Number 40
December 1996

Please note our new Web page address above. Included there are Ascidian News #31-39, plus this edition, #40.

In June we participated in the Italian Embryology Group meetings in northern Italy and greatly enjoyed seeing many Italian ascidiologist colleagues as well as Richard Cloney from Seattle and Michael Thorndyke from London. We also enjoyed meeting many other Italian embryologists for the first time, and we thank the organizer Dr. Paolo Burighel for his wonderful hospitality. From Bressanone we drove to Roscoff to work on Phallusia. Also at Roscoff were Billie Swalla, Bill Jefferies and their students working on Molgula, and Henri and Marie Goudeau and their student continuing work on Phallusia fertilization. We collaborated on some experiments with the Goudeau group and also looked for algae in ascidian tunics. We found the red filamentous Neevea in Phallusia as originally discovered in Phallusia and Ascidiella in Brittany by J. Feldmann many years ago. Jacqueline Cabioch identified the algae for us. On July 2 Charley fell on his right hip and broke the neck of the femur-- spent a week in the hospital at Brest, had surgery to implant two large titanium screws. He had 3 weeks of physical therapy in Roscoff and more when we returned home, and is now doing fine. It was difficult for a while, but with the help of our friends we stayed the full summer in Roscoff. There are 103 new publications cited in this edition of AN. Please send us a copy of all your new publications as soon as they appear; this way you will be assured of being cited in the next AN, and your reprints are a very valuable addition to our reprint file. We always welcome English language Work in Progress articles, meetings abstracts and thesis abstracts.

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

1. Armando Sabbadin, who has made very important discoveries on genetics, colony fusion and fertilization of Botryllus, is retiring after a career spanning four decades at the University of Padua. During this work Dr Sabbadin developed culture methods for Botryllus in the laboratory which are now used throughout the world. His discoveries in genetics of Botryllus first showed us how a marine invertebrate could be used for genetic studies. We join with all of you to wish Armando a long and enjoyable retirement.

2. Billie Swalla and Bill Jeffery have just published a major article on tail formation in Molgula in Science (274:1205-1208, 15 Nov.). This article details research done on the tailed and tailessMolgula species from Roscoff showing the genetic differences between them. The News section devoted over a page to this fascinating article.

3. Laura Corley won the Best Student Paper in the Div. Of Invert. Zool. at the Soc. for Integrative & Comp. Biology (formerly ASZ) in Washington DC last Dec. for her Master's thesis work on "The vascular anatomy of the urochordate Ascidia interrupta in relation to hearbeat reversal, done in Ed Ruppert's lab at Clemson Univ. (see abstract, Amer. Zool. 35:110a, #439.) Laura is now a Ph.D. student with Billie Swalla at Vanderbilt.

4. Gerhard Czihak has about 30 copies of The Sea Urchin Embryo: Biochemistry and Morphogenesis. 1975. Springer-Verlag. Please contact him directly if you wish to buy a copy; the price is $40.00 U.S. His address is Inst. fur Genetik und Entwicklungsbiol., Univ. Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria. email gerhard.czihak@sbg.ac.at

5. It is with great sadness that we report thatDr. Katsuma Dan passed away May 18 at the home of his daughter in Osaka; in the words of Dr. Motonori Hoshi, he "finished in peace his glorious life of 91 years.
Charles Lambert reminisces: I was a student in the Invertebrate Embryology course taught by Katsuma Dan and Arthur Whiteley at the Friday Harbor Laboratories in 1964. I remember the tremendous enthusiasm of Dan in explaining the cleavage a nd early development of echinoderm eggs. His lectures were outstandingly clear and taught all of us beginning graduate students a great deal about how animals develop as well as how science is done. I also remember very clearly him looking through my microscope at dividing sea urchin embryos and being just as enthusiastic as he had been 20 years before when he saw for the first time what I was now seeing. Through the years I saw him again in Woods Hole and Misaki and he was always at work on some fascinating research. Being a student in his class was one of the high points of my entire education. Every day I try to think of development in the same ways that he did and with the same enthusiasm.

