Number 41
May 1997
Here it is spring again and time for another Ascidian News. Please
note that our telephone and fax numbers have changed. We are grateful to
those of you who sent us reprints for our New Publications section (88 new
papers listed in this issue) and abstracts of meeting presentations. Please
send all contributions for AN by e-mail so they can be incorporated without
retyping.
We will be in France from May 31 to August 22, continuing work on fertilization of Phallusia
at the Station Biologique, BP 74, 29682 Roscoff Cedex, France. Tel. 33.2.98.29.23.05;
fax 33.2.98.29.23.24. Our email addresses will remain the same.
*Ascidian News is not part of the scientific literature and should not be cited as such.
1. More ascidian invaders: Our research these days is focussed on the problem of introduced species; we have just finished our spring surveys of southern California harbors. Gretchen has identified several that were mysteries to us, the most recent of which is the Japanese species Symplegma reptans (confirmed by Dr. Nishikawa) and which we observed for the first time in April in Mission Bay, San Diego, where it has undergone an incredible population explosion this spring.
1.Books for sale: We have an extra copy (unbound) of W.G. Van Name's 1945 monograph, The North and South American Ascidians. Bull. Amer. Mus. of Nat. Hist. vol. 84. We would like to sell it for the price we paid, $65 some years ago.
3. It is with great sadness that we report that Dr. John Berrill passed away last October 15, 1996 at the age of 93, peacefully at the home of his daughter Lynn in New Hampshire, with whom he had been living. We never met him but felt that we knew him through our reading of his books and papers and our correspondence; he told us he continued to enjoy reading AN long after he retired. John certainly had a major impact upon several generations of biology graduate students working on ascidian development and all aspects of regeneration. His book on the ascidians of great Britain is still in use in laboratories all over the world. His book on the origin of the vertebrates has influenced our understanding of the similarities and differences in the development of the chordate egg and has had a very large impact upon the questions we ask in developmental biology. His textbooks on animal development have influenced a whole generation of biologists. although they may not be aware of this. He was also instrumental in founding the Society For Developmental Biology, an organization with members from throughout the world and a major impact upon the direction of current research in this field. With his popular books he impacted how we as a society approach environmental concerns; he also influenced at least two biologists to stay close to the sea in all our research. John lived a long fruitful life and made many contributions to science; he will be missed by all of us.
Abbas, S.A., M.B. Hossain, D. vander Helm, F.J. Schmitz, M. Laney, R. Cabuslay, et al. 1996. Alkaloids from the tunicate Polycarpa aurata from Chuuk Atoll. J. Org. Chem. 61:9072.
Acuna, J.L., D. Deibel & C.C. Morris 1996. Particle capture mechanism of the pelagic tunicate Oikopleura vanhoeffeni. Limnol. Oceanogr. 41:1800-1814.
Aiello, A., E. Fattorusso & M. Menna 1996. Low molecular weight metabolites of three species of ascidians collected in the lagoon of Venice. Biochem. System. & Ecol. 24:521-530.
Aizenberg, J., G. Lambert, L. Addadi & S. Weiner 1996. Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates. Advanced Materials 8:222-226.
Arnoult, C., M. Albrieux, A.F. Antoine, D. Grunwald & M. Villaz 1997. A ryanodine-sensitive calcium store in ascidian eggs monitored by whole-cell patch-clamp recordings. Cell Calcium 21:93-101.
Azumi, K. & H. Yokosawa 1996. Humoral factors and cellular reactions in the biological defense of the ascidian Halocynthia roretzi. pp. 43-54 in New Directions in Invertebrate Immunology, ed. vol., ed. by Soderhall, K., S. Iwanaga & G.R. Vasta.
Benslimane, A.F., Y.F. Pouchus, J. Le Boterff, J.F. Verbist, C. Roussakis & F. Monniot 1988. Substances cytotoxiques et antibacteriennes de l'ascidie Aplidium antillense. J. Nat. Prod. 51:582-583.
Bergmann, T., D. Schories & B. Steffan 1997. Alboinon, an oxadiazinone alkaloid from the ascidian Dendrodoa grossularia. Tetrahedron 53:2055-2060.
Biard, J.F., C. Grivois, J.F. Verbist, C. Debitus & J.B. Carre 1990. Origin of bistramide A identified in Lissoclinum bistratum (Urochordata): possible involvement of symbiotic Prochlorophyta. J. Mar. Biol. Ass. U. K. 70:741-746.
Biard, J.F., S. Guyot, C. Roussakis, J.F. Verbist, J. Vercauteren, J.F. Weber, et al. 1996. Lepadiformine, a new marine cytotoxic alkaloid from Clavelina lepadiformis Muller. Tetrahed. Lett. 35:2691-2694.
Biard, J.F., C. Malochet-Grivois, C. Roussakis, P. Cotelle, J.P. Henichart, C. Debitus, et al. 1994. Lissoclimides, cytotoxic diterpenes from Lissoclinum voeltzkowi Michaelsen. Nat. Prod. Lett. 4:43-50.
Biard, J.F., C. Roussakis, J.M. Kornprobst, D. Gouiffes-Barbin, J.F. Verbist, P. Cotelle, et al. 1994. Bistramides A, B, C, D, and K: a new class of bioactive cyclic polyethers from Lissoclinum bistratum. J. Nat. Prod. 57:1336-1345.
Bingham, B.L. 1997. Light cycles & gametogenesis in three temperate ascidian species. Invert. Biol. 116:61-70.
Bruno, J.F. & J.D. Witman 1996. Defense mechanisms of scleractinian cup corals against overgrowth by colonial invertebrates. J. Exp. Mar. Biol. Ecol. 207:229-241.
Cammarata, M., V. Arizza, M. Vazzana & N. Parrinello 1996. Prophenoloxidase activating system in tunicate hemolymph. Ital. J. Zool. 63:345-352.
Cima, F., L. Ballarin, G. Bressa, G.B. Martinucci & P. Burighel 1996a. Embryotoxic effects of organotin compounds on Styela plicata (Tunicata; Ascidiacea). Fresenius Envir. Bull. 5:718-722.
Cima, F., L. Ballarin, G. Bressa, G. Martinucci & P. Burighel 1996b. Toxicity of organotin compounds on embryos of a marine invertebrate (Styela plicata; Tunicata). Ecotoxicol. & Envir. Safety 35:174-182.
Cima, F., L. Ballarin & A. Sabbadin 1996. New data on phagocytes and phagocytosis in the compound ascidian Botryllus schlosseri (Tunicata, Ascidiacea). Ital. J. Zool. 63:357-364.
Cohen, C.S. 1996. The effects of contrasting modes of fertilization on levels of inbreeding in the marine invertebrate genus Corella. Evolution 50:1896-1907.
Cooper, E.L. 1996. The immunology of earthworms and tunicates. Lab Animal Nov. 1996:38-43.
Corbo, J.C., M. Levine & R.W. Zeller 1997. Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589-602.
Dallai, R., P. Burighel, G.B. Martinucci, & N.J. Lane 1997. Scalariform junctions: a revised model. Cell Biol. Intl. 21:23-34.
Davis, A.R. 1996. Association among ascidians: facilitation of recruitment in Pyura spinifera. Mar. Biol. 126:35-41.
Davis, A.R., D.J. Ayre, M.R. Billingham, C.A. Styan & G.A. White 1996. The encrusting sponge Halisarca laxus: population genetics and association with the ascidian Pyura spinifera. Mar. Biol. 126:27-33.
Davis, A.R. & D.J. Campbell 1996. Two levels of spacing and limits to local population density for settled larvae of the ascidian Clavelina moluccensis:a nearest neighbour analysis. Oecologia 108:701-707.
Degnan, B.M., C.N. Souter, S.M. Degnan & S.C. Long 1997. Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus. Dev. Genes & Evol. 206:370-376.
DeLeo, G., N. Parrinello, D. Parrinello, G. Cassara, D. Russo & M.A. DiBella 1997. Encapsulation response of Ciona intestinalis (Ascidiacea) to intratunical erythrocyte injection. 2. The outermost inflamed area. J. Invert. Pathol. 69:14-23.
Esnal, G.B., F.L. Capitanio & L.C. Simone 1996. Concerning intraspecific taxa in Fritillaria borealis Lohmann (Tunicata, Appendicularia). Bull. Mar. Sci. 59:461-468.
Foster, M.P., C.L. Mayne, R. Dunkel, R.J. Pugmire, D.M. Grant, J.M. Kornprobst, et al. 1992. Revised structure of bistramide A (bistratene A): application of a new program for the automated analysis of 2D INADEQUATE spectra. J. Amer. Chem. Soc. 114:1110-1111.
Glardon, S., P. Callaerts, G. Halder & W.J. Gehring 1997. Conservation of Pax-6 in a lower chordate, the ascidian Phallusia mammillata. Development 124:817-826.
Greaves, A.A., A.K. Davis, J.E. Dallman & W.J. Moody 1996. Co-ordinated modulation of Ca2+ and K+ currents during ascidian muscle development. J. Physiol. 497:39-52.
Hirose, E., Y. Saito & H. Watanabe 1997. Subcuticular rejection: an advanced mode of the allogeneic rejection in the compound ascidians Botrylloides simodensis and B. fuscus. Biol. Bull. 192:53-61.
Holyoak, A.R. 1997. Patterns and consequences of whole colony growth in the compound ascidian Polyclinum planum. Biol. Bull. 192:87-97.
Hopmann, C. & D.J. Faulkner 1997. Lissoketal, a spiroketal from the palauan ascidian Lissoclinum voeltzkowi. Tetrahed. Lett. 38:169-170.
Ishida, K., T. Ueki & N. Satoh 1996. Spatio-temporal expression patterns of eight epidermis-specific genes in the ascidian embryo. Zool. Sci. 13:699-709.
Jefferies, R.P.S., N.A. Brown & P.E.J. Daley 1996. The early phylogeny of chordates and echinoderms and the origin of chordate left-right asymmetry and bilateral symmetry. Acta Zool. 77:101-122.
Kang, H.J. & W. Fenical 1997. Aplidiamine, a unique zwitterionic benzyl hydroxyadenine from the Western Australian marine ascidian Aplidiopsis sp. Tetrahed. Lett. 38:941-944.
Kawaminani, S. & H. Nishida 1997. Induction of trunk lateral cells, the blood cell precursors, during ascidian embryogenesis. Dev. Biol. 181:14-20.
Keough, M.J. & P.T. Raimondi 1996. Responses of settling invertebrate larvae to bioorganic films: effects of large-scale variation in films. J. Exp. Mar. Biol. Ecol. 207:59-78.
Kimura, S. & T. Itoh 1996. New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicate Metandrocarpa uedai. Protoplasma 194:151-163.
Koike, I. & t Suzuki 1996. Nutritional diversity of symbiotic ascidians in a Fijian seagrass meadow. Ecol. Res. 11:381-386.
Kozloff, E.N. 1993. Three new species of Stoecharthrum (phylum Orthonectida). Cah. Biol. Mar. 34:523-534.
Lambert, G. 1996. Chapt. 11 - Phylum Chordata: Subphylum Urochordata, Class Ascidiacea. pp. 261-293 in vol. 14 of the Taxonomic Atlas of the Benthic Fauna of the Santa Maria Basin and Western Santa Barbara Channel, Misc. Taxa. Ed. by J.A. Blake, P.H. Scott & A. Lissner. Publ. by Santa Barbara Mus. of Nat. Hist.
Lambert, G. & C.C. Lambert 1996a. Antibodies to echinoid larval spicule proteins cross react with the spicular complex in the ascidian Herdmania momus. Bull. Inst. Oceanogr. Monaco numero special 14: 253-261.
Lambert, G. & C. Lambert 1996b. Spicule formation in the New Zealand ascidianPyura pachydermatina (Chordata, Ascidiacea). Connective Tissue Res. 34:263-269.
Lee, I.H., C.Q. Zhao, Y. Cho, S.S.L. Harwig, E.L. Cooper & R.I. Lehrer 1997. Clavanins, alpha-helical antimicrobial peptides from tunicate hemocytes. FEBS 400:158-162.
Lindquist, N. 1996. Palatibility of invertebrate larvae to corals and sea anemones. Mar. Biol. 126:745-755.
Lindquist, N. & M.E. Hay 1995. Can small rare prey be chemically defended? The case for marine larvae. Ecology 76:1347-1358.
Lindquist, N. & M.E. Hay 1996. Palatability and chemical defense of marine invertebrate larvae. Ecol. Monog. 66:431-450.
Lubbering-Sommer, B., P. Compere & G. Goffinet 1996. Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea). 2. Demonstration of proteins. Tiss. & Cell 28:651-662.
Malochet-Grivois, C., P. Cotelle, J.F. Biard, J.P. Henichart, C. Debitus, C. Roussakis, et al. 1991. Dichlorolissoclimide, a new cytotoxic labdane derivative from Lissoclinum voeltzkowi Michaelsen (Urochordata). Tetrahed. Lett. 32:6701-6702.
McKinney, F.K. 1996. Encrusting organisms on co-occurring disarticulated valves of two marine bivalves: comparison of living assemblages and skeletal residues. Paleobiology 22:543-567.
Monniot, F. & C. Monniot 1996. New collections of ascidians from the western Pacific and southeastern Asia. Micronesica 29:133-279.
