Dual appearances of pigment cells from in vitro cultured embryonic cells of Japanese flounder: an implication for a differentiation-associated clock.

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin-EDTA in Dulbecco's modified Ca(2+)-, Mg(2+)-free phosphate buffered saline and then cultured in vitro using L-15-based fetal calf serum-supplemented growth medium at 20 degrees C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 microns wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 microns) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)