Competence of the animal cap to react with the inductive signal from micromere descendants in the hatching blastula stage of echinoid embryos

Abstract

Micromere signalling is a key to understanding the developmental mechanisms underlying endomesoderm differentiation along the A-V axis in sea urchin embryos. A recent study has shown that micromere activity in inducing endo-mesoderm differentiation of mesomere descendants is, unexpectedly, maximal at the hatching blastula (HB) stage in the echinoids Scaphechinus mirabilis and Hemicentrotus pulcherrimus. This study focused mainly on the timing of emission of the inductive signal by the micromere descendants. The timing of animal cap competent to react to the inductive signal from micromeres was not specifically investigated.In the present study, we examined the competence of mesomere descendants at the HB stage to react to the inductive signal of micromere descendants by producing recombinant embryos of the descendants of mesomeres and micromeres.Adults of the sand dollar Scaphechinus mirabilis and the sea urchin Hemicentrotus pulcherrimus were collected along the shore of Shiraishi Island, Okayama Prefecture and in the vicinity of the Misaki Marine Biological Station, Kanagawa Prefecture, respectively. The fertilised eggs were separated into two groups immediately after removal of the fertilisation membranes. One group was cultured in normal artificial seawater (ASW), and the other in ASW containing 50 mg/ml of rhodamine B isothiocyanate (RITC). At the early 16-cell stage unlabelled embryos were transferred to calcium-free seawater (CFSW) and dissected in the equatorial plane with a glass needle to isolate animal caps consisting of eight mesomeres. Embryos labeled with RITC were also transferred to CFSW at the same stage and dissected to isolate four micromeres. An isolated animal cap and a quartet of micromeres were cultured separately in ASW and recombined at the stage corresponding to the HB of control (undisturbed) embryos.