Naranjo, S.A., J.L. Carballo & J.C. Garcia-Gomez 1996. Effects of environmental stress on ascidian populations in Algeciras Bay (southern Spain). Possible marine bioindicators? Mar. Ecol. Prog. Ser. 144:119-132.
Niermann-Kerkenberg, E. & D.K. Hofmann 1989. Fertilization and normal development in Ascidiella aspersa (Tunicata) studied with Nomarski optics. Helgolander Meeresunters. 43:245-258.
Ogasawara, M., K.J. Tanaka, K.W. Makabe & N. Satoh 1996. Expression of endostyle-specific genes in the ascidian Halocynthia roretzi. Dev. Genes & Evol. 206:227-235.
Okamura, Y., F. Ono & R. Okagaki 1997. Regulation of voltage-gated ion channels during ascidian embryogenesis. Develop. Neurosci. 19:23-24.
Pancer, Z., E.L. Cooper & W.E.G. Muller 1996. A tunicate (Botryllus schlosseri) cDNA reveals similarity to vertebrate antigen receptors. Immunogenetics 45:69-72.
Pancer, Z., U. Scheffer, I. Muller & E.G. Muller 1996. Cloning of sponge (Geodia cydonium) and tunicate (Botryllus schlosseri) proteasome subunit epsilon (PRCE): implications about the vertebrate MHC-encoded homologue LMP7 (PRCC). Biochem. Biophys. Res. Commun. 228:406-410.
Parrinello, N., V. Arriza, M. Cammarata & D.M. Parrinello 1996. Expression and modulation of immunological activities by tunicate hemocytes. pp. 391-405 in Modulators of Immune Responses: The Evolutionary Trail, ed. vol., ed. by Stolen, J.S. et al.
Patil, A.D., A.J. Freyer, R. Reichwein, B. Carte, L.B. Killmer, L. Faucette, et al. 1997. Fasicularin, a novel tricyclic alkaloid from the ascidian Nephteis fasicularis with selective activity against a DNA repair-deficient organism. Tetrahed. Lett. 38:363-364.
Perissinotto, R. & E.A. Pakhomov 1997. Feeding association of the copepod Rhincalanus gigas with the tunicate salp Salpa thompsoni in the southern ocean. Mar. Biol. 127:479-484.
Powell, J.F.F., S.M. Reska-Skinner, M.O. Prakash, W.H. Fischer, M. Park, J.E. Rivier, et al. 1996. Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications. Proc. Natl. Acad. Sci. 93:10461-10464.
Raftos, D. & A. Hutchinson 1997. Effects of common estuarine pollutants on the immune reactions of tunicates. Biol. Bull. 192:62-72.
Reddy, M.V.R., D.J. Faulkner, Y. Venkateswarlu & M.R. Rao 1997. New lamellarin alkaloids from an unidentified ascidian from the Arabian Sea. Tetrahedron 53:3457-3466.
Riisgard, H.U., C. Jurgensen & F.O. Andersen 1996. Case study: Kertinge Nor. Coastal & Estuarine Studies 52:205-220.
Riisgard, H.U., C. Jurgensen & T. Clausen 1996. Filter-feeding ascidians (Ciona intestinalis) in a shallow cove: implications of hydrodynamics for grazing impact. J. Sea Res. 35:293-300.
Robinson, W.E., K. Kustin, K. Matulef, D.L. Parry, X. He & E.K. Ryan 1996. 14C- and 3H-tyrosine incorporation into Ascidia ceratodes tunichrome in vivo. Comp. Biochem. Physiol. 115:475-481.
Sanamyan, K. 1996. Ascidians from the north-western Pacific region. 3. Pyuridae. Ophelia 45:199-210.
Sato, S., H. Masuya, T. Numakunai, N. Satoh, K. Ikeo, T. Gojobori, et al. 1997. Ascidian tyrosinase gene: its unique structure and expression in the developing brain. Developmental Dynamics 208:363-374.
Satou, Y. & N. Satoh 1996. Two cis-regulatory elements are essential for the muscle-specific expression of an actin gene in the ascidian embryo. Dev., Growth & Differ. 38:565-573.
Sawada, H., H. Kawahara, Y. Saitoh & H. Yokosawa 1996. Physiological functions of proteasomes in ascidian fertilization and embryonic cell cycle. pp. 229-232 in Intracellular Protein Catabolism, ed. vol., ed. by Suzuki, K. & J. Bond.
Sawada, H., E. Kodama, M.R. Pinto, R. De Santis & H. Yokosawa 1997. Structure and functions of sperm proteases involved in fertilization of ascidians, Halocynthia roretzi and Ciona intestinalis. J. Repro. & Dev. 43:suppl.
Scippa, S., G. Iazzetti & M. de Vincentiis 1996. Vacuole-containing cells in the body cavity of Phallusia mammillata larvae before and after hatching. Invert. Repro. & Develop. 29:231-234.
Sims, D.W. 1996. A rare record of the salp, Thetys vagina (Tunicata: Thaliacea) from western Scottish waters. J. Mar. Biol. Ass. U. K. 76:833.
Sings, H. & K.L. Rinehart 1996. Compounds produced by potential tunicate-blue-green-algal symbiosis. J. Industrial Microbiol. 17:385-396.
Stoner, D.S. & I.L. Weissman 1996. Somatic and germ cell parasitism in a colonial ascidian: possible role for a highly polymorphic allorecognition system. Proc. Natl. Acad. Sci. 93:15254-15259.
Swalla, B.J. & W.R. Jeffery 1996. Localization of ribosomal protein L5 mRNA in myoplasm during ascidian development. Dev. Genetics 19:258-267.
Tanaka, K.J., S. Chiba & T. Nishikata 1996. Two distinct cell types identified in the ascidian notochord. Zool. Sci. 13:725-730.
Tanaka, K.J. & T. Nishikata 1994. Specification in the primary lineage of ascidian muscle cells analyzed with a specific monoclonal antibody. Mem. Konan Univ. 41:89-97.
Tanaka, K.J., M. Ogasawara, K.W. Makabe & N. Satoh 1996. Expression of pharyngeal gill-specific genes in the ascidian Halocynthia roretzi. Dev. Genes & Evol. 206:218-226.
Tsukamoto, S., H. Kato, H. Hirota & N. Fusetani 1997. Seven new polyacetylene derivatives, showing both potent metamorphosis-inducing activity in ascidian larvae and antifouling activity against barnacle larvae, from the marine sponge Callyspongia truncata. J. Nat. Prod. 60:126-130.
Vervoort, H.C., S.E. Richards-Gross, W. Fenical, A.Y. Lee & J. Clardy 1997. Didemnimides A-D: novel, predator-deterrent alkaloids from the Caribbean mangrove ascidian Didemnum conchyliatum. J. Org. Chem. 62:1486-1490.
Wada, H., P.W.H. Holland & N. Satoh 1996. Origin of patterning in neural tubes. Nature 384:123.
Wada, S., Y. Katsuyama, Y. Sato, C. Itoh & H. Saiga 1996. Hroth, an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis. Mechanisms of Development 60:59-72.
Yamada, A. & H. Nishida 1996. Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi. Dev. Genes & Evol. 206:297-304.
Yasuo, H., M. Kobayashi, Y. Shimauchi & N. Satoh 1996. The ascidian genome contains another T-domain gene that is expressed in differentiating muscle and the tip of the tail of the embryo. Dev. Biol. 180:773-779.
2. Soc. for Integrative & Comp. Biol. annual meeting, Dec. 26-30, 1996. Published in Amer. Zool. 36 (5), 1996.
#260. HOST SPECIFICITY IN THE NEPHROCYCES-MOLGULID SYMBIOSIS.
MB Saffo, Arizona State Univ. West, Phoenix, Arizona.
Nephromyces
is a heterotrophic protist inhabiting universally, and exclusively, molgulid
ascidians. Unusually among horizontally transmitted marine endosymbioses,
its host range is limited to a single taxon. Has Nephromyces co-evolved with molgulids? To supplement rDNA data, I investigated morphological variation and cross-infectivity in Nephromyces from several molgulids. Nephromyces cells do vary morphologically, even when different host species co-occur. In infectivity, Nephromyces shows strict host specificity. Lab-raised, aposymbiotic M. manhattensis, bred from Pacific (San Francisco Bay) populations and inoculated with Nephromyces from 3 other host species (M. citrina, M. occidentalis, and M. provisionalis), remained uninfected. Controls (inoculated with symbionts from SF Bay M. manhattensis) yielded 100% infection rates. Pacific M. manhattensis inoculated with Nephromyces from Atlantic (Cape Cod) M. manhattensis showed early infective cells after 2 weeks, but only sparse, deteriorated cells after 4 weeks, suggesting that Atlantic Nephromyces is only partially compatible with Pacific hosts. These data suggest genetic differentiation in Nephromyces, not only among different host species, but also among geographically isolated populations of a single host species.
#468. MAPPING REPRODUCTIVE CHARACTERS ONTO A MOLECULAR PHYLOGENY AND GEOGRAPHY OF THE BOTRYLLID ASCIDIANS.
S. Cohen, Y. Saito and I. Weissman, Stanford Univ., Calif. and Univ. of Tsukuba, Japan.
Botryllid ascidians show an intriguing diversity in reproductive and developmental
characters, including: the timing and locations of gamete maturation, location
of fertilization and development, length of embryonic development and degree
of maturation of larvae upon hatching, and number of eggs per zooid. Ancestral
states of these traits are determined by mapping them onto a molecular phylogeny
based on small ribosomal subunit nuclear DNA sequence data. The Botryllus clade is ancestral to the Botrylloides
group. There is a trend in the family towards increasing egg size, fewer
eggs per zooid, longer development time and larger larvae upon hatching.
Comparison of these traits with world-wide invasion patterns shows the highly
successful Botrylloides species run counter to predictions on reproductive traits conducive to species introductions.
#497. EVOLUTION OF THE CHORDATES: EXAMINATION OF DEVELOPMENTAL POTENTIAL IN UROCHORDATES.
BJ Swalla, Vanderbilt Univ., Nashville, TN.
There are 3 classes of urochordates or tunicates: thaliaceans, larvaceans
and ascidians. Although there is no fossil record for these organisms, ascidians
comprise by far the largest group with about 3000 described extant species,
allowing comparison of a wide variety of evolved body plans and life histories.
There have been several important vertebrate developmental genes described
recently which appear to have homologous functions during ascidian larval
development. The expression of these genes in ascidian embryos will be reviewed
and compared to the novel maternal genes manx, a zinc finger nuclear protein
and cymric, a nonreceptor tyrosine kinase. In addition, distalless, a gene
that is known to be expressed during appendage formation in a wide variety
of phyla, is shown to be expressed during ampullae and siphon formation in
ascidians. The implication of urochordate appendages which may contain secondary
axes will be discussed with respect to the evolution of paired appendages
in tetrapods.
#504. ELECTRICAL ACTIVITY REGULATES ION CHANNEL EXPRESSION AND CONTRACTILITY DURING DEVELOPMENT OF MUSCLE IN ASCIDIAN LARVAE.
JE Dallman and WJ Moody, Univ. of Washington, Seattle, WA.
Muscle cells of the ascidian, Boltenia villosa,
exhibit 2 distinct phases during differentiation. These phases are created
by expression of voltage-gated ionic currents. During the first phase, just
after neurulation, inward Ca and outward K currents (mediate action potentials)
are first expressed while the inwardly rectifying K current (sets the resting
potential) is transiently absent. An unstable resting potential leads to
spontaneous activity. During the second phase, the return of the inwardly
rectifying K current along with the addition of a Ca-dependent K current
terminates the spontaneous activity. To test the role of spontaneous activity
in muscle differentiation, we blocked activity during the first phase with
the Ca channel blocker Cd. When we compared treated and control cells at
maturity, the treated cells lacked the Ca-dependent K current, while all
other currents were normal. In addition, treated cells were less contractile
than control cells, even though their actin cytoskeleton was similar. These
results suggest that regulation of ion currents causes spontaneous activity
and Ca entry that is required for muscle differentiation.
3. Intl. Soc. of Differentiation 9th Intl. Conf., Pisa, Italy, Sept. 28-Oct. 2, 1996.
#61. VITELLOGENESIS IN ASCIDIANS. L. Manni, M. Della Barbera, G. Zaniolo, P. Burighel. Dipt. di Biol., Univ. di Padova, Italy.
The ovarian oocyte of ascidians possesses one of the most complex set
of envelopes known in the animal kingdom, being surrounded by test cells
encased in its surface, a fibrous vitelline coat, and inner and outer follicular
cells. The size of the oocytes and differentiation of the envelopes seem
to depend on the reproductive mode of the species, which may be oviparous,
ovoviviparous or viviparous. Oviparous species usually produce many small
oligolecithal eggs (about 150 µm), the ovoviviparous comparatively
few, large yolked eggs (up to 720 µm), and the viviparous ones few,
very small alecithal eggs (25 µm). Vitellogenesis was investigated
ultrastructurally in several species representative of the different modes
of reproduction. Two mechanisms of yolk synthesis are involved, according
to the different types of oocyte. In oligolecithal eggs, such as those of
Ciona intestinalis, yolk is accumulated by means of autosynthesis. At the
beginning of vitellogenesis, the oocyte shows signs of intense nucleus-cytoplasm
exchange and nuage, and has numerous mitochondria, annulate lamellae, RER
cisterns and Golgi fields. These organelles co-operate in the synthesis of
yolk, which accumulates in previtellogenic bodies to form progressively larger
and denser granules, mainly distributed in the central area of the oocyte.
