Sea urchin pluteus larvae contain dozens of pigment cells in their ectoderm. These pigment cells are the descendants of the veg2 blastomeres of the 60-cell stage embryo. According to the fate map made by Ruffins and Ettensohn, the prospective pigment cells occupy the central region of the vegetal plate. Most of these prospective pigment cells exclusively give rise to pigment cells. Therefore, specification of the pigment cell lineage should occur at some point between the 60-cell and mesenchyme blastula stage. However, the detailed process of the specification of the pigment lineage is unknown.

When are pigment cells specified? Are cell interactions necessary for the specification? Do founder cells exist? To answer these questions, I treated embryos with Ca2+-free seawater during the cleavage stage and examined the number of pigment cells observed in pluteus larvae. Treatment at 5.5–8.5 h and especially 7.5–10.5 h postfertilisation markedly reduced the number of pigment cells. The decrease was statistically significant. On the other hand, the treatment at 3.5–6.5 h or 9.5–12.5 h never reduced the number of pigment cells. By examining the frequency of the appearance of embryos whose numbers of pigment cells were less than 20, it was also found that the numbers of pigment cells were frequently in multiples of 4. Embryos having 4, 8, 12, 16 and 20 pigment cells were more frequently observed. Statistics indicated that the frequency of appearance was not random. These results indicated that cell contacts are necessary for the specification of pigment cells and that the specification occurs from 7 to 10 h postfertilisation. The results also suggest that the founder cells, if they exist, divide twice before they differentiate into pigment cells.