Cell-cell and cell-matrix interactions responsible for the morphogenesis of the sea urchin primary mesenchyme

Morphogenesis of the skeletal system during development of the sea urchin embryo is based on complex interactions of primary mesenchyme cells (PMCs) with one another and with their environment. To advance the understanding of this process two of its aspects were analyzed: PMC fusion and the interaction of PMCs with underlying extracellular matrix (ECM).; Previous studies have shown that PMCs fuse to form syncytial cables within which skeletal spicules are deposited. The dynamics of PMC fusion was directly observed in vivo by labeling a fraction of PMCs with fluorescent dextran and following dye transfer to unlabeled cells by time-lapse, fluorescence microscopy. Fusion began 2 hours after ingression and progressed in parallel with the assembly of the PMC ring, leading to the formation of a single, extensive syncytium. Fusion assays using isolated, cultured PMC progenitors or embryos to which heterochronic PMCs had been transplanted showed that the capacity of PMCs to fuse is autonomously programmed in the PMC lineage and does not appear to be influenced by external signals. At late developmental stages fusion-competent blastocoelar cells and PMCs were shown to come into contact but did not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. Secondary mesenchyme cells that converted to a skeletogenic fate acquired PMC-specific fusogenic properties and joined the PMC syncytium.; Using an in vitro immunization procedure, a range of monoclonal antibodies (MAbs) was generated against antigens potentially involved in embryonic morphogenesis. Among them, MAb 2.5C4 identified a class of ECM fibers associated with the ectodermal basal lamina. Immunofluorescent staining with MAb 2.5C4 showed the fibers distributed in a vegetal to animal gradient, with a highest concentration at the vegetal pole. They were aligned in an organized pattern converging towards PMC structures. PMC removal resulted in random fiber distribution, suggesting that PMCs are responsible for fiber alignment, possibly through filopodial traction forces. Comparative immunostaining experiments identified one component of 2.5C4 fibers, the protein ECM3, which is similar in primary sequence to chondroitin sulfate proteoglycan (CS-PG) core proteins. The effect of disturbing PG biosynthesis on PMC-fiber interactions was consistent with fibers containing CS-PG and also suggested that PMCs interact with the ECM through multiple mechanisms.