Endocytosis is scarce and the few ovular microvilli establish only occasional
relations with the inner and outer follicular cells. In species with yolked
eggs, like Botryllus schlosseri, early autosynthesis is followed by heterosynthesis,
as shown by the progressively increasing features of endocytosis and exchange
between the oocyte and its environment. Tracers for electron microscopy also
show that molecules coming from the blood are endocytosed by oocytes. The
microvilli are long and polymorphic, and joined by means of gap junctions
to cytoplasmic protrusions of the outer follicular cells. The latter are
more columnar and richer in RER and Golgi fields than in oviparous species,
and seem to be involved in the production of proteins to be passed to the
oocyte. In viviparous species, such as Botrylloides violaceus, dense yolk
granules are absent, although the oocyte produces numerous apparently empty
previtellogenic bodies. Variations in the reproductive strategies of ascidians
therefore seem to involve variations in the mechanism of vitellogenesis and
envelope activities.
#221. DIFFERENTIATION OF THE NEURAL COMPLEX DURING ASEXUAL REPRODUCTION
IN TUNICATES. P. Burighel, G. Zaniolo, L. Manni, Dipt. di Biol., Univ. di
Padova, Italy.
In both the tunicates and the related vertebrates the embryonal neural
tube originates from an ectodermal neural plate. But in tunicates it becomes
completely rearranged when the larva metamorphoses in sessile filter-feeding
zooid. The latter finally possesses a neural complex formed of the cerebral
ganglion and the associated neural gland, a blind sac opened into the pharynx
through the ciliated duct and extended posteriorly with the dorsal strand.
In ascidians, the most studied tunicates, the cerebral ganglion has the unusual
capacity to regenerate completely in few weeks after ablation and the possible
contribution of cells from blood or dorsal strand to the process is under
investigation. Numerous ascidians form colonies which, as single clones,
are constituted of few to thousand genetically identical zooids, each one
derived by asexual reproduction and possessing its own neural complex. Origin
and differentiation of the neural complex was followed in the buds of Botryllus
schlosseri colonies cultured in the laboratory. The early bud derived from
a specialized area of the atrial tissue which folds to form a vesicle covered
by the epidermis. Primordia of atrial and branchial chamber and gut derive
through invaginations and extension of vesicle wall. A tube (neural primordium)
evaginates from dorsal area of the vesicle and extends anteriorly to meet
and open into the branchial chamber rudiment. Posteriorly, the original aperture
is then closed. A laminar roof of mesenchymal cells is interposed between
epidermis and the tube. These cells early show microtubules and cytoplasmic
extensions with scattered junctional areas and differentiate as neurons.
They move ventrally and, together with elements proliferating fom the ventro-lateral
wall of the tube, give rise to the anlage of the cerebral ganglion. In the
latter, cell bodies accumulate in a cortical layer around a fribrous medulla,
from which the peripheral nerves start. Early the neural tube differentiate
forming the anterior ciliated duct and the posterior sac whose wall is of
vacuolated polymorphic cells. Thus in B. schlosseri cerebral ganglion differentiates
early by contribution of elements from blood and neural gland primordium.
3. Italian Embryology Group 42nd annual meeting, Bressanone, June 6-8, 1996. Publ. in Anim. Biol. 5 (2), 1996.
NEURAL COMPLEX DEVELOPMENT IN BOTRYLLUS SCHLOSSERI BUDS. P Burighel, L Manni, & G. Zaniolo, Dipt. di Biol., Univ. di Padova
The neural complex of the ascidian B. schlosseri
(Stolidobranchia) consists of the cerebral ganglion and the dorsally associated
neural gland opening in the prebranchial region through the ciliated duct.
At first, in the oozooid, the nervous complex is formed at metamorphosis,
starting from a primordium to the left of the cerebral vesicle of the motile
tadpole larva; but in every blastozooid it is newly formed. By light and
electron microscopy, we analysed the origin and development of the neural
complex taking the B. schlosseri bud as a model. Colonies of this
ascidian were cultured in the laboratory on glass, and pieces of them were
cut off at opportune stages and processed for study. We refer to the stages
of bud development as proposed by Sabbadin (1955, Boll. Zool. 22:243-263).
Botryllus buds are palleal and originate from two symmetrical regions
of the parental mantle. They soon form a vesicle covered by epidermis with
blood circulating in the interspace. Organ primordia derive mainly from the
inner vesicle, by infoldings of its wall. Early nervous primordium occurs
as tubular evagination from the prospective cloacal region and a mesenchymal
lamina, probably of blood cells, insinuates itself between it and the epidermis.
Neural evagination extends forwards as a dorsal tube which contacts and opens
in the prospective branchial chamber. At the same time its original posterior
aperture closes. The dorsal tube represents the primordium of the neural
gland. In the mesenchymal band, cells begin to differentiate as neurons,
extending cytoplasmic protrusions rich in microtubules and forming junctional
areas. These cells move ventrally to the neural gland to form the anlage
of the cerebral ganglion. However, morphological evidence was obtained to
show that cells proliferating from the ventral and lateral sides of the dorsal
tube also make important contributions to cerebral ganglion formation. When
the bud heart begins beating and the primordia of the branchial stigmata
become recognizable, the neural complex appears in its definitive configuration.
The neural gland is a blind sac with a well-ciliated duct and extends as
a very short dorsal strand. The neurons are arranged peripherally to the
fibrous medulla and form the cerebral ganglion, which sends its nerves at
organ primordia in the various body regions. In conclusion, our observations
show that the neural gland originates as a posterior evagination of the inner
vesicle and that material from this anlage also forms the cerebral ganglion,
with the possible, early contribution of cells from the blood.
DIFFERENTIATION OF TEST CELLS AND VITELLINE COAT DURING OOGENESIS IN THE COLONIAL ASCIDIAN CLAVELINA LEPADIFORMIS. G Zaniolo, M Della Barbara & P Burighel, Dipt. di Biol., Univ. di Padova.
Many studies have dealt with the origin and differentiation of oocytes
and their envelopes in ascidians and, in particular, with the origin and
roles of test cells and the vitelline coat. Test cells differentiate in
close association with oocytes and, although several functions have been
attributed to them during oogenesis, none has ever definitely been demonstrated.
Strong evidence has recently been reported that test cells synthetize and
secrete thin structures called "ornaments" which, during embryo development,
are deposited on the larval tunic, and make it hydrophilic. (Cloney 1996,
Acta Zool. 17:73-78). Test cells are externally covered by the vitelline
coat, the features of which differ among species and which contains the sites
for gamete recognition. Its origin from oocytes has long been debated.
We have now studied the appearance and differentiation of test cells and
the vitelline coat in Clavelina lepadiformis by light and electron microscopy. C. lepadiformis
is an ovoviviparous ascidian, whose branching tubular ovary is continuous
with the single oviduct which opens into the atrial chamber. The ovarian
epithelium is composed of a single layer of cells, joined apico-laterally
by tight junctions and covered by a thin basal lamina towards the stroma.
Early oocytes probably derive from blood cells which segregate within the
ovarian epithelium after crossing the basal lamina. Unlike the contiguous
epithelial cells, they do not establish tight junctions with any type of
cell. They first become surrounded by one layer of primary follicular cells
derived from and continuous with the ovarian epithelium. Later, several dark
and undifferentiated cells insinuate themselves between the primary follicle
cells and the oolemma, and gradually form a discontinuous inner layer. In
the following stage the acellular vitelline coat becomes recognizable as
a homogeneus, fibrous material externally covering several scattered dark
cells which become encased in indentations in the oolemma and represent the
test cells. At this stage, the oocyte possesses many small vesicles of RER
filled with filamentous electron-dense material, several of which closely
adhere to the oolemma. At the beginning of vitellogenesis all the egg envelopes
are well developed (outer and inner follicular cells, vitelline coat and
test cells). In particular, the test cells have giant Golgi with more than
20 piled cisternae with budding vesicles at their borders. In a later phase,
the test cells contain a great number of round secretory granules filled
with homogeneous and/or fibrous content. These granules can fuse one another;
several of them release their fibrous content which intermingles with and
is added to the vitelline coat. At the same time, the vitelline coat assumes
a three-layered aspect, with the middle (central) layer more compact and
dense than the other two; in particular, the inner layer, facing the oolemma,
has a net-like structure, resembling that of the secreted material of the
test cells. Our results suggest that the vitelline coat is built up with
the contribution of oocyte vesicle contents, to which, secretions from test
cells are then added. Nevertheless, because of the large number of test cell
granules at ovulation, it is also possible that, during embryogenesis, the
test cells of C. lepadiformis, like those of other ascidians, secrete hydrophilic ornaments on the larval tunic.
PHAGOCYTE DIFFERENTIATION AND APOPTOSIS IN THE COLONIAL ASCIDIAN BOTRYLLUS SCHLOSSERI. L Ballarin, F Cima & A Sabbadin, Dipt. Biologia, Univ. di Padova, Italy.
Three blastogenetic generations are usually present in colonies of the ascidian Botryllus schlosseri,
namely adult zooids, buds and budlets. At 19 °C adult zooids are weekly
resorbed and replaced by mature buds during the regression phase of the colonial
life cycle. This period is characterised by an intense phagocytosis and a
significant increase in the frequency of circulating macrophage-like cells
is always coupled with a significant decrease in the frequency of circulating
amoebocytes. Hyaline amoebocytes and uni- or multi-vacuolated macrophage-like
cells are the two hemocyte types involved in phagocytosis. Our previous studies
suggest that they represent two functional stages of a single cell type,
the active phagocyting stage being the hyaline amoebocyte which withdraws
its cytoplasmic projections and changes to macrophage-like morphology upon
ingestion of foreign particles. The hypothesis is supported by in vitro
experiments, using short term hemocyte cultures, as test particles appear
inside hyaline amoebocytes after 5 min of incubation whereas they are visible
inside macrophage-like cells after 30 min of incubation. During the resorption
of old adult zooids circulating hemocytes containing apoptotic cells inside
their vacuoles are easily seen. Therefore phagocytes, in a certain stage
of their differentiation pathway must express surface molecules involved
in the recognition of senescent cells. In mammals recognition of apoptotic
cells by circulating macrophages is often mediated by thrombospondin, secreted
by macrophages, which forms bridges between the membrane of senescent cells
and both the receptor for vitronectin and the 88 kD monomer CD36 on macrophage
surface. In order to verify whether a similar mechanism of apoptotic cell
recognition is present also in ascidians, we tested the monoclonal antibody
OKM5, anti CD36, on monolayer of B. schlosseri hemocytes. The results
point to the presence of molecules recognized by OKM5 on the surface of hyaline
amoebocytes. This indicates that hyaline amoebocytes are directly involved
in the recognition of cells undergoing apoptosis thus confirming our hypothesis
that they represent the actively phagocyting cells and suggests that a common
mechanism of apoptotic cell recognition, involving thrombospondin and its
receptors, has been maintained throughout the evolution of chordates.
A MORPHOLOGICAL STUDY OF THE PERICARDIAL BODY IN THE ASCIDIAN CIONA INTESTINALIS.
S Scippa, C Izzo and M de Vincentiis, Dipt. di Genetica, Biol. Gen. e Molec.,
Fac. di Sci. dell'Univ. di Napoli e Stazione Zool. di Napoli.
The
pericardial body is a globular structure present in the pericardial cavity
of some ascidian species. This structure shows granular and large cells (Roule
1884, Ann. mus. hist. nat. Marseille Zoologia Tome II), degenerating cardiac
fibres embedded in an amorphous matrix also containing some hemocytes (Millar
1953. Memoirs on typical British Marine Plants & Animals 35: 1-123),
and degenerating lymphocytes in the middle of the aggregation (Kalk 1970.
Tissue & Cell 2: 99-118). The presence of Cardiosporidium Cionae has
been also reported (Van Gaver and Stephan 1907, C.R.Soc.Biol.Paris 62:556-557).
The present note is a morphological study of the pericardial body of Ciona
intestinalis at the light and electron microscope ((TEM). Cytochemical investigation
at the light microscope revealed the presence of acid and neutral mucopolysaccharides
and collagen fibres. At the electron microscope, numerous blood cells, as
stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes
and globular granulocytes, were found at the periphery of the small-sized
pericardial bodies. The amorphous matrix prevailingly contained degenerating
cells and cardiac fibres. Large cells could be distinguished; they were almost
completely occupied by one or more vacuoles with a generally homogeneous
and strongly electron-dense content. Moreover, at TEM, the cells that, at
the light microscope, appeared as granular cells showed a peculiar ultrastructure.
We observed a sporogonium of a microsporidium containing numerous sporoblasts,
i.e. nucleated cells which will form the lasting spores. The nuclei of these
cells contained electron-opaque zones, probably richer in chromatin. A double
membrane, or pansporoblast, enveloped the plasmodium. The central portion
of the plasmodium showed tubular structures resembling the polar filaments
typical of microsporidium spores. To conclude, the pericardial body of C.intestinalis
is a mass of amorphous material containing degenerating elements surrounded
by numerous blood cells in small-sized pericardial bodies; it is characterised
by the presence of a microspiridium, irrespective of the size. As far as
the formation of the pericardial body is concerned, we suggest that Ciona
might be infected by the sporozoon; the latter would stimulate a reaction
in the blood cells involved in defence mechanisms, which would start producing
the amorphous substance. Alternating contractions of the heart would make
the detachment of fibres and endothelial cells easier due to rubbing of the
heart wall with the pericardial wall.
