Ca²âº influx-linked protein kinase C activity regulates the ß-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.

Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the ß-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of ß-catenin. To this end, we surveyed the expression pattern of ß-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of ß-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of ß-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

Larval arm resorption proceeds concomitantly with programmed cell death during metamorphosis of the sea urchin Hemicentrotus pulcherrimus.

Sea urchins are excellent models to elucidate metamorphic phenomena of echinoderms. However, little attention has been paid to the way that their organ resorption is accomplished by programmed cell death (PCD) and related cellular processes. We have used cytohistochemistry and transmission electron microscopy to study arm resorption in competent larvae of metamorphosing sea urchins, Hemicentrotus pulcherrimus, induced to metamorphose by L-glutamine treatment. The results show that: (1) columnar epithelial cells, which are constituents of the ciliary band, undergo PCD in an overlapping fashion with apoptosis and autophagic cell death; (2) squamous epithelial cells, which are distributed between the two arrays of the ciliary band, display a type of PCD distinct from that of columnar epithelial cells, i.e., a cytoplasmic type of non-lysosomal vacuolated cell death; (3) epithelial integrity is preserved even when PCD occurs in constituent cells of the epithelium; (4) secondary mesenchyme cells, probably blastocoelar cells, contribute to the elimination of dying epithelial cells; (5) nerve cells have a delayed initiation of PCD. Taken together, our data indicate that arm resorption in sea urchins proceeds concomitantly with various types of PCD followed by heterophagic elimination, but that epithelial organization is preserved during metamorphosis.

Larval and juvenile development of the Echinometrid sea urchin Colobocentrotus mertensii: emergence of the peculiar form of spines.

The development of Colobocentrotus mertensii from embryos to larvae and early juveniles was observed to give the first detailed description of larval and juvenile formation and skeletal structures in echinometrid sea urchins. The first larval spicules appeared at the mesenchyme blastula stage, whereas, in many echinoids, spicules were formed after gastrulation. From late eight-armed larva to juvenile, body color of C. mertensii was deep red, which has never been described for any echinoid before. The adult form of C. mertensii is characteristic in that the spines at the aboral side are short, truncated and pavement-like. The first sign of peculiar adult features could be seen in the juvenile spines and adult spines, which are broader than those of closely related Anthocidaris crassispina. The primary podia emerged on the left side of larval body were more stout and thicker in C. mertensii than in A. crassispina. The present study shows that developmental process of larval structure of C. mertensii is in general similar to the A. crassispina and the differences is first seen in juvenile structure including the distribution of pigment spots and morphology of adult spine.

Mechanisms of calcium elevation in the micromeres of sea urchin embryos.

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.

A Novel Substance Localizing on the Apical Surface of the Ectodermal and the Esophageal Epithelia of Sea Urchin Embryos: (egg-surface substance/apical location/larval epithelium/metamorphosis/sea urchin).

A new substance (ES-1) which localizes on the ectodermal and espophageal epithelia of sea urchin embryos was identified by a monoclonal antibody, McA ES-1. McA ES-1 recognized a 175 KDa protein of fertilized and 200 KDa in proteins of unfertilized egg-cortices. By indirect fluorescent antibody staining, ES-1 was found on the plasma membrane of fertilized eggs and in the cortical region of unfertilized eggs. ES-1 was not contained in the cortical granules and remained fixed in the cortex after centrifugation of unfertilized eggs for 30 min at 20,000 g. The polarized localization of ES-1 on the apical surface of ectodermal epithelial cells continued to the metamorphosis. It disappeared from mesenchyme cells and other migrating cells of the gastrula, while ES-1 was reexpressed in the presumptive esophagus to be connected with ectodermal epithelium. This may suggest a functional significance of ES-1 in establishment of cell polarity in the epithelium of larvae. In metamorphosing larvae and adults, the apical localization of ES-1 could no longer be found, and it was found in coelomocytes. From these findings, it is concluded that ES-1 was a novel surface substance of embryos and is probably phagocytosed at metamorphosis.

Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B: (post-cleavage surface movements/cortical microfilaments/egg-surface antibody/cytochalasin B/sea-urchin blastomeres).

Sea-urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg-surface antibody (anti-ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region. The anti-ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti-ES (FITC-Fab anti-ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region. The distributions of pigment granules and NBD-phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti-ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti-ES binding area was concluded to be due to the post-cleavage polarized distribution of submembranous microfilaments in the cortical layer.

Immunocytochemical evidence for the presence of two domains in the plasma membrane of sea urchin blastomeres.

The blastomeres of sea urchin embryos have two surface regions with different properties. Numerous microvilli are present in the apical surface region, while the baso-lateral surface region, either on adjoining adjacent cells or facing the blastocoel, is smooth. When blastomeres are isolated from embryos and stained with fluorescein-isothiocyanate-labelled anti-(egg surface) antibody (anti-ES) prepared against membranes isolated from fertilized eggs, the apical microvillous region fluoresces while the smooth region does not [Yazaki I (1984) Acta Embryol Morphol Exp 5∶3-22]. In order to study quantitatively the 'bindability' of the membrane in the two regions to anti-ES, immunoelectron microscopy was used. Blastomeres isolated from embryos ofHemicentrotus pulcherrimus at the eight-cell stage were treated with rabbit anti-ES serum or pre-immune serum and then with ferritin-conjugated goat anti-(rabbit IgG) for 10 min at 0°C, mainly before fixation. About 10 times (maximally 45 times) more ferritin particles were counted per contour length in the microvillous surface region than in the smooth surface region.These results suggest that the membrane of the blastomeres of sea urchin embryos is a mosaic of two different membrane territories: one represented by the microvillous surface originating from the unfertilized egg, which binds anti-ES, the other by the smooth surface newly organized after the first cleavage, which does not react with anti-ES. The mechanism of segregation of the membrane into these two regions is discussed.

LOCALIZATION OF DYNEIN IN SEA URCHIN EGGS DURING CLEAVAGE.

Detection and localization of dynein in cleaving sea urchin eggs were attempted using antidynein serum (prepared against a tryptic fragment of dynein, Fragment A, of sea urchin sperm flagella) and fluorescein conjugated goat antiserum to rabbit γ-globulin. In both unfertilized and newly fertilized eggs, fluorescence was distributed rather uniformly within the cells but was absent from the nuclei. At prophase, intense fluorescence was observed on both sides of nucleus, suggesting accumulation of dynein in developing asters. From metaphase to anaphase, the whole mitotic apparatus (MA) was stained with the exceptions of the chromosomes and pole areas. Fluorescence then again became dispersed within the eggs. Throughout the mitotic process and cytokinesis, the egg cortex including the cleavage furrow was stained intensely, presumably reflecting the presence of dynein in this region. Similar distributions of fluorescence were obtained with the isolated MAs. Neither non-immune serum nor the antiserum to which Fragment A was absorbed stained the eggs. Little staining was obtained with the antiserum against starfish egg myosin. The results, together with the finding that the chromosome motion in the isolated MAs was completely inhibited by anti-dynein serum, but not with the anti-myosin serum, suggest an active role played by a tubulin-dynein system in mitosis.