Monoclonal Antibodies against Differentiating Mesenchyme Cells in Larvae of the Ascidian Halocynthia roretzi

Mechanisms of cell specification of mesenchyme during ascidian embryogenesis are poorly understood. This is because no good molecular markers have been available to evaluate differentiation of the mesenchyme cells. To obtain molecular markers of mesenchyme differentiation, we established monoclonal antibodies, Mch-1 and Mch-3, that recognize antigens present in the mesenchyme cells of the larva of Halocynthia roretzi. The antigens recognized by both antibodies start to be detectable in the mesenchyme cells at the late tailbud stage. The Mch-3 antibody specifically recognized all mesenchyme cells of the larva, whereas the Mch-1 antibody stained the cells only in the anterior portions of mesenchyme clusters in the trunk region of the larva. The Mch-1 antibody also stained trunk lateral cells. In addition, both antibodies recognized the mesenchyme cells in the ventro-lateral boundary between endoderm and epidermis that are migrating to the anterior head region of the larva. The partial embryos that originated from the mesenchymelineage cells at the 8-cell stage expressed the Mch-1 and Mch-3 antigens. The Mch-1 and Mch-3 antibodies will be useful as immunological probes for studying the specification mechanisms of mesenchyme 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.     

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.     

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.     

Helper Function of Follicle Cells in Sperm-Egg Interactions of the Ascidian, Ciona intestinalis

Studies were made on the involvement in sperm-egg interactions of follicle cells of Ciona intestinalis , which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N-acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA-binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.     

Sperm-egg binding mediated by sperm alpha-L-fucosidase in the ascidian,Halocynthia roretzi

Spermatozoa bind to the vitelline coat in the ascidians and many other animals. The binding of sperm in Halocynthia roretzi is mediated by a sperm alpha-L-fucosidase and complementary-L-fucosyl residues of glycoproteins in the vitelline coat. cDNA clones for alpha-L-fucosidase were isolated from growing testis mRNA. It contained a 1398 bp full-length cDNA insert (HrFuc'ase) that encoded the 466 amino acid residues of H. roretzi sperm alpha-L-fucosidase. A putative signal peptide of 21 amino acid residues proceeded the sequence for the mature protein (M.W. 52.4 kDa). The coding sequence for HrFuc'ase showed 47.7% sequence identity to the human liver fucosidase sequence. The polyclonal antibody was prepared against a lacZ-HrFuc'ase fusion protein expressed in E. coli. The antibody crossed to a 54 kDa protein in sperm on western blotting and inhibited fertilization in a dose dependent manner. These data suggest that sperm-egg binding is mediated by the sperm alpha-L-fucosidase, HrFuc'ase in the ascidian, H. roretzi.     

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.     

Steroidogenesis in the Ovarian Follicle of Medaka (Oryzias latipes, a daily spawner) during Oocyte Maturation

The metabolism of ¹⁴C-labeled steroid precursors by cell-free homogenates of medaka ( Oryzias latipes , a daily spawner) ovarian follicles at 12 different developmental stages was examined using thin layer chromatography (TLC). The radioactive metabolites produced were identified and tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in an in vitro homologous bioassay. When homogenates of follicles isolated during oocyte maturation were incubated with ¹⁴C-labeled 17α-hydroxyprogesterone, 13 metabolites were detected in TLC. Among these metabolites, one metabolite exhibited very high maturation inducing activity by the in vitro bioassay. This metabolite was identified as 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) by its chromatographic mobility in TLC and recrystallization to constant specific activity. 17α,20β-DP production was high in follicles collected between 10 and 6 hr before spawning. A much less biologically active metabolite, 17α,20β-dihydroxy-5β-pregnane-3-one appeared in follicles immediately after the formation of 17α,20β-DP. A similar pattern of steroidogenesis was observed when the follicles were incubated with ¹⁴C-labeled pregnenolone and progesterone. The timely synthesis of 17α,20β-DP in medaka at the onset of oocyte maturation, together with the demonstration that this progestogen is the most potent inducer of oocyte maturation in vitro , provides further evidence that 17α,20β-DP is the naturally occurring maturation-inducing hormone in the medaka. The results also suggest that the conversion of 17α,20β-DP to its 5β-reduced metabolite may be an inactivation process.     

Purification and characterization of beta-N-acetylhexosaminidase from the ascidian, Halocynthia roretzi

beta-N-Acetylhexosaminidase [EC 3.2.1.30] was purified 820-fold from the viscera of Halocynthia roretzi by Sephadex G-200 gel filtration and chromatography on columns of DEAE-Sephadex and CM-Sephadex. The final preparation was sufficiently free from alpha-N-acetylglucosaminidase, alpha-N-acetylgalactosaminidase, alpha- and beta-glucosidases, alpha- and beta-galactosidases, alpha- and beta-mannosidases, and alpha-L-fucosidase, and gave one protein band on disc gel electrophoresis. Two different molecular weight forms which depended upon the pH were observed on Sephadex gel filtration. At pH 7.0, a species with a molecular weight of 170,000 was observed, whereas at pH 4.5, an enzyme of 330,000 daltons was seen. The enzyme was active at pH 4.5 but inactive at pH 7.0. The optimum pH and the Km were pH 4.2 and 1.9 mM for p-nitrophenyl beta-N-acetylglucosaminide and pH 4.0 and 0.9 mM for p-nitrophenyl beta-N-acetylgalactosaminide. The terminal beta-N-acetylhexosamine of glycolipids such as globoside I, GM2, and asialo GM2 was cleaved by the ascidian beta-N-acetylhexosaminidase though GM2 was less susceptible to the enzyme.     

Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi

Cell lineages during ascidian embryogenesis are invariant. Developmental fates of larval mesodermal cells after metamorphosis are also invariant with regard to cell type of descendants. The present study traced developmental fates of larval endodermal cells after metamorphosis in Halocynthia roretzi by labeling each endodermal precursor blastomere of larval endoderm. Larval endodermal cells gave rise to various endodermal organs of juveniles: endostyle, branchial sac, peribranchial epithelium, digestive organs, peripharyngeal band, and dorsal tubercle. The boundaries between clones descended from early blastomeres did not correspond to the boundaries between adult endodermal organs. Although there is a regular projection from cleavage stage and larval stage to juvenile stage, this varies to some extent between individuals. This indicates that ascidian development is not entirely deterministic. We composed a fate map of adult endodermal organs in larval endoderm based on a statistical analysis of many individual cases. Interestingly, the topographic position of each prospective region in the fate map was similar to that of the adult organ, indicating that marked rearrangement of the positions of endodermal cells does not occur during metamorphosis. These findings suggest that fate specification in endoderm cells during metamorphosis is likely to be a position-dependent rather than a deterministic and lineage-based process. Received: 16 June 1999 / Accepted: 16 August 1999     

Self and non-self recognition between gametes of the ascidian,Halocynthia roretzi

Summary The self-sterility ofHalocynthia roretzi from Mutsu Bay, Japan, was examined. This sterility is strict and not a single egg can be fertilized in self-sterile animals. Less than 2% of the animals were self-fertile (with 100% cross-fertility). All heterologous sperm can fertilize all eggs, although there are pairs of individuals in which the coelomocytes recognize each other as self. Eggs deprived of follicle cells cannot be fertilized by either autologous or heterologous spermatozoa. Detached autologous or heterologous follicle cells can reattach to the chorion in calcium-enriched sea water and the reconstituted eggs recover their ability to be fertilized. A mosaic egg can therefore be obtained, which consists of oocyte, test cells and chorion originating from one individual and follicle cells from another. The mosaic egg was used to determine the site of recognition of self and non-self. The results indicate that the recognition resides in the chorion and/or test cells, probably the chorion. The relationship between somatic alloreactivity, previously found in coelomocytes ofH. roretzi, and gamete reactivity is discussed.     

Pigmented Follicle Cells and the Maturation of Oocytes in the Sand Dollar, Dendraster excentricus

Early in the spawning season female D. excentricus can be induced to spawn oocytes in the late stages of oogenesis. Observations with light microscopy indicate that pigment cells migrate in the jelly coat away from the surface of oocytes coincident with the final growth of the oocyte and its maturation. The pigment cells undergo a series of changes in shape and the oocyte elaborates arrays of long microvilli as the cells elevate from the surface of the oocyte. Transmission electron microscopy (TEM) observations of oocytes fixed within the ovary indicate that features of the sequence observed in the spawned oocytes normally take place within the ovary, prior to spawning. Extracts of the pigment cells induce a low level of germinal vesicle breakdown in asteroid oocytes. It is proposed that these cells are homologous to the follicle cells of other echinoderms and are involved in stimulating the maturation of oocytes.     

Establishment of self-sterility of eggs in the ovary of the solitary ascidian,Halocynthia roretzi

When unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, are released naturally they are strictly self-sterile, whereas almost all ovarian eggs isolated after spawning are self-fertile. Self-sterile eggs are prepared within a relatively short period of several hours before the spawning. The morphological changes in ovarian eggs during late oogenesis were studied with special reference to the establishment of self-sterility. Four types of eggs at serial developmental stages were classified according to the morphology of their external envelopes. Self-sterility was established in the last stage, from the ovarian egg type 3 (OVE3) to UFE stages. Ovarian eggs which had become committed to UFE were denoted as full-grown ovarian eggs (FOE). FOE were able to differentiate into self-sterile UFE in vitro, whereas OVE3 could not. Several morphological differences between OVE3 and UFE were found. OVE3 had a germinal vesicle (GV), a type of vitelline coat (VC-OVE3) and no expanded perivitelline space, whereas UFE had completed germinal vesicle break down (GVBD), had another type of coat (VC-UFE) and showed an expanded perivitelline space. There were also some differences in the mode of fertilization between OVE3 and UFE. In UFE, sperm became bound firmly to the vitelline coat and passed through the coat with the help of follicle cells, whereas in OVE3, sperm did not bind so strongly and entered the perivitelline space without the aid of follicle cells. The relationships between the establishment of self-sterility and these morphological and functional changes in ovarian eggs are discussed.