Number 45                                                                                                                                        May 1999

This issue contains a list of the email addresses of many of our Ascidian News subscribers; you will find it near the end of the newsletter, just before the New Publications.  If any of you wish to be added, or if you would like to suggest the addition of any other names, please let Gretchen know.  We have had a number of requests for such a list, and we hope you will find it useful.  If you change your email address, please let us know.  There are a large number of meetings abstracts in this AN, especially from the latest meeting of the Zoological Soc. of Japan.  The New Publications section at the end of the newsletter includes, as always, a large number of excellent new papers on a wide variety of ascidiological topics.  Thank you for the reprints you send us; they are very useful and important to us especially since we have a more limited access to library facilities now.  Please send us your new reprints as soon as they appear, and that way you will be assured of being cited in the next AN.

We spent a very enjoyable and productive 6 weeks in Guam this winter. Gretchen worked on the identification of as many local ascidian species as there was time for, and Charley continued his fertilization studies. Please see the Work in Progress section for a more detailed description.  This summer Charley will teach the Comparative Invertebrate Embryology course at Friday Harbor with Steve Stricker.  Gretchen will continue her identifications of the many ascidians collected in Hawaii and Guam, including a number of problematic forms and probably some undescribed species.  From 13 May to 1 September 1999 we will be at the Friday Harbor Laboratories, 620 University Road, Friday Harbor WA 98250  Phone (206) 543-1484 or (360) 378-2165  Fax: (206) 543-1273.  We will continue with the same email addresses for the summer. Stop by for a visit if you are in the area.

*Ascidian News is not part of the scientific literature and should not be cited as such.

NEWS AND VIEWS

1. Charles and Gretchen Lambert:  During late January and all of February we worked at the University of Guam Marine Laboratory in Mangilao.  Here Gretchen identified many of the local ascidians as part of an on-going Sea Grant study on introduced species in Guam.  Gustav Paulay (the director) and Chris Meyer, his postdoc, were responsible for much of the deeper collecting by snorkel and scuba, while we surveyed the marinas and shallow areas.  This was the first inventory of the ascidians of Guam; they are surprisingly different from other areas, not as rich as Palau but quite different from Hawaii except for the ubiquitous Ascidia (=Phallusia) nigra, A. sydneiensis and Herdmania momus.  Charley continued his fertilization studies begun in Hawaii last November with Ascidia nigra and A. sydneiensis, and has just submitted a manuscript on this work.  Retirement agrees very much with Charley and he is enjoying the opportunity to work on these warm water species while accompanying Gretchen on her taxonomic travels.  We were very fortunate to meet our friend Xavier Turon from Barcelona in Guam. He was working mostly with sponges but collected some very interesting deep water ascidians for Gretchen.  Near the end of our stay Gretchen presented a taxonomic workshop which included live specimens of many of the species.  It was very well received by University of Guam faculty and graduate students. Some of the species are apparently undescribed, including several abundant forms.  We hope to make another trip to Guam next winter.

2.  Rosaria de Santis  desantis@alpha.szn.it    We have now cultures of Ciona intestinalis available.  A major effort of two technologists of the Cell Biology Laboratory, Drs. Paola Cirino and Alfonso Toscano lead to the design of a set-up (tanks, substrates and feeding  systems) for culturing Ciona. The results are quite successful and we can get mature animals in six-eight weeks embryo to embryo. This prompted us to approach the more ambitious project of Ciona mutagenesis that has been developed in our Laboratory in collaboration with Drs. Paolo Sordino and Karl-Philipp Heisenberg, University College, London.  At the moment we are performing the conclusive experiments of the screening and we have already the first set of phenotypes.  We will keep the Ascidian Community informed of our further results.  We presented already the preliminary results at  the EMBO workshop "Reproduction and Development" held in Bergen, Norway, last October.

3. Patrick Frank(a) and Keith O. Hodgson(b) a. Dept. of Chemistry, Stanford Univ., Stanford, CA 94305; b. Stanford Synchrotron Radiation Lab, SLAC, Stanford Univ, Stanford, CA 94309. frank@SSRL01.slac.stanford.edu   Defining chemical species in complex environments using K-edge X-ray absorption spectroscopy: vanadium in intact blood cells and Henze solution from the tunicate Ascidia ceratodes. (The work is still in manuscript form, and we hope to submit it soon for publication.)
A K-edge x-ray absorption spectral fitting approach has been developed to explicate the complex environments of vanadium ions within whole blood cells from the tunicate Ascidia ceratodes. To approach current models of biological vanadium storage, the response of the K-edge x-ray absorption spectrum (XAS) of solution-phase vanadium(III) to changes in [sulfate] and acidity, respectively, was investigated.  At constant pH 1.8, increasing [sulfate] produced systematic effects in the vanadium XAS pre-edge energy region at 5468.8 eV (pre-edge transitions are: 1s->4A2 at 5464.9 eV; 1s-> 4T2 at 5466.9 eV; and 1s->4T1 at 5468.8 eV for 11 different V(III)/sulfate solutions). In contrast, variations in acidity (as pH) at constant [sulfate] produced systematic modification of vanadium pre-edge XAS at 5466.9 eV.  Both sulfate and pH influenced absorption intensity near 5476 eV in the rising K-edge.  The energy position of the V(III) K-edge absorption maximum also serially shifts 0.33 eV/pH unit, from 5483.7 eV at pH 3.0 to 5484.7 eV at pH 0.3.  The K-edge spectra of V(III) in acidic sulfate solutions along with the vanadium K-edge XAS spectra of other appropriate V(III) model complexes were then successfully used to fit the vanadium K-edge XAS spectra of two samples of whole blood cells from the tunicate Ascidia ceratodes, representing 25 and about 37 animals respectively. These population-level fits implied storage of blood cell vanadium(III) ions in four solution regimes: high sulfate/high acid; high sulfate/moderate (pH 1.8) acid; moderate sulfate/moderate acid, and; moderate sulfate/weak (pH 3) acid.  Evidence was found for small amounts of biological vanadyl ion and traces (ca. 2%) of possibly tris-chelated V(III).  For vanadium in Henze solution, the best fit implied a predominant (77%) pH 1.6 acid environment and no detectable V(III)-sulfate interaction, corroborating previous epr and sulfur K-edge XAS results.  Nearly 20% of Henze solution vanadium(III) appeared tris-chelated.  A detailed chemical model of vanadium within intact whole blood cells of the tunicate Ascidia ceratodes is calculated using known the known equilibrium behavior of V(III) and sulfate in acid solution.  The fitting approach is suggested to be generally applicable to elucidating the state of metal ions in a wide variety of complex environments.

