MATRIX METALLOPROTEINASE INHIBITORS DISRUPT SPICULE FORMATION BY PRIMARY MESENCHYME CELLS IN THE SEA-URCHIN EMBRYO

The primary mesenchyme cells of the sea urchin embryo construct an ela borate calcareous endoskeletal spicule beginning at gastrulation. This process begins by ingression of prospective primary mesenchyme cells into the blastocoel, after which they migrate and then fuse to form a syncytium. Skeleton deposition occurs in spaces enclosed by the cytopl asmic cables between the cell bodies. Experiments are described which probe the role of proteases in these early events of spicule formation and their role in the continued elaboration of the spicule during lat er stages of embryogenesis. We find that several inhibitors of metallo proteinases inhibit the continuation of spiculogenesis, an effect firs t reported by Roe et al. (Exp. Cell Res. 181, 542-550, 1989). A detail ed study of one of these inhibitors, BB-94, shows that fusion of prima ry mesenchyme cells still occurs in the presence of the inhibitor and the formation of the first calcite granule is not impeded. Continued e laboration of the spicule, however, is completely stopped; addition of the inhibitor during the active elongation of the spicule stops furth er elongation immediately. Removal of the inhibitor allows resumption of spicule growth. The inhibition is accompanied by almost complete ce ssation of massive Ca ion transport via the primary mesenchyme cells t o the spicule. The inhibitor does not prevent the continued synthesis of several spicule matrix proteins. Electron microscopic examination o f inhibited primary mesenchyme cells shows an accumulation of characte ristic vesicles in the cytoplasm. Gel zymography demonstrates that alt hough most proteases in homogenates of primary mesenchyme cells are no t sensitive to I he inhibitor in vitro, a protease of low abundance de tectable in the medium of cultured primary mesenchyme cells is inhibit ed by BB-94. We propose that the inhibitor is interfering with the del ivery of precipitated calcium carbonate and matrix proteins to the sit e(s) of spicule growth. (C) 1998 Academic Press.