PI3K signaling is required for skeletogenesis in the P. lividus sea urchin embryo.

Phosphatidylinositide 3-kinase (PI3K) belongs to a family of cytoplasmic enzymes, which regulate a wide range of cellular processes, such as cell proliferation, differentiation, migration as well as cancer transformation. The PI3K had already been linked to the skeletogenesis of L. variegatus sea urchin embryo, although its downstream targets have not yet been identified. Here, we analysed the function of PI3K in Paracentrotus lividus embryos by in vivo assays, using the specific inhibitor LY294002 (LY). Mesenchyme blastula embryos were continuously cultured in the presence of increasing concentrations of LY and the development of embryos was monitored at different time intervals. A dose-dependent morphological response was observed 24h after LY addition. At the dose of 40 mM LY, nearly 100% of the embryos completely lacked the skeleton, although the skeletogenic cells appeared to be normally differentiated and arranged, as well as all other embryonic territories. Thus, the inhibition of PI3K activity specifically blocked the deposition of CaCO3 and spicule formation, which was completely reversed after removal of LY from the medium. The expression of genes coding for biomineralization proteins (Pl-SM30, Pl-p16, Pl-p19, Pl-CAN), transcription factors (Pl-Alx1, Pl-SkeT, Pl-FoxO, Pl-jun, Pl-Fra-1), Pl-Pks, Pl-14-3-3 and the newly identified Pl-PI3K, have been evaluated by QPCR in embryos treated with 40 mM LY for 1, 3, 24 and 48h. In the same samples, we further assessed the phosphorylation levels of p38MAPK and ERK by Western Blot. A model describing the role of PI3k signaling in sea urchin skeletogenesis will be shown.