4. Christian Sardet, Station Zoologique, Villefranche sur Mer, 06230 France;  sardet@ccrv.obs-vlfr.fr : There will be an  EEC sponsored Long term course (1 month) organized in June in Roscoff, France about evolution and development. It will include ascidians and there is a web site. It may be still possible to apply; http://www.bio.uu.nl/~embryo/Meetings/EU99.htm

5. Also from Christian Sardet: POST DOC POSITION: DEVELOPMENTAL BIOLOGY RESEARCH UNIT IN VILLEFRANCHE SUR MER.  We are  looking for a post doc to join our laboratory in the context of a Human Frontier network that is just starting and will last 3 years.  The network's focus is on maternal determinants and their identification and localization in the zygotes of Xenopus and ascidians. The participating laboratories are P. Lemaire (Marseille), M.L. King (Miami), R. Elinson (Toronto) and H. Nishida (Tokyo)and ourselves. As far as our lab is concerned the post doc position will be to work on cortices in Xenopus and ascidians (we have preparation of isolated cortices that have kept some functionality (sliding of microtubules etc.)) and we will also isolate mRNAs from cortices  and localize them (low resolution and high resolution in situs with EM).  Some of the work will be done with our partners and will require spending periods of  time in P. Lemaire and/or ML. King and Nishida's lab.  In our laboratory, the post doc will be working directly with Christian Sardet  (ascidian) as well as with Evelyn Houliston (Xenopus). Our laboratory is a Molecular and Cell Biology Unit with 30 members situated in an historical building by the Mediterranean in Villefranche (between Nice and Monaco).  The post doc position could start any day now and would be paid 10000-12000 FF a month and last from 1-3 years.  We will be organizing a workshop gathering the members of the network in May (8-15th) in Villefranche sur mer .  For informations/applications  contact Christian Sardet at ; sardet@obs-vlf.fr

6.  Charles Lambert, Gretchen Lambert and Todd Newberry.  Lights Manual, Intertidal Invertebrates of the Central California Coast,  4th Edition:  We are working on the revision of Don Abbott’s ascidian chapter and key for the 4th edition of this  classic work which is expected to be out in 2000.  Jim Carlton is the Editor in Chief.  We have finished the new check list and are through a third revision of the keys.  We have added several new species because of recent introductions that seem to have naturalized especially in the marinas, and also include some southern California species in the check list, but not the keys, that might be encountered.  Eudistoma purpuropunctatum Lambert, 1991. Ciona  savignyi Herdman, 1882,  Ascidia zara Oka, 1935 Botrylloides diegensis Ritter and Forsyth, 1917, Botrylloides violaceus Oka, 1927, Botryllus schlosseri (Pallas, 1766), and Dendrodoa abbotti Newberry, 1984 have thus far been added to the keys. We would like to hear from any of you if you have found ascidian species that are not in the third (1975) edition or this list of added species. We want to be sure that the keys are truly inclusive of what is out in the real world today.

THE EVOLUTION OF LARVAL MORPHOLOGY IN ASCIDIANS: A PHYLOGENETIC ANALYSIS OF SPECIATION IN THE MOLGULIDAE.  M.S. thesis abstract by Jennifer Lynn Huber.
    The Molgulidae family of ascidians has several species which have independently evolved anural, or tailless, larvae.  This is in contrast to the typical urodele larva that develops in all other families, and contains a notochord, muscle cells and a dorsal hollow nerve cord.  Molecular phylogenies using the 18S rRNA gene and the hypervariable D2 loop of the 28S rRNA gene indicate that species in the family Molgulidae fall into at least four distinct clades, three of which have multiple anural members.  For at least two of these clades, we present clear evidence of a circumpolar tailed ancestor whose distribution has subsequently diminished.  Furthermore, we show that complex life history characters such as viviparity, loss of tail, and ability to self-fertilize are polyphyletic within the Molgulidae, and appear to have evolved independently.  Speciation of the Molgulidae and evolution of tailless larvae appears to have occurred primarily at northern latitudes.  Our analyses suggest that the evolution of tailless larvae may be correlated with the biogeography, rather than the ecology, of adult molgulids.
    This work was written as partial fulfillment of a M.S. degree awarded to Jennifer Huber from Penn State University in the laboratory of Dr. Billie J. Swalla.  A manuscript has been submitted to Evolution describing in detail the molgulid clades.  Meanwhile, Jenn is planning to continue her education at the University of Hawaii, pursuing a Ph.D. in the laboratory of Dr. Mark Martindale.

1. Canadian Soc. of Zoologists, Ottawa, May 8, 1999.

EVIDENCE FOR THE EXISTENCE OF A GnRH-LIKE PEPTIDE IN SENSORY CELLS OF Corella inflata (ASCIDIACEA).  Mackie*, G.O. & R.M. Marx,  Biol. Dept, Univ. of Victoria, Victoria, B.C.
  Previous studies have demonstrated the existence of two novel forms of gonadotropin releasing hormone in ascidians, one of which has been located in a nerve plexus in the dorsal blood sinus and is implicated in control of reproduction and neural regeneration. We now find immunoreactivity to Tunicate I GnRH in sensory cells in the body wall of Corella. The cells appear to derive from neuroblasts produced in the region of the dorsal strand which migrate through blood spaces to subepithelial locations in the  body wall, where they complete their transformation into sensory cells.

2.  Soc. for Experimental Biology meeting, Edinburgh, Scotland, March 23, 1999.

LONG-TERM SURVIVAL OF NEURAL FUNCTION IN DE-BRAINED ASCIDIANS.  G.O. Mackie and  R.C. Wyeth,  Biology Dept., Univ. of Victoria, Victoria, British Columbia.
  Ascidians behave as if they had a coelenterate nerve net in the body wall. Stimulation of one siphon causes contraction of the other after removal or transection of the brain. Some reflex activity persists in Chelyosoma for at least four months after brain removal. The brain does not regenerate and the peripheral innervation does not degenerate. Contrary to previous claims we find that there is no nerve net in the body wall in the sense of a plexus of neurons with cell bodies. The only nerve elements with peripheral cell bodies are scattered sensory elements. The motor neurons have their cell bodies in the brain. We are trying to sort out the sensory and motor pathways in normal and debrained animals. Cholinesterase histochemistry and anti-tubulin immune labeling show a ramifying mass of nerves running between the siphons, some of which bypass the brain and could provide coordinating pathways after brain removal. Suction electrode recordings confirm the propagation of electrical signals between the siphons in debrained animals.. Surgical experiments suggest multiple pathways. It is not yet clear if these pathways involve direct connections between sensory nerves in the periphery, connections between terminal branches of the motor nerves,  or connections between the two.. A recent finding of GnRH-like immunoreactivity in the sensory nerves offers hope of distinguishing sensory from motor elements in mixed bundles and  thus of determining how the animal achieves the functional equivalent of a nerve net.

3.  American Society of Limnology and Oceanography, Santa Fe, NM, Feb. 3, 1999.

LIGHT, DISTRIBUTION, AND LIFE HISTORY ADAPTATIONS OF THE ASCIDIAN, Corella inflata.  Bingham, B.L., A.M. Reitzel, and N.B. Reyns. Western Washington Univ., Shannon Pt. Marine Center, 1900 Shannon Pt. Road, Anacortes, WA.  bingham@cc.wwu.edu
    The ascidian Corella inflata is a common fouling organism in the Puget Sound and the San Juan Archipelago, Washington, USA. Despite its abundance, particularly on floating docks, it is conspicuously absent from areas that receive direct sunlight. We hypothesized that UV irradiation damages exposed individuals and creates the observed distribution. To test this, we exposed C. inflata adults, juveniles, larvae, and embryos to UV irradiation. In laboratory tests and under natural sunlight in the field, UV significantly shortened adult life span. Juveniles died after only 2-3 days in the light. Several hours of exposure were sufficient to decrease larval settlement and metamorphosis. Abnormalities appeared in developing embryos after only 30 minutes of exposure. By selectively filtering natural sunlight, we demonstrated that UV-B wavelengths were most damaging to C. inflata. However, UV-A and visible light also produced significant negative effects. We conclude that C. inflata is sensitive to UV light in all phases of its life history with younger stages being most vulnerable. We suggest that unique life history traits (i.e., time of spawning, brooding behavior, length of larval life) limit exposure and allow C. inflata to persist in its preferred dock habitat despite its UV vulnerability.