1. Zeev Pancer, Immunol. Lab, Marine Pathol. Unit, Center for Biol. Res., CIBNOR, POB 128, La Paz, B.C. S 23000, Mexico. email: pancer@cibnor.mx
Recently I arrived in La Paz, Mexico, for a one-year contract to establish an invertebrate molecular immunology lab. The following are 4 "in press" publications.
a) Pancer, Z, Leuck, J, Rinkevich, B, Steffen, R, Müller, I. and Müller,WEG (1996). Molecular cloning and sequence analysis of two cDNAs coding for anionic trypsinogens from the colonial urochordate Botryllus schlosseri (Ascidiacea). Mol. Mar. Biol. Biotech.
ABSTRACT: Botryllus schlosseri is a colonial marine invertebrate that belongs to the subphylum Urochordata. Previously we analyzed the activity of a serine protease in this species, and cloned a tunicate chymotrypsin-like molecule. In the present study we further analyzed the protease activity of this animal, and found biochemical evidence also for specific trypsin-like activity. Subsequently we utilized a degenerate PCR primer to clone two B. schlosseri cDNAs coding for two different putative trypsinogens, each 243 amino acid long, that differ within the coding region in 42 amino acids and 99 nucleotides. Both clones feature the characteristics of animal anionic trypsinogens. Sequence analysis of the tunicate putative trypsinogens revealed the invertebrate characteristics of three disulfide bridges, and higher similarity to invertebrate than to vertebrate trypsinogens. We therefore propose that the typical characteristics of vertebrate trypsinogens evolved after the divergence of Urochordates and Cephalochordates.
b) Pancer, Z, Cooper, EL and Müller, WEG (1996). A tunicate (Botryllus schlosseri) molecule reveals similarity to vertebrate antigen receptors. Immunogenetics. Brief communication, no abstract.
c) Pancer, Z, Cooper, EL and Müller, WEG (1996). A urochordate putative homolog of human EB1, the protein which binds APC. Cancer Lett. ABSTRACT: The human EB1 protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer. We have cloned a putative EB1 homolog from Botryllus schlosseri (Urochordata, Ascidiacea). The tunicate deduced protein is 287 amino acid long, and is identical with 48% of the residues in human EB1 and 24-25% in two yeast hypothetical proteins. We propose that such a high degree of conservation among EB1 homologs is indicative of an essential regulatory mechanism in eukaryotic cells.
d) Pancer, Z, Scheffer, U, Müller, I and Müller, WEG 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 Comm.
ABSTRACT: Proteasomes are large protein complexes that play a major role in selective degradation of intracellular proteins. Eukaryotes feature 7 different alpha and beta subunits. Two of the vertebrate housekeeping beta-subunits have MHC-encoded homologues, that can substitute the housekeeping counterparts upon interferon-gamma induction. In the present study we report the cloning of invertebrate beta-subunit proteasome epsilon [PRCE], from the marine sponge Geodia cydonium and from the colonial tunicate Botryllus schlosseri. Sequence comparisons revealed that the sponge and tunicate proteins are strikingly similar to vertebrate and yeast PRCEs and their MHC-linked counterparts the PRCCs [also termed LMP7], and to a lesser degree also to archaebacterial proteasome subunit beta. Based on this comparison we suggest that all eukaryotic PRCEs and PRCCs feature a cleavable N-terminal propeptide, including the two mammalian PRCEs which appear to have been wrongly predicted from incomplete cDNAs. Our comparative analysis outlines 25 amino acid positions which appear to be unique for PRCCs, distinct from the corresponding residues in metazoan PRCEs. Fifteen of these positions comprise of amino acids which share no physio-chemical properties among PRCCs and metazoan PRCEs. These distinct PRCC-specific residues are likely candidates to contribute to differences between the specificity of cleavage of vertebrate proteasome assimilated either with the housekeeping PRCE subunit or those with the MHC-encoded PRCC.

2. Rosana Moreira da Rocha, Universidade Federal do Paraná, Setor de Ciências Biológicas, Depto de Zoologia CxP. 19020, 81.531-990 Curitiba, Paraná, Brazil. email:rmrocha@bio.ufpr.br
I would like to tell other ascidiologists the research I have been coordinating in our lab at Universidade Federal do Paraná, Brazil and some collaborative research.
a) "Natural Products from Ascidians and Sponges" by Dr. Roberto Berlinck, Dr. Jose Carlos de Freitas, R. M. Rocha and Dr. Eduardo Haydu. We are screening ascidians and sponges looking for natural substances with pharmacological activity, with some interesting results.
b) "Cellulose content of the tunic of some ascidians" by Dr. Rodolpho Albano and R. M. Rocha. This is a new study that we have just started with the family Styelidae that has a great diversity of tunic structure. Any suggestions will be welcome.
c) "Survey of ascidian species at the Southern coast of Brazil" by R. M. Rocha, graduate student Tatiane R. Moreno and undergraduate student Cinthia M. Nasser. The Brazilian ascidian fauna is very poorly studied and we have yet a lot to do concerning the taxonomy of the group. This study started in 1995 and we presented two posters with new records to the Brazilian list of ascidians.
d) Eudistoma carolinense as a secondary substrate at a rocky shore in southern Brazil for some macrofauna that live between the digitations of the colony" Msc Thesis in progress by Tatiane Regina Moreno. We only know about a similar study at South Africa with Pyura and would like to receive more information about other cases of macrofauna associated to ascidians. Undergraduate student Dailey Fischer is studying the effect of the size of ascidian patches on the associated macrofauna.
e) "The reproductive cycle of Phallusia nigra, Clavelina oblonga and Symplegma rubra at South-Eastern Brazil". A paper presented by RM Rocha with Sergio de Almeida Rodrigues and graduate student Tito MC Lotufo Oct. 1995 at the Cong. Latinoamericano de Ciencias del Mar (COLACMAR), Mar del Plata, AR.

