Inhibitors of procollagen C-terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo.

In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell-free extracts of gastrula-stage embryos of Strongylocentrotus purpuratus contain a procollagen C-terminal proteinase (PCP) activity. A rationally designed non-peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG-HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG-HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG-HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen-cleavage assay. However, FG-HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG-HL1 and FG-HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.

Eggshell formation in Bdelloura candida, an ectoparasitic turbellarian of the horseshoe crab Limulus polyphemus.

The marine triclad Bdelloura candida (Turbellaria) episodically deposits stalked eggshells onto the gill lamellae of the horseshoe crab Limulus polyphemus. Ultrastructural and biochemical analyses indicate that the eggshells consist of primary inner and secondary outer layers. Protein of the primary layer is rich in glycine, aspartate/asparagine, serine, and 3,4-dihydroxyphenylalanine (Dopa). In this regard it resembles the eggshell compositions of other members of the phylum Platyhelminthes. The primary layer appears to be derived from precursors produced in the vitelline cells of the flatworm. Each egg-laying episode consumes all of the precursor-containing vitelline cells. A Dopa-containing protein, vitelline protein 1 (Vp1), has been isolated from B. candida and resembles the primary eggshell layer in its composition. Vp1 has an apparent molecular weight of 34.4 and an acidic pI. Dopa-containing proteins are presumed to be directly involved in the cross-linking reactions that accompany quinone-tanning. The secondary outer layer of the eggshell may serve to adhere the eggshell to the gill. Its amino acid composition is unlike that of the primary eggshell and nothing is known about its precursors.