4. Zool. Soc. of Japan 69th annual meeting, 1998. (Publ. In Zool. Sci. vol. 15, suppl., Dec. 1998)

EXPERIMENTAL ALLOMETRY: HOW DOES METABOLIC RATE CHANGE WITH COLONY SIZE OF ASCIDIANS?    F Nakaya1, Y. Saito2 and T. Motokawa1.  1Basic Biology, Faculty of Bioscience & Biotechnology, Tokyo Inst. of Technol., 2Shimoda Mar. Research Center, Univ. of Tsukuba.
   The allometric relationship between metabolic rate and body size is well established in individual organisms: the metabolic rate increases in proportion to the body weight to the power 0.75. The question why 0.75 is still unanswered. One of the reasons making this problem hard to solve might lie in the difficulties in experimental manipulation of the body size. We studied the relationship between metabolic rate and size of colonial ascidians, because their colony size can be manipulated with ease. The colonial ascidians Botryllus schlosseri and Botrylloides simodensis were used. They were chosen because they form a flat single-layered colony as they grow, thus the effect of three dimensional shape changes can be neglected. We measured oxygen consumption of colonies of various sizes (O.03g-5.0g wet weight) by oxygen electrodes. A clear allometric relationship was found between metabolic rate and colony size: the metabolic rate increased in proportion to the colony weight to the power 0.788 (B. schlosseri ) and 0.815 (B. simodensis ), both of which were statistically not different from 0.75 but different from 1. The effect of cutting a single colony into smaller colonies was studied. A colony reared on a polycarbonate sheet was divided into smaller colonies by cutting it with the polycarbonate sheet attached without tearing the animals from their substrate. The fragmented colonies were kept in open sea water for one week for wound healing. The metabolic rate of fragmented colonies tell just on the regression line of the metabolic fate-size relationship obtained from intact colonies of various sizes.

A CONVENIENT METHOD OF WHOLE MOUNT IN SITU HYBRIDIZATION (WMISH) FOR COMPREHENSIVE SURVEY OF MULTIPLE cDNA CLONES.  T. Minokawa2, M. Ogasawara1, Y. Sasakura1, H. Yamamoto2, H. Nishida2, and K. W. Makabe1.  1: Dept. of Zool., Graduate Sch. of Sci., Kyoto Univ.  2: Dept. of Life Sci., Tokyo Inst. of Technology, Yokohama.  1, 2: "Research for the Future" program
   We constructed an arrayed library of cDNAs for maternal mRNAs in ascidian fertilized eggs.  To survey the localization pattern of each mRNA, the procedure of conventional WMISH protocol is too time-consuming.  To screen a large number of cDNA clones for localized messages in the ascidian egg, we developed a convenient protocol of WMISH. Digoxigenin (DIG)-labeled RNA probes simultaneously synthesized directly from PCR products from a large number of cDNA clones in a relatively short time.  Hybridization and washing were carried out in modified 96-well plates (Silent Screen Plate, Nunc), in which the bottom is sealed with nylon membranes.  The solution in each well was rapidly drained through the membrane by vacuum.  This WMISH system enabled us to survey a large number of cDNA clones in a limited time.

ISOLATION OF cDNA CLONES FOR mRNAs TRANSCRIBED ZYGOTICALLY DURING CLEAVAGE STAGE IN ASCIDIAN EMBRYOS.  T. Miya, H. Nishida.  Dept. of Life Sci., Tokyo Inst. of Tech., Yokohama.
   The ascidian larva consists of relatively small number of tissues, and the cell lineage is well described during embryogenesis.  Most of the blastomeres become tissue-restricted by the 110-cell stage which is just before the onset of gastrulation.  During ascidian embryogenesis, primary muscle, epidermis and endoderm autonomously differentiate, and the processes are mediated by egg cytoplasmic determinants.  We are trying to isolate cDNAs for genes of which expression is directly triggered by maternal determinants during early embryogenesis of the ascidian, Halocynthia roretzi.   We constructed cDNA library of 110-cell embryos. By differential screening using maternal mRNAs and mRNAs from 110-cell embryos, we isolated several cDNA clones for genes of which transcription starts during cleavage stage.

CLASSIFICATION OF HEMOCYTES OF THE ASCIDIAN Halocynthia roretzi by FACS.   M. Kumano, N. Tomita, M. Hoshi.  Dept. of Life Sci., Tokyo Institute of Technology.
   Hemocytes of the ascidian Halocynthia roretzi are believed to play an important role in self-defense system and allo-recognition. Since hemocytes consist of morphologically heterogeneous populations, it is essential to classify them and clarify the roles of each population in self-defense and allo-recognition. The hemocytes were classified into 12 groups by FACS using monoclonal antibodies and dyes. Our classification was compared to the morphological classifications previously reported.  Although tunic cells are thought to be a population of hemocytes that have migrated into the tunic, a  portion of tunic cells show a unique feature, which has never found in hemocytes, by FACS analysis. The contact reaction was quantified by the increase in cell number of smaller population (cell debris) detected by FACS.

THE MECHANISMS RESPONSIBLE FOR THE EXTREMELY LOW PH IN THE VACUOLE OF VANADOCYTES OF THE ASCIDIAN.  T.Ueki1, T.Uyama1, K.Kanamori2 and H.Michibata1.  1Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Grad. Sch. Sci., Hiroshima Univ.; 2Dept. Chem., Fac. Sci., Toyama Univ.
   Ascidians have long been known to accumulate a transition metal, vanadium, in their blood cells. The pH within the vacuole is very low and is correlated with the concentration of vanadium.  Vacuoles of vanadocytes of Ascidia gemmata having the highest level of vanadium of 350 mM exhibit the lowest pH value of 1.86, those of A. ahodori containing 60 mM vanadium have pH 2.67, and those of A. sydneiensis samea containing 13 mM vanadium have pH 4.20. Immunocytological studies suggested that V-ATPases actually function to accumulate protons in the vacuoles.  Here we propose two possible mechanisms to keep the extremely low pH.  One is the dissociation of the water molecules coordinating to vanadium(III) ion accumulated.  The other is the possibility that the V-ATPases in the vanadocytes has unusually high activity of proton pumping.  Both mechanisms may function together in the vacuole of vanadocytes.
 

cDNA SEQUENCE FOR A 100 kDa ANTIGEN REACTED WITH A MONOCLONAL ANTIBODY S8E4 IN VANADOCYTES OF THE ASCIDIAN, Ascidia sydneiensis samea.   Y.Suhama, T.Uyama , T.Ueki, H.Michibata. Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Ascidians have about ten types of blood cells.  Among them, the vanadocyte is a cell characterized by a single, fluid-filled vacuole and has an 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.  A monoclonal antibody S8E4 specifically recognizing a 100 kDa protein in the vanadocyte was produced.  In order to characterize the 100kDa antigen, a gene encoding the antigen was screened using monoclonal antibody S8E4 as a probe from the cDNA library prepared from the blood cells.  A search of sequence databases for similarities detected that the 100kDa antigen was glycogen phosphorylase. S8E4 was confirmed to react with the protein translated from the cloned cDNA in E. coli. Since we have already revealed the existence of enzymes in the pentose phosphate pathway in vanadocytes, we suppose that this glycogen phosphorylase functions to provide substrate G1P.

