adult rudiment, and transmitted to effectors of metamorphosis such as larval and adult tissues. Morpho-genetic, histolytic and histogenic processes progress during metamorphosis to create a juvenile, though direct evidence for the mechanism of induction has not been shown.Glutamine (Gin) induces metamorphosis in larvae of many sea urchin species (Strongylocentrotus intermedius: Naidenko, 1991; Pseudocentrotus depressus: Yazaki & Harashima, 1994; Hemicentrotus pulcherrimus: Yazaki, 1995). We have analysed the metamorphosis of sea urchin larvae using Gln, neurotransmitters and a natural cue (green algae).

Inversion of left–right asymmetry in the formation of the adult rudiment in sea urchin larvae: removal of a part of embryos at the gastrula stage

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).