Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differenti ate to produce spicules. Although several authors have used this model, alm ost nothing is known about the signalling pathways responsible for initiati ng skeletogenesis. In order to investigate the potential involvement of pho sphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtaine d using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not seri ne/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formatio n resumed. Inhibition of tyrosine phosphatases resulted in an increase in t he tyrosine phosphorylation level of two major proteins and a modest decrea se in the expression of the mRNA coding for type I fibrillar collagen. Thes e findings strongly suggest that tyrosine phosphorylation and dephosphoryla tion is required for micromere differentiation and for normal skeletogenesi s during sea urchin embryo development.