1. Ascidian introductions into southern California harbors and marinas. Charles & Gretchen Lambert. Presented at the Calif. Sea Grant Workshop on Nonindigenous Species, San Francisco, Oct. 18, 1996.
When Ritter and Forsyth (1917) assayed the ascidians of San Diego and other southern California harbors, the west coast native species Ascidia ceratodes and Pyura haustor were the dominant ascidians along with numerous Ciona intestinalis (which Ritter and Forsyth considered to be introduced). Since that time many species of ascidians have arrived; some have spread and persist in these harbors. The arrival and persistance of Styela clava and S. plicata have been well documented (Abbott and Johnson 1972 for review); along with Ciona intestinalis they have replaced the native species. Sometime between 1945 and the 1960s Botryllus schlosseri arrived. We have observed the appearance of a number of additional non-indigenous species: Styela canopus (formerly S. partita) was first encountered in 1972 in south San Diego Bay. Microcosmus squamiger, first collected in Alamitos Bay in 1986, is now abundant from San Diego to Ventura. The Japanese species Ciona savignyi, first recorded in Long Beach Harbor in 1985, is now common in all harbors. A long-siphoned species distinct from both the native Ascidia ceratodes and the east coast Ascidia interrupta was first collected from Harbor Island in San Diego Bay in 1983. In 1994 we collected a third species of Ascidia very similar in external morphology to A. ceratodes which remains unidentified Symplegma oceania, first encountered in 1991, is now present in various locations on both sides of San Diego Bay. The east coast ascidian Molgula manhattensis , first collected in San Francisco Bay in the late 1950s (Trason 1959), was found in Long Beach-Newport Harbors in 1984 and is now also in Marina del Rey and Ventura. An apparently very recent introduction is Polyandrocarpa zorritensis, first collected in Oceanside Harbor in 1994 and found to be abundant also in San Diego and Mission Bays. We report on the arrival of these ascidians and their relative abundance in all harbors in southern California from San Diego to Santa Barbara and also make predictions concerning their persistence and further spreading.

2. Basal granular cells in the digestive epithelium of an ascidian. H. Koyama, College of Nursing, Yokohama City Univ., 3-9 Fukuura, Kanazawa-ku, 236 JAPAN. 19th annual meeting of the Japan Neurosci. Soc., July 1996, Kobe. Published in Neuroscience Res. Suppl. 20, 1996.
The presence of basal granular cells was studied in the digestive epithelium of the adult zooids of Polyandrocarpa misakiensis, a budding styelid ascidian. Basal granular cells are usually scattered in the simple columnar epithelium of esophagus, stomach, and intestine. Most of these cells are bottle shape. They have low electron-dense cytoplasm, apical microvilli and/or cilia, rough and smooth ER, Golgi complex, dense bodies. and many small electron-dense granules (100-200 nm in diameter) in the basal part of the cell. At least some of these cells are open type. The basal granular cells in the stomach epithelium are characterized by a low cell height, a nucleus with clefts, one or two cilia and the basolateral part of the cells often bulges into the adjoining cells. Immunohistochemical and immunoelectron microscopic study suggest that some of the basal granular cells contain serotonin-equivalent activity in their small granules. The contents of the basal granules are thought to be released into the haemocoel. Since ascidians have an open circulatory system, the biogenic substances in the secretory granules seem to locally regulate the activity of other gastric epithelial cells, and/or may be transported to the CNS, which neurally or humorally control the digestive organs, although no direct innervations to them have been found. The density heterogeneity among the secretory granules within a single cell may reflect their differences in content, or in the process of maturation of the granules containing same substance. There are morphological similarities between the stomach basal granular cells and intraepithelial primary sensory neurons of amphioxus. Both cell types are bottle shape and have apical cilia surrounded by microvilli, a nucleus with clefts, folded basolateral surface for interlocking in the epithelium. Addendum: I could not find typical basal granular cells in the hind intestine. Instead, several types of non-mucous and non-basal granular cells were found. I tentatively assume that they may be mechanoreceptive.

