Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone Hi kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.The motive force of cleavage in animal cells is generated by the interaction between actin filaments and myosin in the contractile ring (CR) or the contractile arc (1, 2). It has been proposed (3) that induction of the cleavage furrow is mediated by an as-yet-uncharacterized substance that is transported from the mitotic apparatus to the cortical layer via astral microtubules at a specific stage ofcell division. The CR forms rapidly but reduces its volume during contraction and disappears soon after cytokinesis (4). The mechanisms that induce this sequence of events and the nature of the signal that stimulates the furrow formation have not yet been elucidated. One interesting approach to this problem is to identify substances that induce cleavage or a specific stage in the cleavage process.Calyculin A (CL-A), a tumor-promoting substance isolated from the marine sponge Discodermia calyx (5), is a specific inhibitor of protein phosphatase 1 (PP1) (IC50 = 0.5-1 nM) and protein phosphatase 2A (PP2A) (IC50 = 2 nM) in rabbit skeletal muscle (6). It has been shown to induce oocyte maturation in starfish (7) and stimulate contraction of smooth muscle (6), effects also induced by the phosphatase inhibitor okadaic acid (OA). We report here that CL-A induces a CR-like apparatus in unfertilized sea urchin eggs. The simultaneous condensation of chromosomes was also observed. MATERIALS AND METHODSMaterials. The species of sea urchins used in this study were Anthocidaris crassispina, Clypeaster japonicus, Diadema savignyi, Diadema setosum, Echinometra mathaei, Echinostrephus Wheat germ agglutinin (WGA) receptors were visualized by staining with fluorescein-conjugated WGA (1 ,ug/ml; Vector Laboratories). To visualize DNA, the eggs were stained with 4',6-diamidino-2-phenylindole (0.25 ,Ag/ml). The chemicals were dissolved in buffer A. Isolation of egg cortices on protamine-coated glass surface was carried out as described (9). The sample was examined with a Nikon Optiphot microscope equipped with ordinary epifluorescence optics or confocal optics (MRC-00; Bio-Rad). Triton X-100 for 10 min on ice. The, egg suspension was passed through a nylon mesh (50 u...

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.

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