Initial stages of calcium uptake and mineral deposition in sea urchin embryos

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.