3. 6th Intl. Cong. on Cell Biol. & 36th Amer. Soc. for Cell Biol. annual meeting, San Francisco, December 7-11, 1996.

689. Mechanism of an evolutionary change in muscle cell differentiation in ascidians with different modes of development. T Kusakabe, BJ Swalla, N Satoh, WR Jeffery.
We have investigated the mechanism of an evolutionary change in ascidian muscle cell development. The ascidians Molgula oculata and M. occulta are closely-related spp. with different modes of development.M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into modified tailless larvae lacking differentiated muscle cells. MocuMA 1 is a single copy, larval-type muscle actin gene of M. oculata. MocuMA 1 mRNA first appears in the prospective muscle cells during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA 1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA 1a and MoccMA 1b were isolated from an M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products non-functional. Expression of MoccMA 1a/lacZ and MoccMA 1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMA 1b. These results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.

690. Myogenic determinants in the ascidian egg. J Chenevert & C. Sardet, URA 671 CNRS/UPMC, Mar. Cell Biol., Station Zoologique, 06230 Villefranche-sur-Mer, France.
The fertilized ascidian egg contains cytoplasmic domains of distinct cytoskeletal and organelle composition in which determinants of developmental fate are localized. One of these domains, the myoplasm, contains molecules which specify muscle development in the cells which inherit it after cleavage. We wish to identify these muscle determinants and to elucidate their mechanism of localization and segregation. We have undertaken two approaches. The first is to isolate ascidian homologs of the myogenic bHLH factors. Our first clone, "PMF", encodes a myoD-like protein. It is expressed transiently during embryonic development, starting prior to gastrulation at about the 64 cell stage. Spatially, PMF expression is confined to the myoplasm-containing blastomeres, which are those which will give rise to the muscle cells of the tadpole tail. Analysis of the in vivo function of PMF is underway. Since PMF message is absent in the egg, it is unlikely to be the primary determinant in the myoplasm. We are therefore searching for other factors, of the MyoD and MEF2 families, which could function upstream of PMF. An alternative approach to identify determinants relies on function rather than homology. Egg messages are microinjected into nonmuscle blastomeres and are screened for their ability to promote a myogenic fate in the embryo. Positive clones are amplified and purified. Our model is that the myoplasm-localized determinant, of as yet unknown identity, acts to turn on PMF in the cells which inherit it. Future work will address the mechanism of this activation.

691. Localization and movement of cytoplasmic domains in ascidian zygotes. F Roegiers & C Sardet. [address same as above]
In the egg of the ascidian Phallusia mammillata , fertilization triggers the formation and reorganization of distinct cytoplasmic domains in two phases. A first phase occurs shortly after fertilization and is triggered by the activating Ca++ wave (Roegiers et al., Development 121, 1995), the second occurs after the completion of meiosis and depends on the displacement and growth of the large sperm aster. Two main domains are relocalized, the mitochondria-rich myoplasm, and a domain of endoplasmic reticulum (ER) accumulation that forms during the first phase. The myoplasm, labelled with DiO(C2)3, ER with DiIC16(3), and microtubules labelled with rhodamine, were observed in vivo using a confocal microscope. During the second phase of relocalization, both the ER domain and the myoplasm migrate into the center of the sperm aster and with it away from the membrane surface. This movement depends on the presence of microtubules. Before this bulk movement and immediately following meiosis, the second phase begins with a transient protrusion ("vegetal button"). The vegetal cortex then begins to vibrate. This is followed by a surface contraction wave just prior to the onset of mitosis. None of these surface movements appear to be microtubule dependent, as all of the events occur in the presence of 1.2 uM nocodazole. In treated eggs the sperm aster does not form, the second phase of ooplasmic segregation is incomplete, and the eggs fail to undergo mitosis.

