A Monoclonal Antibody Specific to Embryonic Trunk-Lateral Cells of the Ascidian Halocynthia roretzi Stains Coelomic Cells of Juvenile and Adult Basophilic Blood Cells

In spite of extensive knowledge on the structure and function of ascidian blood cells, little is known about their embryological origin. In the present investigation, the developmental fate of trunk lateral cells (TLCs) was explored using a specific monoclonal antibody. TLCs comprise a group of undefined embryonic cells of the ascidian Halocynthia roretzi , which arise from the A7.6 blastomeres of a 64-cell embryo. The antigenicity first appeared at the middle tailbud stage in a pair of TLC-clusters situated lateral to the brain stem of the bilaterally symmetrical embryo. The position and number of stained cells did not change during later embryogenesis until hatching. After hatching, the stained cells were found in the entire trunk region of the swimming larva. After metamorphosis, cells that expressed the antigen were present within the coelom and within the tunic layer of the juvenile. In addition, the antibody stained adult basophilic blood cells. These observations suggest a relationship of this group of embryonic cells with the prospective blood forming mesenchymal cells.     

Differentiation Expression in Blastomeres of Cleavage-Arrested Embryos of the Ascidian Halocynthia roretzi

The question whether two or more different genetic programs are expressed in the common cytoplasm of single blastomeres or the expression of one genetic program somehow excludes expression of the other, was analyzed by assessing the occurrence of a muscle-specific and two epidermis-specific antigens in cleavage-arrested blastomeres in early embryos of the ascidian Halocynthia roretzi . Blastomeres which had been arrested in 1- to 4-cell stages expressed only the epidermis markers. Arrested 8-cell to 32-cell embryos produced both epidermis and muscle markers, but each cell expressed only one program of differentiation, even though some possessed the potential to express both. The differentiation expressions followed their cell lineages. These results indicate that at least in this experimental system differentiation markers of the two different cell-types are expressed exclusively.
A distinct order was noticed in expression of the two epidermis markers in a single blastomeres; a marker is always superiorly expressed, whereas the other appears only when the superior marker is expressed.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.     

Pattern of Segregation of Mitochondria into Muscle Lineage Cells during Embryogenesis of the Ascidian Halocynthia roretzi

Unequal partition of preexisting egg cytoplasmic components is one of possible cues to produce various types of cell in development. Segregation during ascidian embryogenesis of mitochondria into muscle lineage cells is a well-known example of cytoplasmic localization and segregation. In this study, using a monoclonal antibody specific to mitochondria, we re-examined changes in the distribution of mitochondria during oogenesis and embryogenesis of the ascidian, Halocynthia roretzi . The quantification method of the relative amount of the mitochondria-specific antigen revealed differences in the amount of mitochondria contained in four blastomere-pairs of an 8-cell embryo; the primary muscle lineage B4.1-pair contained about 40% of the total amount of mitochondria, while the secondary lineage b4.2- and A4.1-pairs contained about 23% and 20% respectively, and non-muscle lineage a 4.2-pair about 17%. In addition, it was shown that the total amount of mitochondria-specific antigen in the embryo remained constant throughout H. roretzi embryonic development. These results suggest that preferential segregation of preexisting mitochondria causes the characteristic distribution pattern of mitochondria within the embryo.     

Phorbol Ester Induces Elevation of the Vitelline Coat of Eggs of the Ascidian Halocynthia roretzi

Elevation of the vitelline coat of eggs of the ascidian, Halocynthia roretzi , was induced by 12-O-tetradecanoylphorbol-13-acetate or phorbol-12, 13-didecanoate, but not by their 4-epimers. After elevation of the vitelline coat in this way, eggs were unable to undergo cleavage on addition of sperm. This elevation of the vitelline coat by phorbol esters was inhibited by specific inhibitors of trypsin-like enzyme, calmodulin, phospholipase A₂, and protein kinase C. In association with elevation of the vitelline coat, a trypsin-like enzyme was released from the eggs. The properties of this enzyme were similar to those of the enzyme released by calcium ionophore, which also induced elevation of the vitelline coat, suggesting that similar exocytosis of intracellular granules occurred in both cases. Calmodulin, possibly involved in the elevation process, was isolated from eggs and characterized.     

Opsonin-independent and -dependent Phagocytosis in the Ascidian Halocynthia roretzi: Galactose-specific Lectin and Complement C3 Function as Target-dependent Opsonins

To clarify the molecular mechanisms of phagocytosis, we have been preparing monoclonal antibodies that inhibit phagocytosis by the hemocytes of the ascidian Halocynthia roretzi. A monoclonal antibody, RA5, inhibited the phagocytosis of non-treated sheep red blood cells (SRBCs) and yeast cells. It was demonstrated that the phagocytosis by the hemocytes was enhanced by pretreatment of target cells, SRBCs or yeast cells, with H. roretzi plasma. However, the RA5 antibody was unable to inhibit the phagocytosis of plasma-treated target cells. These results strongly suggest that the molecule recognized with the RA5 antibody is involved in the opsonin-independent phagocytosis. Western blot analysis showed that this antibody recognized a 200 kDa protein in H. roretzi hemocytes. On the other hand, flow cytometry analyses showed that a galactose-specific lectin (Gal-lectin) and complement C3 (AsC3), present in H. roretzi plasma, can bind to SRBCs and yeast cells, respectively, to enhance the phagocytosis of the respective target cells. Thus, H. roretzi hemocytes undergo opsonin-independent and -dependent phagocytosis, and Gal-lectin and AsC3 both function in the opsonin-dependent phagocytosis.     