METAL ION AFFINITY WITH A VANADIUM-ASSOCIATED PROTEIN EXTRACTED FROM THE VANADIUM-RICH ASCIDIAN, Ascidia sydneiensis samea.   Y.Matsumura, T.Uyama, T.Ueki and H.Michibata. Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Ascidians are known to accumulate vanadium selectively at extremely high concentration in their blood cells.  The highest level of vanadium ions was reported to be in excess of 350 mM such level has never reported in living organisms.  This phenomenon has attracted the interest of many investigators between biology and chemistry as a peculiar mechanism of uptake.  We previously identified three kinds of vanadium-associated proteins (VAPs) which are co-fractionated with vanadium ions using a technique of anion exchange column.  We considered that VAPs are connected with the peculiar mechanism of vanadium uptake.  By equilibrium dialysis methods, we showed that the VAPs specifically bound to vanadium(V) ions and the affinity of VAPs to vanadium(V) ions is much higher than that of BSA or that of proteins at each purification step.

VANADIUM ION AFFINITY OF BLOOD CELL COMPONENTS IN Ascidia sydneiensis samea.
T.Fukumitsu, T.Uyama, T.Ueki and H.Michibata. Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Ascidians are known to accumulate vanadium selectively at extremely high levels in their blood cells.  In the process of the accumulation, vanadium-binding proteins are expected to have important functions.  We previously identified several vanadium-binding proteins in the soluble fraction of vanadocytes, but no such proteins in the insoluble fractions have been found.  The purpose of the present study is to examine the affinity of organella and proteins in insoluble fractions to vanadium ions. After blood cell homogenates were separated by ultracentrifugation, using equilibrium dialysis methods, we found that the membrane fraction containing nuclear membranes and plasma membranes, and the vacuolar membrane fraction had a strong affinity to vanadium ions.

EXPRESSION OF RECOMBINANT VANADIUM-ASSOCIATED PROTEINS (VAPS) IN E. coli.
S.Kawano, T.Ueki, T.Uyama and H.Michibata. Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Ascidians, known as sea squirts, accumulate extremely high levels of vanadium(III) ions in their blood cells. From the blood cell extracts, we have recently identified several low molecular weight proteins which are co-purified with vanadium after the anion exchange column fractionation.  We designated them as vanadium-associated proteins (VAPs).  The cDNAs encoding 12.5kDa and 15kDa VAPs have been cloned and sequenced completely.  In this study, we cloned each of them into an expression vector and transformed into E. coli.  Expression of the fusion protein was induced by the addition of IPTG. The proteins extracted from the bacteria were analyzed by SDS-PAGE.  The fusion proteins were detected by the polyclonal antibodies against VAPs on the western blots. Thus, 12.5kDa and 15kDa VAP fusion proteins were successfully synthesized in the bacteria.

PREPARATION OF MONOCLONAL ANTIBODIES SPECIFIC TO VACUOLAR MEMBRANE PROTEINS OF ASCIDIAN VANADOCYTES.   T.Uyama, S.Nomura, T.Ueki, & H.Michibata.  Mukaishima Mar. Biol. Lab., Fac. Sci. and Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Vanadocyte, vanadium-containing blood cell, is specified to accumulate and reduce vanadium in ascidian blood cells.  Although vanadium is in the +5 oxidation state in sea water, the accumulated vanadium is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuole of the vanadocyte.  However, it is unclear how vanadium ions are transported into cytoplasm from serum and into vacuole from cytoplasm in the accumulation and reduction process.  In this experiment, therefore, we tried to raise monoclonal antibodies specific to the vacuolar membrane proteins of the vanadocyte in vanadium-rich ascidian, Ascidia sydneiensis samea. As the result, a hybridoma cell line which secretes a monoclonal antibody, designated V2C3, specific to the vacuolar membrane of the vanadocytes has been established.  Immunoblotting analysis showed that the V2C3 monoclonal antibody was revealed to react with a vacuolar membrane protein of about 130 kDa.

REDUCTION OF VANADIUM BY NADPH UNDER AEROBIC AND ANAEROBIC CONDITIONS.   T.Kinoshita1,T.Uyama1, K.Kanamori2, T.Ueki1 and H.Michibata1.  1Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ., Hiroshima. 2Dept. Chem., Fac. Sci., Toyama Univ.
   Ascidians are known not only to accumulate high levels of vanadium in the vacuole of the vanadocytes, vanadium-containing blood cells, but also to reduce the accumulated vanadium to V(III) via V(IV). Since vanadium is dissolved in V(V) in seawater, some reducing agents must participate in the reduction process in the vanadocytes.  Recently, we found that enzymes of the pentose phosphate pathway exist specifically in the vanadocytes.  The finding suggested a possible participation of NADPH in the reduction of V(V) to V(IV), since enzymes in the pentose phosphate pathway are known to produce NADPH.  The present experiment is designed to examine whether V(V) is reduced to V(IV) by NADPH in vitro with the final aim being to prove the intrinsic participation of NADPH in the reduction of vanadium in the vanadocytes of ascidians. As the result, it revealed that NADPH can reduce V(V)-EDTA to V(IV)-EDTA but cannot do V(V) in the form of ortovanadate both under aerobic and anaerobic conditions.

CLONING OF TRANSKETOLASE FROM VANADOCYTES IN THE ASCIDIAN, Ascidia sydneiensis samea.   K.Yamamoto, T.Ueki, T.Uyama, and H.Michibata.  Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Ascidians are known to accumulate extremely high levels of vanadium(III) ions in the vacuole of their blood cells (vanadocytes).  During the characterization of some antigens recognized by monoclonal antibodies specific to vanadocytes, we have revealed that glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are expressed in the vanadocytes.  Those enzymes belong to the oxydative pathway of the pentose phosphate pathway, which convert NADP+ to NADPH.  In this study we isolated a cDNA encoding transketolase, one of the enzymes in the non-oxydative pathway of the pentose phosphate pathway, expressed in the vanadocytes. Our result strongly supported that pentose phosphate pathway exists and functions in the vanadocytes.

PREPARATION OF MONOCLONAL ANTIBODY AGAINST GIANT CELLS OF Ascidia sydneiensis samea.   N.Ishimura, T.Uyama, T.Ueki and H.Michibata.  Mukaishima Marine Biol. Lab., Fac. Sci. and Marine Molec. Biol., Graduate Sch. Sci., Hiroshima Univ.
   Ascidians are known to accumulate high levels of vanadium in the vacuole of one of blood cells, the vanadocytes.  Blood cells are morphologically classified into at least ten types.  Among them, giant cells ranking with vanadocytes are known to have several particular features. The giant cell is the biggest cell having approx. 80um in diameter in ascidian blood cells.  The large portion of the cell body is occupied by a vacuole containing a lot of mucopolysaccharides.  The vacuole has extremely low pH as seen in the vanadocytes.  In this present experiment, by immunizing mouse with vacuolar membrane fraction of blood cells, we successfully obtained several strains of hybridoma cells which produced antibodies recognizing the vacuolar membrane of both vanadocytes and giant cells.  The size and the localization of the antigens were determined by western blotting.