COMPARATIVE AND EXPERIMENTAL ANALYSES OF THE ROLE OF EXTRAEMBRYONIC
TEST CELLS IN THE DEVELOPMENT OF ASCIDIAN LARVAE. R Cloney, Dept. of Zool.,
Univ. of Washington, Seattle, WA.
During early oogenesis in solitary ascidians each ovarian oocyte becomes
enclosed within extensions of the ovarian epithelium known as the outer follicular
epithelium. Gradually, accessory cells of maternal origin (probably hemoblasts)
are recruited into the compartment around each oocyte. A vitelline coat
is secreted around each oocyte. Some accessory cells, outside the vitelline
coat, differentiate as inner follicular cells. Others, beneath the vitelline
coat, in close contact with the oocyte, become test cells. The function
of the test cells has been an enigma since the middle of the 19th century.
The popular assumption that they transfer substances into oocytes has not
been substantiated. In many ascidians the test cells synthesize and store
submicroscopic particles or filaments (called ornaments) in vacuoles. Seven
types have been identified in 8 families of ascidians (Cloney 1994). Many
species with "multigranular ornaments" contain silica (Monniot et al.1992a).
In species with ornaments the test cells deposit them on the outer cuticular
layer of the larval tunic (C1) before hatching. The presence of these ornaments
has been invaluable in tracing the secretions of test cells by TEM. The
test cells of some species (ascidiids and Ciona intestinalis) do not
synthesize ornaments. Instead, they attach themselves firmly to C1. The
test cells of nearly all species are metachromatic. Their vacuoles contain
acidic (usually sulfated) glycosaminoglycans (Monniot et al.1992b)
or, based upon recent analyses, sulfated polysaccharides (M. Pavao, pers.
comm.). I infer that the secretions of test cells are negatively charged
regardless of the presence or absence of ornaments. What happens if ascidian
larvae are deprived of test cells or their secretions before hatching? Embryos
that develop normally and hatch in the presence of test cells (controls)
are always hydrophilic and never become trapped at the surface of SW. When
demembranated neurula are cultured alone or in small numbers in 10 ml of
SW (without test cells, follicle cells or the vitelline coat), C1 does not
form fins; the larvae are sticky, hydrophobic and cannot swim. But when
demembranated neurulae are cultured in large groups (30-80) in 1 ml of SW,
many form fins with normal morphology. They are not sticky, they swim normally,
but are also hydrophobic, as demonstrated with 7 solitary species (Cloney
1990; Cloney & Hansson 1996). These larvae easily become trapped at the
SW surface and cannot reenter. The test cells normally deposit a negatively
charged hydrophilic "finishing coat" on the larval tunic. The significance
of the diverse types of ornaments and silica (when present) is unknown.
In Molgula pacifica, there is no swimming larva and no outer cuticular
layer (C1); development is direct and test cells are completely absent (Cloney,
1995). Apparently, the test cells were eliminated during the evolution of
this species when they had no significant function. (Refs: Cloney, R 1990.
Acta Zool. 71:151-159; Cloney, R 1994. pp. 77-95 in Reproduction & Development
of Marine Invertebrates. Wilson, Stricker, & Shinn, eds., Johns Hopkins
Univ. Press; Cloney, R 1995. Acta Zool.76: 89-104; Cloney, R & Hansson,
LJ, 1996. Acta Zool. 77:73-78; Monniot, F et al.1992a. Mar. Biol. 112:283-292; Monniot, F et al.1992b. Biochem. Systematics & Ecol. 20:541-552.)
ON THE SENSORIAL FUNCTION OF ASCIDIA MALACA ADHESIVE PAPILLAE.
M Gianguzza*, G Dolcemascolo*, U Fascio° & F De Bernardi°. *Inst.
of Biol., Univ. of Palermo; °Dept. of Biol., Univ. of Milano, Italy.
At the anterior end of the cephalenteron the swimming larvae of Ascidia malaca
have 3 papillae that secrete an adhesive substance which allows the larvae
to attach. Observations at the light microscope level reveal that papillae
are cone-shaped, are arranged at the vertices of a triangular field and their
anterior part ends in a hyaline cap. Ultrastructural (TEM) observations evidenced
also, in the apical part of the hyaline cap, a mass of fine granular moderately
electron-dense material which probably corresponds to the adhesive substance
secreted by the papillae. Cells forming the papilla body are ovoid and elongated
and can be distinguished ultrastructurally as types "A" and "B". A cells,
whose ultrastructure is characteristic of secretory cells, are considered
to produce the adhesive substance. Their cytoplasm presents a well developed
RER, a Golgi in active synthesis phase and numerous granules in the apical
region which undergo ultrastructural modifications; the material resulting
from this process is later poured into the matrix of the hyaline cap (Gianguzza
& Dolcemascolo, Eur. Arch. Biol. 105:51-62, 1994). Type B cells are ovoid
and elongated and their main feature is the presence of several microvilli
and a single cilium contained in a sort of "pocket" of the cytoplasmic membrane.
Microvilli originate at the basis of the cell membrane, stretch all along
the hyaline cap and sometimes come out of the same cap. Another characteristic
of B cells is the long bundles of microtubules running parallel to the major
cell axis; their presence was visualized through the use of tannic acid during
fixation (cf. Hayat, "Stains and Cytochemical Methods" Plenum , 1993). The
ultrastructure of B cells and, above all, the presence of cilia and microvilli
let us hypothesize that the papillae somehow possess also a sensorial function
which allows them to perceive environmental stimuli. This hypothesis was
confirmed by confocal microscopy using antitubulin fluorescent antibodies.
It was possible to evidence, in the larval cephalenteron, a wide network
of innervations connecting the cerebral vesicle to the adhesive papillae.
The ultrastructure of nervous fibres connecting the cerebral vesicle to
the papillae could be seen with TEM. Ultrathin sections evidenced long bundles
of microtubules running parallel to the axis of the fibre itself. Papillae
being innervated confirm the hypothesis that they could exercise also a sensorial
function through activity of B cells, which may perceive external environmental
stimuli through their long microvilli and the cilium. The perception of these
stimuli would create a signal causing A cells to secrete the adhesive substance
allowing the larvae to attach to a substratum.
THE ADHESIVE PAPILLAE OF THE SOLITARY ASCIDIANS. F. De Bernardi,
U. Fascio, S. Gropelli & C. Sotgia. Dept. of Biology, Univ.of Milano.
The swimming larvae of the solitary ascidians belonging to the Ascididae
family bear 3 anterior, simple, coniform adhesive papillae arranged at the
vertices of a isosceles triangular field. They secrete adhesives that are
used to effect a transitory settlement at the beginning of metamorphosis.
They are formed by the anterior epidermal cells and are induced together
with the nervous system by the A 4.1 cells and by their descendants. In the
method of both induction and differentiation the adhesive papillae may be
considered homologous to the cement gland of the Amphibia Anura. The adhesive
papillae of newly hatched Phallusia mamillata
larvae examined at the SEM appear covered by the tunic. About 1-2 hours after
hatching the tunic becomes fenestrated over the central part of the papillae
and numerous bulb-ended microvilli protrude throughout the holes. The ultrastructure
of this type of papillae as described for Ascidia malaca (Gianguzza
& Dolcemascolo 1994, Eur. Arch. Biol. 105: 51-62) revealed two types
of elongated cells: peripheral secretory-type cells and central cells with
micovilli and bundles of microtubules along the major axis of the cells,
probably with sensorial function. We performed immunofluorescence experiments
with an anti-(-tubulin monoclonal antibody (clone 2-28-33; Crowther &
Whittaker 1990, J. Neurobiol. 23:280-292) specifically reacting with axonal
microtubules. Only the central sensory cells of the papillae were stained
and the axons appeared to arise from the proximal end of these cells. These
axons form a long nerve reaching the brain vesicle. Branches of the same
nerve appear to connect the basal end of the peripheral secretory cells.
By the confocal laser microscope we were able to follow the position of the
papillary nerve both in Phallusia mamillata and in Ascidia malaca.
A dorsal nerve connects the two dorsal papillae to a dorsal position of the
brain vesicle. Another nerve connects the ventral papilla to a more ventral
position of the brain vesicle. Dorsal papillae are also reached by axons
coming from a distal position of the nerve and vice-versa. The central cells
of the adhesive papillae could be primary sensory neurons and they may have
both chemosensory and mechano-sensory function. The presence of two papillary
nerves may be related to a heterochronic development of the thirds papilla.
ACTIVATION OF ASCIDIAN EGGS INVOLVES TWO SEPARATE CALCIUM DEPENDENT PATHWAYS.
C. Lambert1,3, G. Lambert1,3, A. Mc Dougall4, L. Robert1, L. Lucio1, and
C. Goode2 . Depts. Biol.1 and Chem/Biochem2 , Calif. State Univ., Fullerton,
CA 92834-6850; Sta. Biol., Pl. Georges Teissier, Roscoff, France3 ; Sta.
Zool., Villefranche-sur-Mer, France4.
At fertilization ascidian eggs release a membrane-linked N-acetylglucosaminidase
which is a principal block to polyspermy (Lambert 1989, Development 105:415-420)
and also undergo cortical contractions which redistribute specific territories
in the zygote (Sardet et al., 1989, Development 105: 237-250). We have examined
both events in , Ascidia ceratodes eggs in California and Phallusia mammillata
eggs in France. We use the fluorogenic substrate 4-methylumbelliferyl N-acetylglucosaminide
to assay enzyme activity released and light microscopy to determine the cortical
contractions. Both processes are inhibited by BAPTA-AM and thus involve
increase in internal calcium concentrations. Glycosidase release requires
extracellular calcium but the contractions do not when stimulated with ionomycin.
We have detected the increase in internal calcium that precedes the cortical
contractions in calcium green-loaded Phallusia mammillata eggs in
the confocal microscope. We have thus far been unable to detect any change
in intracellular calcium when we activate the eggs with ryanodine, which
results in glycosidase release without the contractions ( Lambert et al.,
1994, Growth Develop. & Differ. 36, 133-139). The glycosidase is released
seconds after insemination but the contractions occur several minutes later.
The calcium ionophore ionomycin induces cortical contractions within a minute
in contrast to the 4.5 minutes required after insemination. This suggests
that a multi-step pathway involving calcium is involved in the contractions
but that the glycosidase release pathway is more direct (McDougall et al.,
1995, Zygote 3: 251-258). The tyrosine kinase activator dimethylbenzanthracene
(DMBA, Archuleta et al. 1993, Proc. Natl. Acad. Sci. 90, 6105-6109) causes
glycosidase release without the contractions at 20(M. This release can be
inhibited by the tyrosine kinase inhibitors genistein at 10(M and tyrphostin
A-23 at 200(M. The tyrphostin blocks fertilization-induced glycosidase release
without affecting sperm motility but genistein does not.. This is to be
expected as genistein and tyrphostins affect different parts of the tyrosine
kinase pathway (Robert et al. 1995, Mol. Biol of the Cell 6: 431a). Incubation
of intact ascidian eggs in sea water containing the fluorogenic PLC substrate
4-methylumbelliferyl-phosphocholine discloses that they have a cell surface
phospholipase C (PLC) which may be involved in release of the glycosidase.
PLC activity is increased by DMBA, and we have detected on western blots
an electrophoretic band that is phosphorylated in activated eggs but not
phosphorylated in tyrphostin-treated eggs. Possibly this cell surface phospholipase
is activated by tyrosine phosphorylation which then releases the lipid linked
glycosidase. The cortical contractions would then be activated by an internal
phospholipase which could generate inositol-tris-phosphate to release calcium
from the endoplasmic reticulum (Dale 1988, Exp. Cell. Res. 177: 205-211).
Thus activation of the ascidian egg involves two calcium dependent processes
which may be under independent controls. The glycosidase release may have
tyrosine kinase involved in its activation but the cortical contractions
are initiated by a different trigger.
4. Zool. Soc. of Japan 67th annual meeting, 1996. (Publ. in Zool. Sci. ;some abstracts were edited slightly to conserve space.)
INVOLVEMENT OF PROTEASOME AND SPERMOSIN IN FERTILIZATION OF THE ASCIDIAN, CIONA INTESTINALIS
H. Sawada1, M. Rosaria Pinto2, and R. De Santis2. 1Dept. Biochem., Fac. Pharm.
Sci., Hokkaido Univ., Sapporo; 2Dept. Cell Dev. Biol., Stazione Zoologica
'Anton Dohrn', Naples, Italy.
It is currently proposed that sperm trypsin-like proteases (spermosin
and acrosin) and proteasomes (20S and 930-kDa) are involved in fertilization
of the stolidobranch ascidian, Halocynthia roretzi. However, whether or not spermosin and proteasomes are involved in fertilization of the phlebobranch ascidian, Ciona intestinalis,
is not yet known. Here, we found that two proteasome inhibitors (MG115 [Z-Leu-Leu-Nva-H]
and MG132 [Z-Leu-Leu-Leu-H]) and two spermosin inhibitors (Z-Val-Pro-Arg-H
and Dns-Pro-Arg-H), but neither acrosin inhibitor (leupeptin) nor cysteine
protease inhibitor (E-64-d), inhibited the fertilization of C. intestinalis
in a concentration-dependent manner. These results indicate that spermosin
and proteasome may play a key role also in fertilization of the eggs of C. intestinalis.
