Endocytosis in primary mesenchyme cells during sea urchin larval skeletogenesis

The sea urchin larval embryo elaborates two calcitic endoskeletal elements called spicules. Spicules are synthesized by the primary mesenchyme cells (PMCs) and begin to form at early gastrula stage. It is known that the calcium comprising the spicules comes from the seawater and we wish to further consider the mode of calcium transport from the extracellular seawater to the PMCs and then onto the forming spicules. We used PMC in vitro cultures, calcein, fluorescently labeled dextran, and fluorescently labeled Wheat Germ Agglutinin (WGA) to track calcium transport from the seawater into PMCs and spicules and to determine how molecules from the surface of PMCs interact with the incoming calcium. Labeling of PMC endocytic vesicles and forming spicules by both calcein and fluorescently tagged dextran indicate that calcium is taken up from the seawater by endocytosis and directly incorporated into spicules. Calcein labeling studies also indicate that calcium from the extracellular seawater begins to be incorporated into spicules within 30min of uptake. In addition, we demonstrate that fluorescently labeled WGA and calcein are taken up by many of the same endocytic vesicles and are incorporated into growing spicules. These findings suggest that PMC specific surface molecules accompany calcium ions as they enter PMCs via endocytosis and are incorporated together in the growing spicule. Using anti-spicule matrix protein antibodies, we pinpoint a subset of spicule matrix proteins that may accompany calcium ions from the surface of the PMCs until they are incorporated into spicules. Msp130 is identified as one of these spicule matrix proteins.