EVOLUTIONARY HISTORY OF FREE SWIMMING AND SESSILE LIFESTYLES IN UROCHORDATES AS DEDUCED FROM ONTOGENY PHYLOGENY.   H. Wada. SMBL, Kyoto University, Shirahama-cho, Wakayama.
   Whether the ancestral chordates were free-swimming or sessile is a longstanding question that still remains to be settled.  Vertebrates and amphioxus are free-swimming, but the most basal chordate subphylum (the urochordates) includes both sessile and free-swimming species.  Molecular phylogenetic analyses on 18S rDNA of urochordates  revealed a close relationship between salps and doliolids, and paraphyly of the ascidians.  An early divergence of larvaceans, which show a tadpole-like bodyplan throughout life, is also supported from the analyses.  I also present evidence that the ascidian tadpole larva possesses a highly organized neural tube, with traces of segmentation, dorsoventral differentiation and subdivision into regions homologous to fore- and midbrain, anterior hindbrain and posterior hindbrain and spinal cord, respectively.  Together with the fact that larvacean neural tube is also segmentally organized, it is likely that the ancestral chordates already possessed a complicatedly organized neural tube.  It is unlikely that such a highly organized neural tube evolved solely for use during larval stages, especially considering that the main function of extant ascidian larvae is simply to find a place to settle and metamorphose.  Based on these observations, a free-swimming ancestor for chordates is more likely and more parsimonious than a sessile ancestor.  The evolutionary history of various lifestyles in chordates from this ancestral form is proposed.

SURVEY OF GENETIC MOLECULE MARKER OF Ciona intestinalis.   S. Kano1, S. Chiba2, N. Satoh1.  1Dept. of Zool., Graduate Sch. of Sci., Kyoto Univ.; 2Dept. of Biol., Konan Univ., Okamoto, Hisgashi-Nada-Ku, Kobe.
   Little genomic information about a solitary ascidian, Ciona intestinalis have stored. To obtain available marker sequences, RAPD markers which show genetic polymorphisms were surveyed by RAPD-PCR and AP-PCR method. In the present study, we report that reliable ones were converted to STS markers and investigated their inheritance between generations. We also report that some RAPD markers revealed genetic differences between wild population of both the Ocean side and the Sea of Japan side. These RAPD markers and STS markers are useful to progress in genetics of this species.

MUSASHI HOMOLOGUE IN THE ASCIDIAN, H.roretzi, IS EXPRESSED IN THE NEURAL TISSUE OF THE EMBRYO.   T. Kawashima, Y. Sasakura, M. Ogasawara, and K.W. Makabe.  Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ.
   Musashi gene is required for development of adult external sensory organs in Drosophila neural development. Mouse-Msi-1, musashi homologue in mouse, is essential for neural development and differentiation. We have found Hrmsi-1, a gene homologous to the Drosophila Musashi, from a fertilized egg - cDNA library of H.roretzi.Hrmsi-1 mRNA is 1.5kb in length and contains two RNA - binding motifs which are common to musashi family members. By whole mount in situ hybridization, maternal transcripts were detected in all blastomeres.  Zygotic gene expression was seen predominantly in the neural tissue of the embryo. The Hrmsi-1 protein may be involved in neural pattening in ascidian.

A HNF-6 HOMOLOGOUS GENE IS INVOLVED IN NEUROGENESIS IN ASCIDIAN EMBRYOGENESIS.   Y. Sasakura and K. W. Makabe.  Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ.
   HNF-6 is a novel class of transcription factor which contains single cut domain and homeodomain.  To know the function of HNF-6 in ascidian development, we isolated a cDNA of HNF-6 homolog from a gastrula-cDNA library of the ascidian Halocynthia roretzi.  The cDNA is about 3.5kb in length, and the predicted amino-acid sequence contains a cut domain and a homeodomain.  Whole-mount in situ hybridization revealed that HNF-6 homolog trancripts first appeared at the neural plate stage in the small regions of neural plate.  At the tailbud stage, the transcripts were detected in some parts of neural tissues including the brain.  The mRNAs of HNF-6 homolog was microinjected in the fertilized eggs of H. roretzi, and the malformation of the morphology was observed.  In addition, the expression of the neural marker gene HrTBB2 was altered.  Above results suggest that the HNF-6 homolog functions in the development of neural tissues.

Ciona BRACHYURY GENE TARGETS.   K. Hotta1. H. Takahashi1. N. Satoh1. A. Erives2. M. Levine2.  Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ., Kyoto.1?ADept. of Mol. Cell Biol.,Univ. of California, Berkeley, USA2
  When a fusion gene construct in which the promoter of Cifkh (Ciona forkhead) was fused with CiBra (Ciona Brachyury) coding sequence was injected into Ciona eggs by electroporation, CiBra ectopic expression was induced in the endodermal strand cells of tailbud-stage embryos. This CiBra ectopic expression was used to isolate candidate CiBra target genes. Subtractive hybridization method, thus far, yielded about 900 cDNA clones for CiBra downstream gene candidates. Sequencing of both 3'and 5' regions and dot blot hybridization demonstrated that, among them, 528 clones are independent and activated by CiBra overexpression. In situ hybridization revealed that several genes are expressed specifically in notochord cells of the embryo.

ISOLATION AND ANALYSIS OF AN ASCIDIAN MATERNAL T-BOX GENE, AS-MT
N. Takada1 , K.Tagawa2+ , H. Takahashi2 , N. Satoh2.  1 Dept. of Biol., Fac. of Edu., Mie Univ., 2 Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ.; + Present address: Dept. of Biochem. and Mol. Biol., Univ. of Texas MD Anderson Cancer Center, Texas, USA
   The T-box genes encode transcriptional factors that contain DNA binding region called T-domain. An ascidian egg is a typical mosaic egg, and cell differentiation and morphogenesis are determined dependent on prelocalized egg cytoplasmic factors.  In this study, taking note of T-box gene, we characterized maternally expressed T-box gene ( As-mT ).  As-mT cDNA consisted of 3819bp and encoded a polypeptide consisting of 891 amino acids with T-domain.  Its transcript was distributed almost evenly within the embryo until about 110-cell stage.  Zygotic expression was not detected. Furthermore, the injection of As-mT mRNA resulted in delay of gastrulation and tissue differentiation. This result suggests that As-mT is involved in temporal control of the early development.

ORIGIN AND EVOLUTION OF THE PHARYNGEAL GILL AND Pax1/9 RELATED GENES
M. Ogasawara, N. Satoh.  Dept. of Zool., Grad. Sch. of Sci., Kyoto Univ., Kyoto.
  The pharyngeal gill is an organ key to an understanding of the molecularmechanism underlying the origin and evolution of chordates.   We focused onthe Pax1/9 related genes (Pax1 and Pax9) that encode transcription factors and are expressed in the pharyngeal pouch of higher vertebrates.  In this study, we isolated cDNA clones of Pax1/9 related genes from urochordates (HrPax1/9 and CiPax1/9) and hemichordate(PfPax1/9).  These Pax1/9 related genes are expressed only in the adult pharyngeal gills in these species, suggesting its function in the formation of this organ.   Therefore, these Pax1/9 related genes may serve as probes for further analysis of molecular mechanisms involved in the formation and evolution of the pharyngeal gill.

INDUCTION OF SPAWNING BY INJECTION OF GONADOTROPIN-RELEASING HORMONE (GNRH) INTO COELOM OF AN ASCIDIAN Halocynthia roretzi.   K. Terakado.  Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa.
   Recently, two new froms of GnRH were identified in a tunicate by Powell et al. (1996).  They speculated that GnRH ppeptides may be released into the bloodstream to act directly on the gonads for gonadal development and spawning.  We injected GnRHs into the body cavity of the ascidian Halocynthia roretzi to examine the ability to spawn during the breeding season.  Several types of GnRHs were used, and each given at dose of 1ng, 10 ng and 100ng/gr of body weight.  It is known that, in "day-type" of H. roretzi, spawning naturally occurrs when individuals are maintained under light condition for 4-5 hrs following maintenance in dark condition, but not under continuous light.  The injected animals were all placed under illumination by fluorescence lights.  The injected animals significantly spawned sperm initially and then eggs.  Control animals also began to spawn after 4-5 hrs of light exposure.  In excised and GnRH-treated gonads, no spawning was observed.  These observations suggest that GnRH may induce spawning in the ascidian, but not directly.