THE ROLE OF INOSITOL 1,4,5-TRISPHOSPHATE-INDUCED CALCIUM RELEASE IN ASCIDIAN EGGS AFTER FERTILIZATION
M. Yoshida1, R. Deguchi2, M. Morisawa2, K. Mikoshiba134. 1Mol. Neurobiol.
Lab., Tsukuba Life Science Center, Inst. of Phys. and Chem. Res., Tsukuba,
2Misaki Mar. Biol. Stn., School of Sci., Univ. of Tokyo, Miura, 3Dept.
of Mol. Neurobiol., Inst. of Med. Sci., Univ. of Tokyo, Tokyo, and 4Mikoshiba
Calciosignal Net Project, ERATO, JRDC, Tokyo.
In ascidian eggs, fertilization initiates a sequence of events including
exit from meiosis, rearrangement of cortical and cytoplasmic domains with
egg shape modification (ooplasmic segregation), first and second polar body
extrusion, etc., and the elevation of intracellular Ca2+ is thought to initiate
these processes. In the ascidian Ciona savignyi,
transient Ca2+ wave just after fertilization and following repetitive Ca2+
oscillation are observed. In somatic cells, there are two mechanisms of
the elevation of intracellular Ca2+; inositol 1,4,5-trisphosphate (IP3) -induced
Ca2+ release (IICR) and Ca2+ -induced Ca2+ release (CICR). To study the
role of IICR and CICR upon egg activation, we first examined the presence
of IP3 receptor (IP3R), which mediated IICR, in eggs of ascidians. Monoclonal
antibodies (mAb) against the type I IP3R recognized a protein band in C. savigyi
membrane preparations by Western blots. Then we investigated the role of
IICR by determining the effects of microinjected mAb 18A10, which binds to
IP3R and inhibits IICR. The antibody did not inhibit the Ca2+ wave and
egg shape modification, but inhibited the Ca2+ oscillation and 1st cell
division. It indicates that IICR is essential for ascidian egg activation.
CALCIUM DYNAMICS DURING FERTILIZATION AND MATURATION DIVISION IN THE ASCIDIAN, CIONA INTESTINALIS.
K. Kyozuka1, G.L. Lusso2, E. Tosti2 and B. Dale2. 1Asamushi Mar. Biol. Stn.,
Tohoku Univ, Aomori and 2Div. of Cell Biol., Stazione Zoologica 'Anton Dohrn',
Naples, Italy.
Changes in [Ca2+]i during fertilization and meiosis in Ciona intestinalis
oocytes were measured using the fluorescent calcium indicator. An initial
large transient increase of [Ca2+]i followed by periodic changes of [Ca2+]i
took place during egg contraction and first meiotic division. After the
first polar body extrusion, Ca2+ oscillations took place again during the
second meiotic division. Histone H1 kinase activity was high at metaphase
I and metaphase II of meiosis. When heparin-injected oocytes were fertilized,
the initial increase of [Ca2+]i occurred, however, subsequent Ca2+ oscillations
were suppressed. Microinjection of BAPTA into the oocyte at telophase I
of meiosis inhibited both the Ca2+ oscillations and the increase of histone
H1 kinase activity at metaphase II. Protrusion of the second polar body
was also inhibited. These results indicate that activity of metaphase promoting
factor, estimated from the activity of histone H1 kinase, is regulated by
[Ca2+]i. Periodic changes of [Ca2+]i are necessary for the egg contraction
and protrusion of polar bodies during meiosis.
DISTRIBUTION OF MITOCHONDRIAL LARGE rRNA IN EARLY ASCIDIAN EMBRYOS
T. Oka1, R. Amikura2, S. Kobayasi2, H.Nishida1. 1Dept.of Life Sci., Tokyo Inst. Tech.,
Yokohama; 2Inst. Biol.Sci., Univ. of Tsukuba, Tsukuba.
Mitochondorial large ribosomal RNA(mtlrRNA) had been identified as a cytoplasmic
factor that is involved in poll cell formation in Drosophila embryos. mtlrRNA
is a component of germ plasm, and transported out of mitochondria. We examined
the distribution of mtlrRNA in ascidian embryos by in situ hybridization.
Different distribution between mitochondria and mtlrRNA was observed in the
posterior-vegetal blastomeres at the 8-cell stage.
EXPRESSION ANALYSIS OF ENDODERM-SPECIFIC ALKALINE PHOSPHATASE GENE IN ASCIDIAN EMBRYOS.
G. Kumano and H. Nishida. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
In the previous meeting, we reported that we had purified endoderm-specific
alkaline phosphatase and determined its N-terminal amino acid sequence in
larvae of the ascidian, Halocynthia roretzi.
In this meeting, we present the results of molecular cloning of the enzyme
and the analysis of its temporal and spacial expression during embryogenesis.
We sequenced the 3024-bp long cDNA clone of alkaline phosphatase, containing
the complete open reading frame and polyadenylic acid. The expression of
mRNA is observed in endoderm cells at tailbud stage.
THE RAS SIGNALING PATHWAY IS ESSENTIAL FOR NOTOCHORD INDUCTION IN ASCIDIAN EMBRYOS.
Y. Nakatani1, H. Koide2, Y. Kaziro2 and H. Nishida1. 1Dept. of Life Sci.
and 2Dept. of Biol. Sci., Tokyo Inst. Technol., Yokohama.
bFGF can induce notochord differentiation in ascidian embryos. Generally,
bFGF signals are transmitted via membrane-bound receptor tyrosine kinases
(RTKs). Ras is a small GTP-binding protein and play an important role in
RTK signaling. Microinjection of dominant negative Ras (RasN17) protein
into fertilized eggs blocked notochord differentiation. When the notochord
precursor blastomere was co-isolated with the inducer blastomere and then
RasN17 protein was microinjected, notochord formation was suppressed. The
notochord precursor blastomeres microinjected with RasN17 protein were treated
with bFGF. Many of them failed to develop notochord features. These results
suggested that the Ras signaling pathway is involved in notochord induction
during ascidian embryogenesis.
TERMINAL FATE OF THE TRUNK LATERAL CELLS OF ASCIDIAN LARVAE.
T.Hirano and H.Nishida. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
To study the origin of adult tissues of the ascidian Halocynthia roretzi,
we traced the cell fates by intracellular injection of horseradish peroxidase
(HRP) into identified blastomeres at 110-cell stage. Descendants of the injected
cells were histochemically detected at juvenile stage. We have reported that
blood cells of juveniles are derived from trunk lateral cells of the larvae.
Further detailed analysis revealed that trunk lateral cells give rise to
body-wall muscle cells (in an oral siphon and a longitudinal mantle) and
cells around 1st and 2nd gill slits, as well as blood cells.
ULTRASTRUCTURE OF THE CENTROSOME ATTRACTING BODY (CAB) THAT IS INVOLVED IN UNEQUAL CLEAVAGE IN ASCIDIAN EMBRYO
T.Iseto and H.Nishida. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
CAB is a structure that exists in the cortex of the posterior blastomere
which shows successive unequal cleavage in ascidian embryos. It has been
suggested that CAB attracts the centrosome and the nucleus with microtubules,
thus it causes unequal cleavage. We observed CAB with transmission EM.
CAB is a homogeneous electron-dense region that directly lines the plasma
membrane. Some microvilli are observed in the surface area of the blastomere
where CAB is just beneath the membrane.
INFLUENCE OF VEGETAL CELLS ON THE ANTEROPOSTERIOR PATTERNING OF THE NEURAL TUBE AND EPIDERMIS OF THE ASCIDIAN EMBRYO
S. Wada, Y. Ueno and H. Saiga. Dept. Biol., Fac. Sci., Tokyo Metropolitan Univ.
Previously we reported that homeobox genes, Hrdll-1, Hroth, HrHox-1 and Hrcad-1 of the ascidian Halocynthia roretzi
are expressed in the neural tube and epidermis of the embryo with a distinct
expression domain. The expression domains of these genes align in the order
as described above along the anteroposterior axis, showing the presence
of the anteroposterior patterning in these tissues. To understand the mechanism
of anteroposterior patterning, we examined the involvement of vegetal cells
in this patterning mechanism by cell ablation experiments. Embryos in which
various sets of vegetal blastomeres were ablated using a sharpened needle
at early cleavage stages were cultured until the middle tailbud stage and
then gene expression was examined by whole mount in situ hybridization.
We have found that expression pattern of above genes in the ectodermal
tissues is severely disrupted in the operated embryos. This suggests that
in the ascidian embryo, influence of vegetal cells plays an important role
in patterning of the neural tube and epidermis along the anteroposterior
axis.
ISOLATION AND ANALYS OF A cDNA CLONE CONSPECIFIC TO TEST CELLS IN CIONA INTESTINALIS.
K. Takamura, S. Ooyabu, Y. Yamauchi, Y. Inoue, M. Hashimoto, N. Ise and Y.
Yamaguchi. Div. Biotech., Fac. of Engineering, Fukuyama Univ.
We isolated a cDNA clone for an antigen recognized by UA165 monoclonal antibody which was specific to test cells of C. intestinalis.
This cDNA clone was about 600bp in length and showed high homology to sequence
coding C`-half region of calreticulin of other animals. However, its 3`-region
was very different from that of other animals. We also isolated another
type of calreticulin homologue, which showed higher homology, by 3`-RACE
method. These results suggest that C. intestinalis has at least two
types of calreticulin homologue and we think that they seem to be different
in their localization and function. Now, we try to isolate full-length cDNAs
and analyze by in situ hybridization and Nothern blot analysis.
TWO CIS-REGULATORY ELEMENTS ARE ESSENTIAL FOR THE MUSCLE-SPECIFIC EXPRESSION OF THE ACTIN GENE IN THE ASCIDIAN EMBRYO
Y. Satou and N. Satoh. Dept. Zool., Grad. Sch. of Sci., Kyoto Univ.
Muscle cells in the anterior and middle part of the ascidian larva tail differentiate
in a highly autonomous manner. Because this autonomy is due to so-called
muscle determinants in the myoplasm of fertilized eggs, there is a genetic
cascade for muscle cell differentiation in ascidian embryos, which begins
with muscle determinants and ends with the expression of muscle-specific
structural genes. We approached the molecular nature of the muscle determinants
going upstream of the cascade by analyzing transcriptional control mechanisms
of muscle-specific structural genes. The HrMA4a actin gene, a muscle specific
structural gene, has been analyzed for this purpose. It has been demonstrated
that a short upstream sequence up to -103 base pairs from the transcription
start site is sufficient for the muscle specific expression of HrMA4a. We
performed a more detailed analysis using the beta-galactosidase genes as
a reporter. We demonstrated here that, within the proximal region (-103
~ -66) of HrMA4a gene, there are two short sequences essential to the muscle
specificity of HrMA4a promotor, one is 9-bp long (5`-TCGCACTTC-3`) and the
other is 13-bp long (5`-GTGATAACAACTG-3`).
EXPRESSION OF ENDOSTYLE-SPECIFIC GENES IN THE ASCIDIAN HALOCYNTHIA RORETZI
M. Ogasawara1, K. J. Tanaka2, K. W. Makabe1, N. Satoh1. 1Dept. Zool., Grad.
Sch. of Sci., Kyoto Univ., and 2Dept. of Biol., Fac. of Sci., Konan Univ.,
Kobe.
Notochord, nerve cord, pharyngeal gill and endostyle are structures key
to understanding of the molecular mechanisms underlying the origin and evolution
of chordates. We made an ascidian endostyle cDNA library and isolated two
endostyle-specific genes, HrEnds1 and HrEnds2 by differential screening of
the library. Predicted amino acid sequences of these genes suggested that
both genes encode novel secreted proteins. Transcripts of these genes were
detected in the endostyle of 1-month young adult under our in situ hybridization
condition. These genes may serve as probes for further analyses of molecular
mechanisms involved in formation of the endostyle.
ISOLATION OF PHARYNGEAL GILL-SPECIFIC GENES IN THE ASCIDIAN HALOCYNTHIA RORETZI.
K. J. Tanaka1, M. Ogasawara2, K. W. Makabe2, N. Satoh2. 1.Dept. of Biol.,
Fac. of Sci., Konan Univ., Kobe and 2.Dept. of Zool., Grad. Sch. of Sci.,
Kyoto Univ.
Pharyngeal gill-slit, endostyle and notochord are key
structures to understanding mechanisms underlying the origin and evolution
of chordate. We made an ascidian pharyngeal gill cDNA library and isolated
cDNA clones for two pharyngeal gill-specific genes (HrPhG1 and HrPhG2) by
differential screening of the library. Transcripts of these genes were detected
in pharyngeal gill wall a few days after metamorphosis. This expression pattern
was retained to adult stage. These genes may serve as useful probes for further
analysis of molecular mechanisms involved in the formation of the pharyngeal
gill in chordates.
CLONING OF Y-BOX PROTEIN GENE FROM THE ASCIDIAN, CIONA INTESTINALIS.
