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 (Pa ralichthys olivaceus) are cultured in vitro to examine the development al fate of their pigment cells in relation to establishment of bilater ally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin-EDTA in Dulbecco's modified Ca2+ Mg2+-fr ee phosphate buffered saline and then cultured in vitro using L-15-bas ed fetal calf serum-supplemented growth medium at 20 degrees C, numero us 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 (abou t 70 mu m wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 mu m) and overwhelmingly higher in cell density than the first, doe s so about 1 month after plating. The timing of their appearances in v itro is in good accordance, respectively, with that observed for the l arvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period co rresponding to the completion of metamorphosis. Light microscopic exam inations disclose that each group of pigment cells is composed of blac k melanophores and reflecting leucophores, and that the population den sity of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are di stributed in the skin of the larvae of this species are scarcely obser ved in culture in vitro. Because of their dual synchronous appearances with about I month interval under the similar culture conditions, and because of their low proliferative activity during the periods from t he first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their dif ferentiation. Light and electron microscopic immunocytochemistry on cu ltured cells using the HNK-I antibody, which marks avian migratory neu ral crest cells, both disclose that the antibody cross-reacts with all these pigment cells, and that a certain number of immunoreactive unpi gmented cells exist even at the time of the second appearance of pigme nt cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to meta morphosis, at which they start to differentiate under control of a clo ck presumably set during neurulation.