LYSIS OF TUNIC LUMINOCYTES IS ESSENTIAL FOR LUMINESCENCE IN A LUMINESCENT ASCIDIAN?   Euichi Hirose1 and Kazuyoshi Chiba2.  1Dept. Chem. Biol. & Mar. Sci., Fac. Sci., Univ. of the Ryukyus, Nishihara, and 2Dept. Biol. Col. Sci., Ochanomizu Univ., Tokyo.
   One type of tunic cell (free cells distributed in the tunic) is the light source in the luminescence of Clavelina miniata.  Strong luminescence occurs in the tunic, when the tunic or tunic cells treated with cell-lytic condition (picking the tunic or hypertonic/hypotonic treatment).  High concentration of potassium ion is known to induce luminescence, but the amount is about one 80th of that induced with hypotonic treatment. Ionophores, A23187 and valinomycin, also induce luminescemce at very high concentration (0.2Ð0.01 mM), but the light amount is only one half to one eighth of that induced with potassium ion treatment.  These low amounts of luminescence may indicate that luminescence is produced from spontaneous cell lysis but not the ionic effects in these treatments.  Tunic luminocytes contain acidic vesicles that may be concerned with luminescence.  However, since luminescence was not induced with anmonium ion treatment, neutralization of the vesicles is not sufficient for the luminescence induction.  In C. miniata, cell lysis may be essential for luminescence even in natural conditions.

EXPRESSION AND FUNCTION OF A RETINOIC ACID RECEPTOR HOMOLOG IN BUDDING TUNICATES.   Y. Tsuchida, S. Fujiwara, K. Kawamura and T. Yubisui.  Dept. of Biol., Fac. of Sci., Kochi Univ., Kochi.
   Retinoic acid (RA) is an endogenous regulator of cell differentiation and morphogenesis in developing buds of the tunicate Polyandrocarpa misakiensis.  We examined the localization of a tunicate homolog of retinoic acid receptor (PmRAR) by whole mount in situ hybridization. The signal was first detected in the epidermis at the proximal-most area of developing buds soon after isolated from the parent. Then, the atrial epithelium and mesenchymal cells including the glomerulocyte of epidermal origin were stained weakly. By the stage where the gut rudiment formed, the PmRAR signal disappeared from any tissue of developing buds, but it reappeared on the pharynx of adult animals. Recombinant PmRAR protein was bound in vitro to affinity-purified PmRXR protein. This binding was enhanced by 13-cis-RA that has been shown to have the highest activity to induce the secondary bud axis. Our results suggest strongly that, like vertebrate RARs, PmRAR acts as a heterodimer together with PmRXR, while it binds 13-cis-RA rather than all-trans-RA. We suggest that one of functions of PmRAR in the epidermis is to regulate gene expression of aldehyde dehydrogenase, a potent RA synthase.

G-PROTEIN  FAMILY IN ASCIDIAN, Halocynthia roretzi.  T.Iwasa, K.Kanehara, A.Watari, N.Ohkuma, M.Tsuda.  Dept. of Life Sci., Fac. of Sci. Himeji Inst. of Technology, Hyogo.
   Heterotrimeric G proteins form a family of signal transducing molecule from cell surface receptor to cytoplasmic effector.  The family was divided several class according to their amino acid sequence identity, and also to their effector.   Recently, it became evident that G proteins occupy a large part of an interconnecting signaling pathways in living cells.  In order to investigate how such signaling networks are formed during the developmental stages and how it works in an organism, we studied the G protein family of an ascidian larva.   A well characterized cell lineage of the ascidan larvae makes it a simple model system for studying the differentiation and organization of a signaling network of an organism.  As an first step, we tried to characterize all G proteins participating in a signaling network of the ascidian larvae.  We isolated five different  clones of G protein a subunit (Ga) from a cDNA library (kindly provided from Y. Okamura) of an ascidian larva, Halocynthia roretzi.  One was a Gn, a novel Ga previously reported by us. The other three classes were found to be Gi, Gq, and Gs class based on their amino acid identity.  Comparing the full length predicted amino acid sequence, the remaining one showed a low degree of identity with any Ga class. We tentatively termed this as G?.  Two distinct cDNA clones of Gi and G? were isolated.  They were identical in a coding sequences, but different in 3' non-coding region.  Northern blot analysis of these Ga message revealed that these Ga subunit genes, although they were obtained from cDNA library of larva,  were found in all adult tissues studied.  In situ hybridization of these Ga messages in the tadpole larva shows the spatial distribution of these messages in different tissues.

GROWTH OF THE LARVAE OF THE SOLITARY ASCIDIAN Ciona IMPLANTED IN THE TUNIC OF THE ADULT.   T. Numajiri, T. A. Nomaguchi*, and H. Fujisawa.  Fac. Educ., Saitama Univ. and *Tokyo Metropol. Inst. Gerontol.
   Growth of an ascidian larva implanted in the tunic of the adult was investigated in vivo and in vitro.  The materials used were the ascidian Ciona intestinalis and C. savignyi obtained at Kiba, Yokohama and Futtsu around Tokyo Bay.  The implanted larvae produced by self-fertilization were able to metamorphose into juveniles in the tunic of their parents, whereas their metamorphosis was blocked at the caudal or acaudal stage in the tunic of non-parental adults.  The growth of larvae produced by cross-fertilization was almost suppressed at the caudal or acaudal stage in the tunic of both parental and non-parental adults.  The implanted larvae were enveloped in a capsule formed in the tunic by the recipient (larva implanted adult).  The capsules enclosing the dead larvae became shrunken or the larvae was ejected from the body of the adult.  Growth of the larvae was also arrested in vitro within the larval stage by blood cells from the non-parental adults.  These results indicate that blocking of the growth of implanted larvae is dependent on an immunogenetic interaction between the donor (larva) and the recipient.

THE RELATION BETWEEN PROTOCHORDATE ABD VERTEBRATE TROPONIN CS
H. J. Yuasa and T. Takagi.  Biol. Inst., Grad. Sch. Science, Tohoku Univ.
   The troponin C (TnC) gene of the ascidian and amphioxus, both belong to Protochordata, is a single copy gene.  On the other hand, mammal and birds possess two TnC genes, fast skeletal and slow/cardiac TCs.  In this study, we determined the cDNA of two TnC isoforms from the frog and the lamprey.  Thus it is supposed that the presence of two TnC isoforms is unversal among vertebrate species, and that the gene duplication might have occurred at a vertebrate ancestor after the protochordate/vertebrate divergence.  The distribution of introns in the TnC genes of protochordate and vertebrate are identical, except 4th intron.  One possibility is to assume that the 4th intron sliding had occurred prior to the gene duplication.  Another possibility is that the difference of 4th intron positions arised from the evolutionary independent gain of the intron.  If so, there might  have been two lineages of the TnC, the protochordate-type and the vertebrate-type.