M. R. Wada, N. Tanimoto, Y. Ohtani and T. Nishikata. Dept. of Biol., Fac. of Sci., Konan Univ., Kobe.
Maternal
mRNAs localized in the egg cytoplasm are very important to early embryogenesis.
The cytoplasmic determinant which plays a key role in the differentiation
mechanism in the ascidian egg are suggested to be an mRNA. Recently, the
Y-box proteins in the Xenopus oocyte were proved to be RNA-binding
proteins which prevent the RNA from translation. Therefore, we isolated the
cDNA of the Y-box protein homolog from Ciona intestinalis gonads.
This Y-box protein in the ascidian egg might be a good clue to identify
the molecular nature of the cytoplasmic determinant.
INHIBITION OF HATCHING IN THE ASCIDIAN HALOCYNTHIA RORETZI BY NARAIN ISOLATED FROM A MARINE SPONGE JAPSIS SP.
H.Kato, K. Matsumura, M. Nagano, S. Tsukamoto, H. Hirota, and N. Fusetani. Biofouling Project, ERATO, JRDC, Yokohama.
Enzymes involved in hatching of ascidians have not been well-studied.
Previously we reported that narain isolated from a marine sponge Japsis sp. inhibited hatching in the ascidian H. roretzi
at a concentration of 10 uM. Narain did not inhibit the enzme reaction of
commercially available trypsin and crude hatching enzyme, but inhibited dissolution
of isolated fertilization membrane by them. Therefore, narain inhibited neither
a trypsin-like proteinase activity in hatching enzyme nor its secretion.
MORPHOLOGY OF THE BROODING ORGAN OF AN UNDESCRIBED BOTRYLLID ASCIDIAN COLLECTED FROM SHIKINE ISLAND OF IZU ISLANDS
M. Okuyama and Y. Saito. Shimoda Marine Res. Center, Univ. of Tsukuba, Shimoda.
There are two major genera, Botryllus and Botrylloides, in the family Botryllidae. An unknown botryllid collected from Shikine Island was thought to be a Botryllus, as it shows the feature of Botryllus
in the arrangement of gonads, that is, the ovary is situated anterior to
the testis. However, we found that a zooid of this botryllid forms a brood
pouch as a brooding organ for its embryo. The brood pouch formation is one
of the important features of Botrylloides. Histological studies on
brood pouch formation in this botryllid show that the pouch is formed by
extension of a part of the branchial sac. Thus, brood pouch formation in
this botryllid is quite different from that of Botrylloides, in which
the pouch is always formed by invagination of the atrial epithelium. On the
other hand, it is known that a kind of brooding organ (not a brood pouch)
is formed from the branchial sac in Botryllus sexiens. This undefined botryllid might have a close relationship with B. sexiens rather than the species of Botrylloides.
CORRELATION BETWEEN ADHESIVE PAPILLAE DEGRADATION AND TAIL RESORPTION OR TRUNK DIFFERENTIATION DURING METAMORPHOSIS OF CIONA INTESTINALIS.
Y. Sato, M. Morisawa. Misaki Marine Biol. Sta., Univ. of Tokyo, Kanagawa.
In metamorphosis of ascidian tadpole larva, sequential degradation of
the adhesive papillae (APBD) on its tip, tail resorption (TR) and differentiation
of trunk occur. In Halocynthia
in which the larval body is vertically cut off at various places before APBD,
TR of posterior fragment of larva never occurs, but TR occurs when the amputation
is carried out after APBD, suggesting that APBD is a signal for TR (Numakunai,
1967). In Ciona, APBD in the anterior part of fragments of amputated
larvae occurred at any timing (before or after APBD) and in any other part
of larval body. Furthermore, TR occurred in the posterior fragments of amputated
larvae in any timing and any amputation, suggesting that contrary to Halocynthia, APBD is not essential for TR in Ciona.
When the trunks of larvae were cut off anterior to brain vesicle (BV), both
the posterior part of the amputated fragments including BV and tail and the
anterior part of the trunk without BV were not fully differentiated. When
the larvae were cut off posterior to the BV or mid part of the tail at any
stage, the anterior fragments with BV or with BV and tail completely differentiated
and became juveniles. These suggest that induction of the trunk differentiation
does not need the part posterior to the BV.
METAMORPHOSIS-INDUCING COMPOUND FROM ASCIDIAN HALOCYNTHIA RORETZI ADULTS AND LARVAE
S. Tsukamoto, H.Kato, H. Hirota, and N. Fusetani. Biofouling Project, ERATO, JRDC, Yokohama.
H. roretzi
larvae do not metamorphose in fresh seawater (SW) when they are maintained
at low densities in laboratory. Metamorphosis was induced, however, by addition
of adult- or larvae-conditioned SW. The presence of metamorphosis-inducing
compound(s) in the conditioned SW was confirmed by partial purification followed
by physicochemical characterization. We have succeeded in isolation of 1mg
of the metamorphisis-inducing compound from 2L of larvae. The same compound
was detected in unfertilized eggs, larvae, and adult tunic, but not in other
adult tissues.
EXPRESSION AND FUNCTION OF C-TYPE LECTINS IN THE ASCIDIAN POLYANDROCARPA MISAKIENSIS.
S. Ohmori, J. Matsumoto, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
The activity of calcium-dependent (C-type) galactose- binding lectin is
required for the aggregation of multipotent mesenchymal stem cells, called
hemoblasts, around the morphogenetic region in the developing bud of the
ascidian Polyandrocarpa misakiensis. We isolated two closely related cDNA clones encoding C- type lectins, named TC14-1 and TC14-2, from P. misakiensis.
TC14-1 encodes a galactose-binding lectin TC-14, which had been previously
purified from this species. TC14-1 and TC14-2 mRNAs were detected in hemoblasts.
The amount of the mRNAs was increased during bud development. Two other
C-type lectins, TCA18 and TCA15, were purified from this species, which had
the activity to make cultured ascidian cells form large aggregations. Amino
acid sequences of them were distinct but similar to those of TC14-1 and TC14-2.
These results suggest that several types of C-type lectins synergistically
act as a stem cell-aggregation factor in the developing bud of this species.
MOLECULAR CLONING OF ALDEHYDE DEHYDROGENASES THAT ARE EXPRESSED IN THE DEVELOPING BUD OF THE ASCIDIAN, POLYANDROCARPA MISAKIENSIS.
N. Harafuji, S. Fujiwara, K. Kawamura and T. Yubisui. Dept. of Biol., Fac. of Sci., Kochi Univ.
Retinoic acid is an endogenous determinant of anteroposterior axis in the developing bud of Polyandrocarpa misakiensis.
Since the activity of aldehyde dehydrogenase (ALDH) is gradually enhanced
in the morphogenetic region of the bud, local activation of this enzyme
is thought to be one of the earliest events of bud development. Previously,
we isolated 2 clones of the fragments of Polyandrocarpa ALDH genes
that were expressed both in the adults and buds. But changes in the amount
of mRNAs were not obvious during bud development. These results suggested
that there was another ALDH gene which had not yet been cloned and was specifically
expressed in the developing bud. We isolated several clones of ALDH cDNA
fragments by RT-PCR, from the total RNA purified from developing buds. Amino
acid sequences deduced from some of the cDNA clones were similar to those
of mammalian ALDHs.
ALLOREACTIVITY IN THE TUNIC OF A SOLITARY ASCIDIAN HALOCYNTHIA RORETZI.
T. Ishii. Biol. Lab., College of Education, Akita Univ.
Self or nonself recognition is well known in some species of ascidians. A colonial ascidian Aplidium yamazii,
which lacks tunic vessels, shows self or nonself recognition. In this species,
it is strongly suggested that phagocytic tunic cells play an important role
in allorecognition and rejection. On the contrary, the solitary ascidian
Halocynthia roretzi shows self or nonself recognition in hemocytes.
In mixed-hemocyte-incubation (MHI-assay), alloreactivity appears in most
nonself combinations. I wondered if allorecognition occurred as quickly also
in the tunic of H. roretzi. Tunic fragments were fused in alloreactivity
positive combination. After a day or two, in each of them, there was invisible
response to the naked eye unlike MHI-assay. Histological observation also
showed no particular response in the tunic in allogeneic combinations. In
the tunic, alloresponse may be occurring slowly or there may be a different
manner existing in the tunic.
TWO TYPES OF ALLO-REJECTION REACTIONS IN COLONY SPECIFICITY IN THE COMPOUND ASCIDIAN SYMPLEGMA REPTANS
M. Shirae and Y. Saito. Shimoda Marine Res. Center, Univ. of Tsukuba, Shimoda.
Colony specificity has been studied mainly in botryllid ascidians. We
previously reported that the allo-rejection reaction of Botryllus scalaris
is different from allo-rejection reactions of other botryllids. In order
to understand the phylogenetic relationship among those rejection reactions,
we examined colony specificity of Symplegma reptans that was thought to have a common ancestor with botryllids. In S. reptans,
two types of rejection reactions were observed among the combinations of
incompatible colonies. In one type, allo-rejection begins with the aggregation
of blood cells after vascular fusion between two colonies, followed by the
disintegration of vascular vessels in the fusion area. In the other type,
rejection begins with the infiltration of blood cells from the tips of ampullae
(vascular termini at the colony periphery) into the tunic without vascular
fusion, followed by their disintegration there. In comparison with allo-rejection
of botryllids, the former type resembles the allo-rejection reaction of B. scalaris and the latter resembles that of Botryllus schlosseri.
CELL-TYPE SPECIFIC MONOCLONAL ANTIBODY AGAINST HAEMOCYTES OF THE ASCIDIAN HALOCYNTHIA RORETZI.
N. Tomita and M. Hoshi. Dept. of Life Sci., Tokyo Inst. Technol., Yokohama.
Hemocytes of the ascidian Halocynthia roretzi
are known to exhibit allogeneic reaction called 'contact reaction'. The ascidians
have several types of hemocytes, yet it remains unclear what type participates
in each step leading eventually to the contact reaction. Besides morphological
features, specific cell markers are required for the classification and identification
of hemocytes. We raised a monoclonal antibody (mAb) that recognized only
one type of hemocytes, namely the giant cells. The antigenicity was localized
in the cell surface of the intact giant cell. Western blot analyses indicated
that the mAb recognized a protein of more than 500kDa. The giant cell-specific
mAb had no effect on the contact reaction.
ANALYSIS OF CELL SURFACE ANTIGENS INVOLVED IN ALLOGENEIC CYTOTOXIC REACTION BY HEMOCYTES OF THE ASCIDIAN HALOCYNTHIA RORETZI.
M. Suzuki1, R. Kondoh 1, H. Ohba1, K. Tabaka2, and J. Chiba1. 1Dept. of
Biol. Sci. and Technol., Sci. Univ. of Tokyo, Noda, and 2Dept. of Biol.,
Nihon Univ. Sch. of Med., Tokyo.
Hemocytes from H. roretzi
undergo rapid lysis in vitro when those derived from different individuals
come in contact with each other. We have prepared monoclonal antibodies
(mAbs) against membrane fractions of hemocytes from H. roretzi and
selected mAbs that inhibit this allogeneic cytotoxic reaction (ACR). Two
groups of mAbs that significantly inhibit ACR, named CRB1 and CRB2, were
found to react with different antigens in membrane fractions of hemocytes
by Western blot analysis; CRB1 was reactive with many glycoproteins of high
molecular weight (62, 129 kDa and more) and CRB2 was with similar those of
47, 58, 80, 100, 115 kDa and more. The ability of the protease to destroy
the antigenic activity seem to support the possibility that both mAbs recognize
a peptide epitope. The epitope recognized by CRB1 and CRB2 wre detected
on the same 30, 38, 44, 49, 68, 86, and 98 kDa peptides after treatment of
the membrane fractions with N-glycanase. In addition, a competitive binding
ELISA using combinations of biotinylated and native CRB1 and CRB2 revealed
that their binding to the deglycosylated membrane fractions was mutually
competitive. These results indicate that the epitopes are located very close
to each other on the peptide, and carbohydrate chain(s) attached through
N-glycoside linkage exist(s) near the antigenic determinant and influence(s)
the reaction with both mAbs. Enhancement of ACR by the addition of carbohydrates
such as chondroitin sulfate in the reaction media and inhibition of ACR by
pretreatment of hemocytes with heparinase suggest that carbohydrates on glycoproteins
apparently involve in the cellular recognition process in ACR by H. roretzi
hemocytes as known in natural cytotoxicity in higher vertebrates, The mAbs
probably inhibit ACR through blocking of this process.
ARE THE VIRIFORM CELLS OF HALOCYNTHIA RORETZI HEMOCYTES?
S. Ohtake1, T. Sawada2, M. Dan-Sohkawa3, and K. Tanaka. 1Dept. of Biol.,
Nihon Univ. Sch. of Med., Tokyo, 2Dept. of Anat., Yamaguchi Univ. Sch. of
Med., Ube, & 3Dept. of Biol., Osaka City Univ.
There has been an argument on the hemocytic nature of the viriform cells of H. roretzi.