Temporal and Spatial Expression of a Gene for the Nuclear Protein Hgv2 in Embryos and Adults of the Ascidian Halocynthia roretzi

The pHgv20 cDNA clone encodes an ascidian embryonic nuclear protein, Hgv2, that is closely related to the amphibian histone-binding protein N1. Genomic Southern blot analysis revealed the presence of two or more genes that hybridize with the Hgv2 probe under high-stringency conditions, although it remains to be determined whether or not each of them is actively expressed. On Northern blots prepared from embryos, a single, 2.3-kb Hgv2 mRNA was detectable during early stages of embryogenesis. The amount of Hgv2 mRNA gradually decreased after the 64-cell stages. Northern, Western and immunohistochemical analyses showed that the Hgv2 protein was not expressed exclusively in the oocyte: small amounts of the 2.3-kb mRNA and of the 83-kDa Hgv2 protein were detectable in the branchial sac of adult organisms. Weak but specific immunohistochemical staining was observed in the spermatocytes and/or spermatogonia. An Hgv2-specific antiserum reacted specifically with the 83-kDa protein on the Western blot of the testis. These results suggest that Hgv2 functions not only in embryonic cells but also in sperm precursor cells and some somatic cells.     

Ascidian Larvae: The Role of Test Cells in Preventing Hydrophobicity

Abstract Ascidian test cells co-differentiate on the surface of each ovarian oocyte beneath the vitelline coat. They become vacuolated and later occupy the perivitelline compartment of each egg and embryo. In some species, their vacuoles contain submicroscopic granules or filaments called ‘ornaments’ and acidic glycosaminoglycans. These test cells deposit their products on the surface of the larval tunic in late embryogenesis. In these species, the test cells are lost at hatching. In other species, the test cell vacuoles contain acidic glycosaminoglycans, but no ornaments. In these species, the test cells attach to the larval tunic and probably secrete acidic glycosaminoglycans. We deprived the embryos of seven species of ascidians of their test cells and vitelline coats during midembryogenesis. After completing their development, the larvae of both kinds of species were hydrophobic. They easily become trapped on the surface of sea water in cultures. Normal larvae (controls), bearing test cell secretions, are hydrophilic and never become trapped. We infer that negatively charged secretions of the test cells make normal larvae hydrophilic. Some molgulids with direct development have no test cells, no fins and no swimming larva. We reason that the test cells of these species may have been lost during evolution because they no longer had an important role in preventing hydrophobicity.     

Storage of retinal in the eggs of the ascidian,Halocynthia roretzi

Retinoids in the eggs of the solitary ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Retinal was the almost exclusive retinoid (>99%), and the concentration of retinal was 25.9-40.1 (30.6 on average) ng/mg of protein. The egg retinal consisted of four isomers: all-trans (50.9%), 9-cis (6.8%), 11-cis (20.4%) and 13-cis (21.9%). The presence of retinal in the eggs of this ascidian is a characteristic shared with the wide range of oviparous vertebrates, although the isomer composition differs between ascidian eggs and vertebrate eggs; in vertebrate eggs, almost all the retinal is in the all-trans form. The egg retinal was bound to a protein complex via a Schiff base linkage. The electrophoretic characteristics of the protein complex were similar to that of egg yolk proteins of oviparous vertebrates. The results presented in this study strongly suggest that, as is found with oviparous vertebrates, retinal in the ascidian eggs is the essential mode of retinoid storage, and is the precursor of photoreceptive pigment chromophores and retinoic acid during development.     

Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Myogenic Events in Compound Ascidian Larvae

SYNOPSIS. Comparative studies on the compound ascidians Distapliaoccidentalis and Diplosonta macdonaldi indicate that larvalmyogenesis is divisible into three periods. In the prolifnativephase, the presumptive muscle cells divide by mitosis, assumespecific relationships to the notochordal and epidermal rudimentsof the embryo, and assemble the synthetic machinery to supportthe ensuing period of myogenesis. In the synthetic phase, thedifferentiating muscle cells increase in volume, establish thecontractile apparatus, and augment the cisternae of agranularsarcoplasmic reticulum. The sarcoplasmic reticulum and sarcolemmaform local adhesions that foreshadow structural couplings. Inthe elaborative phase, the caudal muscle cells refine the contractileapparatus, envelop it with perifibrillar cisternae of sarcoplasmicreticulum, and anchor it to the sarcolemma at the transversecellular boundaries. Dyadic couplings link the perifibrillarsarcoplasmic reticulum to the sarcolemma. Subsarcolemmal specializationsappear at future synaptic sites shortly before hatching.     

Scanning Electron Microscopic Observations of the Mesenchyme Cells in the Larvae of the Starfish Pisaster ochraceus

The are two morphological types of mesenchyme cells, bipolar and multipolar, and both are derived from the tip of the archenteron at the gastrula stage. Some of the cells form larval muscle and others may be involved in the stomodeum and coelomic pouch formation.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.