IDENTIFICATION OF THE MYOPLASMIC COMPONENTS WHICH INTERACT WITH MYOPLASMIN-C1.   Y. Shibata and T. Nishikata.  Fac. of Sci., Konan Univ., Kobe.
   In the ascidian embryo, the myoplasm, which is thought to contain muscle determinants,  has a critical role in larval muscle formation. Myoplasmin-C1 is a component of the myoplasm and is thought to be important for the muscle cell differentiation. It is firmly associated with the egg cytoskeleton.  The sequence analysis revealed that the myoplasmin-C1 could form coiled-coil structures on both ends and might be a tethering molecule.  In this study, using 6xHis-myoplasmin-C1 fusion proteins synthesized with pET-28(+) vector, we searched for the molecules which can bind to the myoplasmin-C1. This in vitro binding assay revealed that one of the myoplasmic component, p58, could interact with myoplasmin-C1.

MTR1; A NOVEL PROTEIN WHICH RELATED TO THE OOPLASMIC SEGREGATION OF THE ASCIDIAN Ciona intestinalis.   R. Nakamori, A. R. Murakami and T. Nishikata.  Fac. of Sci., Konan Univ., Kobe.
   In the fertilized ascidian egg, cytoplasmic domain called myoplasm is thought to contain cytoplasmic determinants for the larval muscle cell differentiation. The myoplasm is segregated into the appropriate region of the egg by the movement of microtubules during ooplasmic segregation.  MTR1(microtubule related antigen 1), which is recognized by a monoclonal antibody, shows a similar localization pattern to that of microtubules. But, these two localization patterns are not identical. Moreover, MTR1 and microtubule have different relative molecular mass. During ooplasmic segregation, MTR1 localized between the myoplasm and the egg cortex. This result suggested that the MTR1 related to the segregation movement of the myoplasm.

VIRIFORM CELL SEEN AS A NATIVE TUNIC CELL OF Halocynthia roretzi THROUGH ANALYSIS OF GENOMIC DNA.   T. Abe, S. Ohtake, F. Shishikura, and K. Tanaka.  Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo.
   Viriform cells crowd close to the epidermis and move freely in the tunic matrix.  There has been an argument on whether viriform cells are natural or parasites.  In order to find out, we examined their genomic DNA fingerprints by RAPD.  Tunic pieces of H. roretzi were placed in a culture dish containing artificial sea water.  Viriform cells migrated from the cut surface and formed clusters exclusive of other cell types on the substratum. The cells were collected from the clusters in high purity of more than 95%.  The genomic DNA was isolated from mantle cells, hemocytes and viriform cells by proteinase K/phenol method. When the genomic DNA was used as templates for polymerase chain reaction (PCR) with 10-mer oligonucleotide primers (OPA-9 and OPA-18), fingerprints similar to those of mantle cells, hemocytes and viriform cells from an individual used.    It is known that viriform cells constituted the major population in the tunic matrix but are rare in the hemolymph and not found in other tissues.    It is concluded that the viriform cell is a native cell and not a parasite of H. roretzi and that it is a kind of tunic cell.

THE STRUCTURE OF "WHITE ROOTS" AND THE DISTRIBUTION OF VIRIFORM CELLS IN THE ROOTS OF Halocynthia roretzi.   Shin-Ichi Ohtake1, Teruhisa Ishii2, and Kunio Tanaka1.  1Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo. 2Dept. of Natural & Envl. Sci., Fac. of Educ. & Human Studies, Akita University, Akita.
  Halocynthia roretzi is a sessile marine animal.  Animals reared in laboratory aquaria send out some new "white roots" (attachment villi) from the tunic surface around the marginal zone of the animals within a month.  We observed new white and old roots by light microscopy and electron microscopy.  The new roots consist of the tunic cuticle, tunic matrix and some tunic vessels.  In the tunic matrix, viriform cells are prominent.  Many viriform cells distributed around the tunic vessels (epidermis) were especially distinct.  These findings are basically the same in the old roots.  The density of viriform cells is, however, higher in the new roots.  The cells crowded at an area just under the cuticle where subcuticle is formed later.  But the cells were absent from the developed subcuticle layer of the old roots.  We find that the viriform cell is a native cell and not a parasite of H. roretzi and believe it is a kind of tunic cell.  This suggests that viriform cells may play an important role in tunic formation and the new root development of this ascidian.

IDENTIFICATION OF GELSOLIN-POSITIVE CELLS IN ASCIDIAN TADPOLE LARVA AS EPIDERMAL NEURONS.   Y. Ohtsuka1, Y. Okamura1 and T. Obinata2.  1Natl Inst. of Biosci. and Human Technology Agency of Industrial Sci. and Technology M.I.T.I., Tsukuba; 2Dept. of Biol., Fac. of Sci., Chiba Univ.
   We previously reported that gelsolin, an actin filament severing and capping protein, was detectable in the cells within epidermis during early embryogenesis.  These cells possessed cilia which extended into larval tunic.  We assumed that they are epidermal sensory neurons as judged by morphological characteristics  TuNaI is neuron-specific voltage-gated sodium channel, and its expression in tadpole was very similar to that of gelsolin.  In this study, to confirm that the gelsolin-positive cells are epidermal sensory neurons, we performed two-color in situ hybridization using both gelsolin- and TuNaI-specific riboprobes.  Both signals were detected in the same cells withinepidermis, whereas the signal of troponin T, a marker for larval tail muscle, was detected in a pattern distinct from the gelsolin-signal under the same condition.  These results indicate that the gelsolin-positive cells in epidermis are epidermal neurons, and that transcription of gelsolin gene scarcely occurs in the larval muscle, although gelsolin was abundant in the adult body wall muscle.

IDENTIFICATION OF THE INDIVIDUAL MOTOR NEURONS IN ASCIDIAN LARVAE
T. Okada1,  Y. Katsuyama1,  F.Ono, Y. Okamura 1,2,3.  1Lab of Cell Biochem., NIBH.,Tsukuba, 2Univ.of Tokyo; 3PRESTO., Japan Sci. and Technology Corp., "Intelligence and Synthesis".
   We previously showed that the motor neurons of ascidian larvae originate from the A-line. However, the exact number and position of motor neurons remains unknown. In this study, we identified motor neurons in larvae of Halocynthia roretzi by expressing GFP protein under control of a neuron-specific promoter.  We microinjected the plasmid, containing GFP gene linked to the native synaptotagmin promoter, into a blastomere at the 8 and 16 cell stage. We observedGFP signals at the larval stage with epifluorescense microscope. Wefound three motor neurons consecutively lined up in the caudal neural tube.  Judging from the distribution of co-injected lineage tracer, these were the most three anterior cells of the neural tube derived from A5.2. We called these neurons moto-A, -B, and -C in order from anterior to posterior. Moto-A extends the axon onto the ventral muscle cells. On the other hand, moto-B and C innervate the dorsal muscle cells. Electronmicroscopic observation was also performed on these neurons.  We also found that some neurons originate from A5.1 in the region just posterior to the sensory vesicle. These neurons extend their axons toward motor neurons, suggesting that they are interneurons.

THE MOLECULAR PHYLOGENY OF THE GENUS Halocynthia Verrill.  T. Kakuda.  Reseach Inst. for Integrated Sci., Kanagawa Univ.
   On the coast of Japan, five species of the genus Halocynthia Verrill are found.  The phylogenetic relationships among three species of the genus Halocynthia was inferred by the amino acid sequences in the mitochondrial cytochrome b gene.  In this study, 120 amino acids of pertical cytochrome b gene were sequenced from five individuals in each species, and a phylogenetic tree was constructed using Styela clava Herdman as an outgroup.  The phylogenetic tree revealed H. roretzi (Drasche) and Halocynthia aurantium (Pallas) are more closely related than the synonym of H. hispida (Herdman).  This shape of tree constructed from sequence data was much like the one constructed from the data of restriction fragment length polymorphism (RFLP) (Kakuda '97).  However, proportion of base-pair substitution of sequence data was far larger than the rate of RFLP data.