We examined their distribution, behavior and surface features in vitro by
light microscopy (LM) and by transmission and scanning electron microscopy
(TEM, SEM). When cone-shaped pieces of the papilla of the animal were placed
in a culture dish containing seawater, viriform cells formed a donut-shaped
cluster exclusive of other cell types on the substratum facing the cut surface
of each cone. When observed under SEM, their surface was rough. They attached
to the substratum with their heads and tails. Under LM and TEM, viriform
cells concentrated in the tunic matrix adjacent to the epidermis of the papilla.
They constituted the major population in this area among the several cell
types found in the tunic matrix. Interestingly, viriform cells showed no
contact reaction to allogeneic viriform cells nor to allogeneic hemocytes.
These features suggest that they are not hemocytes but a kind of tunic cells,
or they may even be protozoan symbionts which live in the tunic of the ascidian.
BIFURCATING REACTION PATHWAY OF AN ASCIDIAN 58 KDA PROTEINASE INHIBITOR TOWARD TRYPSIN
T. Abe, F. Shishikura, S. Ohtake and K. Tanaka. Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
A 58 kDa proteinase inhibitor from the hemolymph plasma of an ascidian, Halocynthia roretzi,
inhibits activities of serine proteinases and is considered as a member of
the serpin superfamily. SDS-PAGE and immunoblotting analysis showed that
the inhibitor forms an equimolar SDS-stable complex with trypsin as well
as other inhibitory serpins. Further investigation on the interaction of
the inhibitor with bovine pancreas trypsin was performed. As the result of
the relation between the [I] / [E] ratio and the inhibitory activity, another
inhibitory path on trypsin was considered: The 58 kDa inhibitor was cleaved
by trypsin. The reaction may proceed along two divergent paths: one is the
formation of the stable complex and the other is the breakdown of the inhibitor.
When a reaction was started by the addition of trypsin to the mixture of
the inhibitor and MCA-substrate, the relatively rapid initial velocity decreased
to slower steady-state rate after 10 min. It was therefore suggested that
the ascidian 58 kDa inhibitor was a slow-binding inhibitor.
RAPID INCREASE IN NUMBER OF VANADOCYTES IN THE VANADIUM-RICH ASCIDIAN, ASCIDIA SIDNEIENSIS SAMEA, BY THE NEUTRALIZATION OF SOME ACIDIC COMPARTMENTS.
Y. Nose, M. Hayashi, T. Uyama, Y. Moriyama and H. Michibata. Mukaishima Marine Biol. Lab., Fac. of Sci., Hiroshima Univ.
Ascidians are known to accumulate high levels of vanadium in their blood
cells. Among about 10 types of blood cells, signet ring cells are vanadium
accumulating cells called vanadocytes. They contain vanadium in highest levels
of about 350 mM, corresponding to 10,000,000 times higher than that in seawater.
Recently, we found that, in the vanadium-rich ascidian Ascidia sydneiensis samea,
the number of vanadocytes in the body fluid increased dramatically when the
ascidians were immersed in seawater containing 10 mM or 20 mM NH4Cl for 20
hrs (Hayashi et al., 1996). NH4Cl is known to neutralize the acidic compartments
in biological membrane systems. Therefore, this phenomenon might be caused
by the neutralization of the acidic compartments in ascidian. To examine
this possibility, we incubated A. sydneiensis samea with several salts,
ionophores and inhibitors for ion-pumping ATPases. The ionophores which
dissipate delta pH and/or delta psi in membrane systems, and the inhibitors
for V-ATPase and F-ATPase caused an increase in number of vanadocytes, about
3-5x higher than the control. These reagents neutralized several acidic compartments
in the ascidian body, which we think is the key to the increase in number
of vanadocytes.
PARTIAL cDNA CLONING ENCODING THE VANADIUM ASSOCIATED PROTEIN EXTRACTED FROM BLOOD CELLS OF ASCIDIA SYDNEIENSIS SAMEA.
T. Adachi, T. Kanda, T. Uyama, Y. Moriyama, and H. Mitibata. Mukaisima Mar. Biol. Lab., Fac. of Sci., Hiroshima Univ.
We extracted a vanadium-associated protein (VAP) from the blood cells of the ascidian, Ascidian sydneinensis samea.
VAP was estimated to associate with vanadium at an approximate ratio of 1
mol : 1 mol. SDS-PAGE and polyclonal antibody against VAP (anti-VAP) , revealed
that VAP is composed of at least three types of peptides, 12.5kDa, 15kDa
and 16kDa, whose proteins are hydrophilic and localized in the cytoplasm
of the vanadocytes. In the present experiment, based on the partial sequence
of amino acids of VAP, PCR primers were designed and the cDNA sequence encoding
the 15kDa peptide of VAP was partially determined.
PREPARATION OF A MONOCLONAL ANTIBODY THAT REACTS WITH A 100kDa PROTEIN IN VANADOCYTES OF THE ASCIDIAN, ASCIDIA SYDNEIENSIS SAMEA
Y. Suhama 1, K. Takamura 2, T. Uyama 1, Y. Moriyama 1 and H. Michibata 1.
1 Mukaisima Mar. Biol. Lab., Fac. Sci., Hiroshima Univ. 2 Dev. of Biotechnol.,
Fac. of Engin., Fukuyama Univ., Hiroshima.
Ascidians have about ten types of blood cells. Among them, vanadocyte
is cell characterized by a single, fluid-filled vacuole and has the unique
and unusual functions of containing both extremely high levels of vanadium
ions in the +3 oxidation state and sulfate ions under pH 2 in the vacuole.
V-ATPase has already been revealed to be present in the vanadocyte and a
monoclonal antibody S4D5 recognizing the vanadocyte specifically was produced.
In the present experiment, the other monoclonal antibody reacted with a
100 kDa hydrophilic protein was produced and the antigen confirmed immunohistologically
to be present in the vanadocyte.
BIOLUMINESCENCE AND LUMINOCYTE IN A COLONIAL ASCIDIAN CLAVELINA MINIATA. 1. ULTRASTRUCTURES OF THE LUMINOUS TISSUE (TUNIC)
E. Hirose1, M. Aoki2, and K. Chiba3. 1Biol. Lab., Col. Bioresource Sci.,
Nihon Univ., Fujisawa; 2Shimoda Mar. Res. Ctr., Univ. of Tsukuba, Shimoda,
and 3Dept. Life Sci., Tokyo Inst. Technol., Yokohama. In the colonial
ascidian Clavelina miniata, physical stimulations
induce strong luminescence in the tunic. We describe here the tunic cell
morphology and distribution in the tunic which is a luminous tissue of this
species. Three types of tunic cells are morphologically discriminated as
morula-like tunic cells, tunic phagocytes, and tunic granulocytes, and they
correspond respectively to the Type I, Type II, and Type III cells described
by Aoki et al. (1989). Tunic phagocytes and tunic granulocytes occasionally
contain phagosomes and elongated bacteria of unique forms. These bacteria
are also distributed outside the tunic cells. To identify the luminous cell
(luminocyte) exactly, it was necessary to record luminescence in vitro.
We succeeded in transferring the tunic cell onto a glass slide. Each of
three types of tunic cells described above are also discriminable under in
vitro condition.
BIOLUMINESCENCE AND LUMINOCYTE IN A COLONIAL ASCIDIAN CLAVELINA MINIATA. 2. IDENTIFICATION OF THE LUMINOCYTE.
K. Chiba1, M. Hoshi1, and E. Hirose2. 1Dept. Life Sci., Tokyo Inst. Technol.,
Yokohama; 2Col. Bioresource Sci., Nihon Univ., Fujisawa.
In Clavelina miniata,
tunic is luminous tissue, where morula-like tunic cells, tunic phagocytes,
and tunic granulocytes are distributed. Because cell density in the tunic
was so high , it had been difficult to determine which type of cells were
involved in bioluminescence. To solve this problem, we isolated tunic cells
and transferred them onto glass slides. Tunic cells on glass slides responded
to hypotonic seawater and showed luminescence as had been observed in the
tunic. We monitored the luminescence with a photon-counting camera connected
to a microscope and found that only the tunic phagocytes luminesced. Similar
results were obtained when each cell was punctured with microneedle; tunic
phagocytes luminesced immediately after destruction, but morula-like tunic
cells and tunic granulocytes did not show any luminescence. Thus we conclude
that the tunic phagocyte is the luminous cell, named luminocyte.
VISUALIZATION OF CELLULOSE SYNTHESIZING ENZYME COMPLEXES ON PLASMA MEMBRANE OF EPIDERMAL CELLS IN ASCIDIANS.
S. Kimura and T. Itoh. Wood Research Institute, Kyoto Univ.
Cellulose synthesizing enzyme complexes (= TCs) have been found in the plasma membrane of epidermal cells in the ascidians Metandrocarpa uedai, Halocynthia roretzi and Perophora japonica,
by using freeze-fracture replication techniques for electron microscopy.
In plant cells, celluloses are synthesized by TCs on their plasma membranes.
The present results suggest that the participation of the TCs in cellulose
synthesis is a universal phenomenon both in plant and animal kingdoms. The
TCs of ascidians consist of two types of membrane subunits: large particles
(14.5 nm in diameter) on the periphery and small subunit particles (7.2 nm)
filling its inside. The TCs are the linear type (ca. 200 nm in length and
80 nm in width), They are often connected with the termini of microfibrils.
PECULIAR GONADAL STRUCTURE IN THE SIMPLE STYELID ASCIDIAN SERIOCARPA SP. FIRSTLY COLLECTED FROM JAPANESE WATERS.
T. Nishikawa, Grad. School of Human Inform., Nagoya Univ.
In the Ascidiacea, the tunic is usually separable more or less easily
from the mantle except in siphonal areas. Rarely, however, a small part
of tunic is mingled complicatedly with adjoining area of mantle (sometimes
involving gonads) to form a complex, as is known in the genus Seriocarpa.
I made a detailed observation of serially thin-sectioned or whole specimens
clearly assignable to this genus, collected from sandy bottoms of Omura Bay
and the Seto Inland Sea, ca. 25m deep. In the specimens, up to 14 mm long,
a small patch of tunic protruded inward to enclose thinly and almost completely
ca. a dozen globular hermaphroditic gonads individually, situated in two
rows beneath the endostyle. Each gonad projected from the mantle body outwards
with enveloping thin mantle epidermis, and opened into the peribranchial
cavity with short ducts. In the gonadal structure, the present specimens
were rather similar to S. rhizoides Diehl, 1969 from the North Atlantic and the Banda Sea, of the known 3 congeners.
IDENTIFICATION OF NEURONAL CELLS BY DETECTING ION CHANNEL MRNA IN THE LARVA OF THE ASCIDIAN, HALOCYNTHIA RORETZI.
T.Okada1, Y.Okamura2,3. 1Ushimado Marine Lab., Univ.of Okayama; 2Lab of Cell Biochem., NIBH., Tsukuba; 3Univ.of Tokyo.
TuNaI is a neuronal sodium channel expressed in the larva of the ascidian, Halocynthia roretzi.
We identified TuNaI positive neuronal cells by the combined method of whole-mount
in situ hybridization with serial sectioning. TuNaI positive cells were found
in: 1)brain vesicle and the related region, 2) neck neural tube, 3) papilla,
4) epidermal sensory neurons. Double staining with DAPI confirmed location
of nucleus for respective TuNaI signal. Intense signals in the posterior
part of 1) correspond to a pair of large neurons which lie at the ventral
region. We studied the cell lineage of TuNaI positive cells by microinjecting
cell lineage tracer, dextran biotin, into each blastomere at the 8 cell and
16 cell stage. Double staining of TuNaI in situ hybridization and HRP-staining
showed TuNaI positive cells in 1) and anterior part of 4) were derived from
a4-2 blastomere. On the other hand, TuNaI positive cells in 2) were derived
from A5-2, probably corresponding to cell soma of motor neurons. We also
compared the expression pattern of TuNaI with that of TuNaII, another putative
ascidian sodium channel gene.
FINE STRUCTURE OF NERVE FIBERS IN CEREBRAL GANGLION OF THE ADULT ASCIDIAN, HALOCYNTHIA RORETZI.
S. Kajiwara, Dept. Biol., Fac. Educ., Iwate Univ., Morioka.
Scanning and transmission EM studies were made on the cerebral ganglion of the solitary ascidian, Halocynthia roretzi. The cerebral ganglion of the adult H. roretzi
(weights about 150g) has a cylindrical shape. Nerve fibers in the cortex
of the ganglion run parallel to the long axis of the ganglion. I found many
varicosities, 10 (m in diameter, of the fibers by TEM. They resemble cell
bodies of the ganglion by SEM, but they have no nuclei and other organelles
particular to the soma. On the other hand, the medulla consisted of nerve
fibers which ranged from 0.3 to 1.0 (m diameter.
APPLICATIONS OF NEURONAL TRACERS ONTO THE CEREBRAL GANGLION OF THE ASCIDIAN, HALOCYNTHIA RORETZI.
H.Koyama, Coll. Nurs., Yokohama City Univ.
The organization of the cerebral ganglion was studied in the solitary ascidian Halocynthia roretzi
by the application of the neuronal tracers horseradish peroxidase, cobalt
conjugated with wheat germ agglutinin (Co-WGA), and carbocyanine fluorescent
dyes (DiI, DiA etc.). Of these, only Co-WGA and carbocyanine fluorescent
dyes were effective. For the application of carbocyanine dyes, the cerebral
ganglia were isolated with the four main nerve trunks attached and fixed
in 4% paraformaldehyde in 0.1M PBS. Dyes were applied directly as powder,
or dissolved in DMSO/EtOH. In about 4 weeks, dyes were transported to about
half the length of the ganglion. When viewed with an excitation filter of
330-385 nm, the neuronal somata of the ganglion fluoresced brightly, but
with a filter for DiI (520-550 nm), autofluorescence was negligible. In
horizontal sections of the cortex, there were columns of soma arranged longitudinally.