THE ROLE OF A VITELLINE COAT COMPONENT, HrVC70, IN FERTILIZATION OF THE ASCIDIAN, Halocynthia roretzi.   E. Tanaka, T. Abe, H. Yokosawa and H. Sawada.  Dept. Biochem., Grad. Sch. of Pharm. Sci., Hokkaido Univ.
   Although mature eggs of several ascidians including Halocynthia roretzi and Ciona intestinalis, which are hermaphroditic animals, are self-sterile, immature oocytes and acidic seawater-pretreated mature oocytes of these animals are self-fertile.  We previously reported that a 70-kDA component of the vitelline coat of H. roretzi (HrVC70) appears to be specifically attached to the vitelline coat during oocyte maturation and this molecule is released from the isolated vitelline coat by treat ment with 1mM HCl.  In the present study, we investigated the binding ability of self- or non-self-sperm to the Affi-Gel 10 agarose beads that were coupled with components released from the vitelline coat under acidic conditions.  We found that the number of nonself-sperm bound to the beads is significantly larger than that in the case of self-sperm, suggesting that HrVC70 is a candidate for a self-nonself recognition molecule in the fertilization of H. roretzi.  cDNA cloning of HrVC70 revealed that it is made up of 12 EGF-like repeats, which are similar but not identical to one another.

LOCALIZATION AND VITELLINE COAT-DEGRADING ACTIVITY OF SPERM PROTEASOMES IN FERTILIZATION OF THE ASCIDIAN Halocynthia roretzi.   H. Sawada, Y. Takahashi, T. Abe, E. Tanaka,  and H. Yokosawa.  Dept. Biochem., Grad. Sch. of Pharm. Sci., Hokkaido Univ.
   We previous ly reported that the sperm 20S proteasome and 26S-like high molecular mass proteasome are involved in sperm binding to and penetration through the vitelline coat, respectively, of the eggs of the ascidian, Halocynthia roretzi.  Here, we investigated the localization and vitelline coat-degrading activity of the proteasomes during fertilization.  Ascidian sperm were treated with NHS-LC-biotin and extracted with CHAPS.  Avidin-agarose chromatography of the extract showed that about 40% of the total activity was adsorbed to the column, suggesting that a part of the proteasome is exposed to the sperm cell surface.  About 70% of the total sperm proteasome activity was detected in the hydrophobic fraction by fractionation with Triton X-114.  In addition, Suc-Leu-Leu-Val-Tyr-MCA-hydrolyzing activity, which is inhibited by MG115, was localized in the sperm head and was activated by alkaline treatment, which can induce the ascidian sperm reaction.  It was found that the purified 26S-like proteasome digested the vitelline coat 70kDa component (VC70) via the ubiquitin pathway.  Immunocytochemistry using anti-multi-ubiquitin antibody revealed that the vitelline coat was ubiquitinated during fertilization.

CIS-REGURATORY ELEMENTS SPECIFICALLY CONSERVED IN THE VERTEBRATE TYROSINASE GENE AFFECT EXPRESSION OF ASCIDIAN TYROSINASE FAMILY GENES OF A PROTOCHORDATE Halocynthia roretzi.    R. Toyoda1, S. Sato1, T. Numakunai2, T. Gojobori3, K. Ikeo3 and H. Yamamoto1.  1Biol. Inst. Yohoku Univ.; 2Marine Biol. Stn. Tohoku Univ., Asamushi; 3Natl. Inst. Genet., Mishima.
   Tyrosinase family genes are expressed in pigment cells and determine the vertebrate coloration.  In tadople larvae of a Japanese ascidian, Halocynthia roretzi, the tyrosinase gene is also expressed specifically in pigment cells.  To carry out a phylogenetic study on the regulation of tyrosinase gene expression, we cloned a putative tyrosinase gene and a gene encoding tyrosinase-related protein (TRP) from H. roretzi.  A deletion series of the 5' region of these genes were fused to a lacZ reporter gene and microinjected into fertilized eggs of H. roretzi.  Pigment cell specific expression was obtained with as little as 250 base pairs of 5' flanking sequence of the tyrosinase gene.  In vertebrates, there are several cis-elements playing important roles in expression of tyrosinase family genes.  Surprisingly, none of those vertebrate cis-elements were found in the corresponding ascidian upstream region.  Nonetheless, we found that cis-elements are active if introduced into the ascidian fertilized eggs.  These results suggest a functional conservation of developmental mechanism of pigment cells.

PHOTO-SENSITIVE NEURON IN THE CEREBRAL GANGLION OF ASCIDIAN, Ciona savignyi.
H. Tsutsui and Y. Oka.  Misaki Marine Biol. Station, Univ. Tokyo.
   Biochemical and histochemical evidence has implicated that the cerebral ganglion of adult ascidian may be a light-sensitive organ.  However, there has been no direct electrophysiological evidence to prove this possibility.  Here, we recorded membrane potentials from neurons on the ventral side of the 'isolated neural complex preparation' of adult ascidian, Ciona savignyi using intracellular microelectrodes.  Resting potentials of the neurons were -30~-60mV, and some of the neurons showed various types of voltage responses to light stimuli.  The voltage responses could be categorized as follows: 1) transient hyperpolarization, 2) transient depolarization, 3) high frequency discharges, 4) off-responses with discharges and sustained depolarization, and combinations of these.  Among these types, type (1) was the majority.  Voltage responses of neurons of type (1) showed all-or-none properties to different stimulus duration, and response amplitude decreased during hyperpolarizing DC current injections.

A MUSCLE-LIKE STRUCTURE OF Amphioxus NOTOCHORD.   K. Kubokawa1, K. Terakado2, and S. Kimura3.  1Ocean Research Inst., Univ. Tokyo; 2Dept. Regul. Biol., Fac. Sci., Saitama Univ.; 3 Dept. Biol., Fac. Sci., Chiba Univ.
   The notochord of Amphioxus has been shown to be derived from striated muscle cells by electron microscopy (Flood 1968; Welsch 1968).  The present detailed observations revealed that the striation of Amphioxus notochord differs from that of muscle.  An SDS PAGE examination showed the presence of muscle myosin heavy chains and of actin.  An actin antibody cross-reacted with the actin band.  It is to be mentioned that pieces of glycerinated notochord did not contract at all on addition of MgATP and Ca 2+.  The unique striated structure of Amphioxus notochord will be discussed in detail.

5. Amer. Soc. for Cell Biol. meeting (San Francisco Dec12-16, 1998)

LOCALIZATIONS AND MYOGENIC DETERMINANTS IN THE ASCIDIAN EGG. J Chenevert,F Roegiers, C Sardet. Station Zoologique, Villefranche sur Mer, 06230 France.  Molec Biol of the Cell(1998) Vol 9 supplement,abst 354.

PHASES OF CORTICAL AND CYTOPLASMIC REORGANIZATIONS IN THE ASCIDIAN ZYGOTE. Sardet, C., F Roegiers, C Djediat, R Dumollard, C Rouviere, P Dru, & J Chenevert.  Abstract L12 late abstracts poster session

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