DiI-labeled fibers ran between these columns in longitudinal bundles.
A NOVEL G-PROTEIN a-SUBUNIT GENE EXPRESSED IN THE LARVA OF HALOCYNTHIA RORETZI.
T. Iwasa, T. Azuma, M. Ohkuma , and M. Tsuda. Dept. of Life Sci., Fac.
of Sci., Himeji Inst. of Technol., Harima Science Garden City, Hyogo.
In order to clarify the signal transduction system of the ascidian
larva, we investigated G-proteins expressed in the larva by PCR methods.
Using degenerate primers corresponding to conserved amino acid sequences
of vertebrate G protein a subunits, we successively cloned several classes
of a subunit using a cDNA library of ascidian larva as a template. We screened
the cDNA library with the PCR fragment which showed a similarity to Go-class.
The deduced amino acids sequence was composed of 357 amino acids. Although
the deduced sequence exhibits the highest similarity to Goa, the sequence
lacks a consensus cysteine residue at C-terminal, which is a pertussis toxin
target. The phylogenetic tree inferred from the deduced amino acid sequence
shows the protein belongs to the Gia-class, and branched from Goa. The deduced
amino acid sequence was different from the conserved amino acid sequences
within Goa-class. These results indicate that the Ga protein obtained here
is a novel one. Northern blot analysis showed that the message is expressed
after the 64-cell stage, and the amount increases with developmental stage.
In situ hybridization revealed that the message is expressed in a restricted
number of cells of the brain region at the early tailbud stage of ascidian
larva.
PRESENCE OF PROLACTIN-LIKE SUBSTANCE IN SECRETORY GRANULES IN THE NEURAL COMPLEX CELLS OF A PROTOCHORDATE, HALOCYNTHIA RORETZI.
K. Terakado, M. Ogawa*, K. Inoue, K. Yamamoto** and S. Kikuyama**. Dept.
of Regul. Biol. and *Molec. Biol. and Biochem., Fac. of Sci., Saitama Univ.
Urawa and **Dept. Biol., Sch. of Educ., Waseda Univ. Tokyo.
There is growing evidence that the vertebrate adenohypophysis originates
from neuroectoderm. This suggests that equivalents of pituitary cells, if
any, in protochordates may exist in derivatives of neuroectoderm. The neural
complex in ascidians is known to be derived from embryonic neural tube.
Electron microscopical studies on the structure of these organs in Halocynthia roretzi
were performed with particular attention to their secretory systems. We
found that the cells scattered along the dorsal strands as well as neural
cells in the cerebral ganglion contain electron-dense secretory granules
of varying sizes. Immunoelectron microscopical studies of these granulated
cells using antiserum against bullfrog prolactin revealed that some of these
cells contain prolactin-like substance within the secretory granules of 100-250
nm in diameter. The cells belonging to the neural grand and dorsal strand
neither contained electron-dense granules nor showed immunoreactivity. The
results suggest that phylogenetic progenitors of the adenohypophyseal cells
in ascidians may exist in the cerebral ganglion and along the dorsal strand.
EXPRESSION OF GELSOLIN DURING DEVELOPMENT AND METAMORPHOSIS OF ASCIDIAN.
Y. Ohtsuka and T. Obinata. Dept. of Biol., Fac. of Sci., Chiba Univ.
Gelsolin is an actin filament severing and capping protein. It is detectable
in a variety of tissues of higher vertebrates, particularly abundantly in
platelets, macrophages and muscle tissues. Its activity is regulated by Ca2+
and phosphoinositides. Previously, we found that gelsolin is present abundantly
in ascidian body wall muscle and determined its cDNA entire sequence. In
this study, we examined expression of gelsolin during ascidian development
to clarify its role in morphogenesis. Immunoblot analysis showed that gelsolin
expressed maternally but its amount gradually increased after the neurula
stage. When expression in embryos was examined by in situ hybridization and
immunofluorescence staining with the whole mount specimens, gelsolin was
detected in neural cells and unidentified cells beneath the epidermis in
larva, and the cells in tunic during metamorphosis. When mantle was formed,
gelsolin became detectable abundantly in body wall muscle.
800 kDa PROTEINS IN SMOOTH MUSCLE CELLS OF THE ASCIDIAN HALOCYNTHIA RORETZI.
N. Iwai, T. Iino, K. Sawaya and Y. Nakauchi. Dept. of Biol., Fac. of Sci., Yamagata Univ., Yamagata.
It has been reported that the mantle of the ascidian Halocynthia roretzi
is constructed with multinucleated smooth muscle cells (Terakado, K. &
Obinata, T., 1977, 1982, 1987; Shinohara,Y. & Konishi, K. 1982), and
that these muscle cells contain vertebrate striated muscle-type proteins,
e.g. troponin (Toyota, N. et al. 1979) and connectin (titin) (Nakauchi, Y.
& Maruyama, K. 1992). Some high molecular (~ 800 kDa) protein bands
are seen in SDS gel electrophoresis pattern of a total SDS extract of the
mantle muscle cells of H. roretzi. It is known that a polyclonal
antibody against chicken skeletal muscle nebulin does not cross react with
these peptides. We applied the preparation methods of rabbit 1,200 kDa
fragment of a-connectin (Matsuura, T. et al. 1991) and crayfish kettin-like
protein (Maki, S. et al. 1995) to isolate the ~ 800 kDa proteins of H. roretzi
smooth muscle cells. We investigated the relations between these proteins
and 1,200 kDa fragment of rabbit or kettin-like protein of crayfish.
GENOMIC STRUCTURE OF PROTOCHORDATE TROPONIN Cs.
H. J. Yuasa and T. Takagi. Biol. Inst., Grad. Sch. of Sci., Tohoku University, Sendai.
Troponin C (TnC) is one of three components of troponin, a main regulator
of striated muscle contraction, and two isoforms have been identified in
mammalian and avian muscles. One is fast skeletal TnC (sTnC) which is expressed
in fast skeletal muscle, and the other is slow/cardiac TnC (cTnC) which is
present in heart muscle and slow skeletal muscle, and two isoforms were encoded
by distinct genes. We have determined two distinct cDNAs of TnC isoforms
from the ascidian, Halocynthia roretzi.
These two isoforms did not correspond to vertebrate TnCs. One was expressed
in larval striated muscle and the other in body-wall smooth muscle and heart
muscle. From the results of Southern blot analysis and genomic sequencing,
these isoforms were encoded by a single gene and expressed by alternative
selection of a third exon. The localization of intron was identical to vertebrate
cTnC except the 5th intron, corresponding to the 4th intron of vertebrate.
We also determined the genomic structure of amphioxus, Brachiostoma lanceolatum
TnC. In amphioxus, alternative splicing between larva and adult seemed not
to be involved. The positions of introns were identical to vertebrate sTnC
except 4th intron.
GENETIC VARIABILITY OF MTDNA IN HALOCYNTHIA RORETZI; DIFFERENCE BETWEEN THE COAST OF JAPAN SEA AND PACIFIC OCEAN.
T. Kakuda and A. Hino. Dept. Biol. Sci., Kanagawa Univ. Hiratsuka.
Halocynthia roretzi
is distributed around the coast of Hokkaido, Honshu Island and Korean Peninsula.
Because of differences in the breeding season and the start time of spawning,
H. roretzi is classified into three types A, B, C. Only type C occurs
all around Honshu Island. Over 150 individuals of 10 populations of H. roretzi
that were collected from the shores of Hokkaido and Honshu Island were studied
by RFLP and restriction site analysis of mtDNA using 13 restriction enzymes.
From these analyses, we found some individuals which showed polymorphic
pattern after digestion by Hinc II, Hpa I, Ban II, Eco T22I and Xba I, and
finally defined 17 halotypes. Sugashima (Ise Gulf) population had no polymorphism.
In this area, oyster farming has existed for 10 years. It seems that a
small population of H. roretzi was introduced with juvenile oysters
and spread their habitat to the Sugashima area. Even if we calculated without
this population, the frequencies of polymorphism of the Japan Sea population
were higher than that of the Pacific Ocean populations.
DIFFERENTIATION IN THE PYURID ASCIDIAN HALOCYNTHIA HISPIDA (HERDMAN) AS VIEWED FROM MITOCHONDRIAL DNA
T. Kakuda1, A. Hino1, T. Numakunai2 and T. Nishikawa3. 1Dept. of Biol. Sci.,
Kanagawa Univ., Hiratsuka; 2Mar. Biol. Stn., Fac. of Sci., Tohoku Univ.,
Asamushi; 3Grad. Sch. Human Informatics, Nagoya Univ.
In the Japanese population of Halocynthia hispida,
two forms are recognizable based on the difference in density of tunic spines.
Although these forms have been considered only an individual variation,
our present results strongly suggest their genetic differentiation. We extracted
and purified mtDNA from both forms, which were collected at Shimokita Peninsula,
and digested by 8 restriction endonucleases. After fragment pattern analysis,
we estimated the genetic divergence (Nei & Li 1979) between the two forms
to be 4.2/100. This value is far larger than the value within the reproductively
isolated three types of H. roretzi, but almost equal to the value between
H. roretzi and H. aurantium (Kakuda et al., 1995).
Cephalochordata: EXPRESSION OF MUSCLE ACTIN GENES DURING DEVELOPMENT OF AMPHIOXUS AND MEDAKA.
R. Kusakabe1, T. Kusakabe2, N. Satoh1, N. D. Holland3, L. Z. Holland3 and
N. Suzuki2, 1Div. of Biol. Sci., Grad. Sch. of Sci., Kyoto Univ.; 2Div. of
Biol. Sci., Grad. Sch. of Sci., Hokkaido Univ., Sapporo; 3Scripps Inst. of
Oceanog., UC San Diego, California, USA.
To investigate the evolution of muscle differentiation mechanisms in
chordates, we isolated actin cDNAs from amphioxus and medaka and examined
their expression patterns and phylogenetic relationships. It was revealed
that amphioxus has a muscle actin similar to the vertebrate muscle actin.
However, the amphioxus muscle actin has characteristic amino acid residues
that are not conserved in other vertebrate-type muscle actin genes, suggesting
that the amphioxus muscle actin gene diverged early in evolution. Transcripts
of this muscle actin gene appears in the somites before the notochord formation
and the myotomal expression continues throughout larval development. The
amphioxus muscle actin gene is also expressed in smooth muscle cells associated
with gill slits. Medaka was also found to possess an actin gene that resembles
the mammalian skeletal muscle actin gene. In contrast to the amphioxus muscle
actin gene, medaka muscle actin gene begins to be expressed when CNS development
proceeds considerably. The expression is restricted to somites destined
to differentiate to skeletal muscle. Since this medaka actin gene was not
expressed in heart, we predict the existence of a family of multiple muscle
actin genes in medaka, including a cardiac muscle actin gene. Further analysis
on differential expression of multiple actin genes during embryogenesis will
provide insights into the evolution of developmental mechanisms in chordates.
HABITAT POPULATION AND ENVIRONMENT IN AMPHIOXUS, BRANCHIOSTOMA BERCHERI
K. Kubokawa1, M. Watanabe1, N. Azuma2 and M. Tomiyama3. 1 Ocean Res. Inst.,
Univ. of Tokyo, 2 Toyohashi Univ. of Tech. & Sci., Arch. 3
Japanese amphioxus (Branchiostoma bercheri)
is on the endangered species list, its preferred habitat lost by environmental
pollution; its population gradually decreases in Japan. However, the population
and its environment has not been surveyed at all in Japan. We collected
amphioxus in various seasons and characterized its natural environment.
Since we found a high population density of amphioxus in the fields around
Mikawa Bay, we analyzed their growth and gonadal development by collecting
them once a month from May to November in 1995. In May, 1996, precise studies
were performed on the population density of amphioxus and other benthos,
submarine sediment and topography, water quality and a video recording at
the same area by using the research boat, Tansei-maru, belonging to the Ocean
Research Institute. The distribution of body length in every month showed
two peaks which described more than two years' life. The testis and ovary
matured in July and disappeared in September. The number of mature males
was always higher than females. Habitat selection was severely dependent
on sand diameter. However, the water quality, chlorophyll and plankton
quantities were not different between a habitat and a non-habitat plot.
Hemichordata: THE EARLY DEVELOPMENT OF THE HAWAIIAN HEMICHORDATE (ENTEROPNEUST) PTYCHODERA FLAVA
K. F. Tagawa1, T. Humphreys2, N. Satoh1. 1Dept. of. Zool., Kyoto Univ.; 2Kewalo Marine Lab., Univ. of Hawaii, Honolulu.
The phylogenetic status of hemichordates (enteropneusts and pterobranchs
) has long been disputed. This animal has recently attracted attention with
relation to the origin of chordates. We discuss deuterostome evolution
towards chordates by examining the ontogeny and phylogeny of hemichordates.
As the first approach, we describe the early development of the Hawaiian
acon worm Ptychodera flava.