2842. A shark family tyrosine kinase, localized in the myoplasm, is implicated in axis formation and muscle determination in ascidians. BJ Swalla. [similar to abstracts below]

3715. Liposomal delivery of inositol 1,4,5-triphosphate activates sperm of the sea squirt, Ascidia ceratodes . FE Garrett & RA Koch, Dept. of Biol.Sci., Calif. State Univ., Fullerton, CA 92834.
Sea squirt (Ascidia ceratodes) sperm cells are useful for studying cell signaling in fertilization. A rise in intracellular calcium ion (Ca) concentration triggers many cellular events and is required for sperm activation in all organisms studied. Based on previous work, we propose that sea squirt sperm activation is dependent on Ca entry from the external sea water via G protein-regulated opening of Ca channels or Ca release from non-mitochondrial internal stores via inositol 1,4,5-trisphosphate (IP3) gated-Ca channels. Mitochondrial translocation was the assay for sperm activation, and fluorescence of the Ca-sensitive dye fura-2 and octadecyl rhodamine B (R18) were measured in bulk samples by spectrofluorimetry. Liposomes, used to deliver impermeable agents, were made by dialyzing a mixed micellar solution of distearoyl PC : distearoyl PE : cholesterol (2:1:1 mole ratio) against HEPES-buffered KCl (740mM) at pH 8.0 in the Mini Lipoprep (Sialomed, Inc.). Chloroform was the solvent and sodium cholate (22.4 mg/ml) was the detergent. We produced liposomes with a mean diameter of 98.2±15.4 nm as determined by laser-light scattering and verified by freeze-fracture analysis. Liposome-sperm fusion was monitored by a lipid-mixing assay using R18-loaded liposomes (8-10 mole-percent, R18 to total phospholipid). Fusion of R18-liposomes with live sperm cells produced a concentration-dependent rise in fluorescence intensity as R18 self-quenching was reduced upon membrane mixing. Ca-loaded (10mM) liposomes, used to test whether liposomes could deliver a membrane-impermeable agent, were found to cause a time-dependent increase in sperm activation compared to sperm-only and empty-liposome controls. IP3-loaded (1mM) liposomes also caused a time-dependent increase in sperm activation, a response that could be blocked by the presence of heparin (125 mg/ml) in IP3-loaded liposomes. We conclude that: (1) liposomes can be used to deliver impermeable agents into small, live sperm cells and (2) IP3-dependent Ca release from internal stores is sufficient to activate sperm. (Support: NIH R15-HD28229 to RAK and NIH MBRS SO6-GM08258 for FEG).

4. Soc. for Dev. Biol. annual symposium, Patterning Mechanisms in Development. Vanderbilt Univ., Nashville, TN, May 30-June 4, 1996. Dev. Biol. 175:369-402.

Molecular analysis of ascidian development and evolution. B.J. Swalla, Vanderbilt Univ., Nashville, TN 37235.
Evolutionary changes in developmental genes may profoundly influence larval morphology. These changes are readily seen in ascidian larvae, which function primarily as a dispersal mechanism for the species. Ascidians are the simplest chordate, containing a tail with a dorsal nerve chord, a notochord, flanking muscle cells, and a ventral endodermal strand. We have isolated maternal genes involved in specfying the chordate body plan by subtractive hybridization between two sister species with dramatically different larval forms. Three of these genes, manx, lynx, and cymric, are expressed in the species that makes a larval tail, but not expressed in a sister species that has a tailless (anural) larve. Manx appears to be a zinc finger transcription factor involved in specifying body plan, cymric is a shark family tyrosine kinase with 5 ankyrin repeats that is localized in the myoplasm and may be involved in specifying muscle cells. These maternal genes may interact with p58, a unique protein localized in the myoplasm. The integrity of the myoplasm appears to be important for both body plan specification and tissue differentiation in ascidian larvae because it is disrupted in those species in the Molgulidae family that have evolved tailless development. Supported by NSF grant IBN-9304958.

Cymric, a shark family non-receptor tyrosine kinase, is expressed differentially in ascidian species with different larval phenotypes. A.R.W. Hatmaker1, K.M. Suling1, B.J. Swalla1, and W.R. Jeffery2. 1Vanderbilt Univ., Nashville, TN 37235 USA. 2Section of Molec. & Cell. Biol., Univ. of Calif., Davis, CA 95616.
Cymric (or uro-1) was identified in a subtractive screen of gonad cDNAs from two closely related ascidian species. Southern blots show that cymric is present in M. oculata , which has a tailed larva, but the cymric gene cannot be detected in M. occulta , which has a tailless larva. The cymric gene encodes a 3 kb mRNA which is expressed during oogenesis in M. oculata . The cymric protein encodes a non-receptor tyrosine kinase (TK) with an N-terminal region containing two SH2 domains flanking five ankryin repeats and a C-terminal tyrosine kinase, separated by a short proline rich sequence, which may be an SH3 binding site. Thus, the ascidian cymric gene is the third described member of the SHARK family of non-receptor Tks, which also includes the hydra HTK 16, Drosophila shark. In situ hybridization shows that cymric mRNA is expressed in M. oculata oocytes, and is expressed in presumptive muscle cells during development in M. socialis , another tailed species. Supported by NSF grant IBN-9304958.

A shark family tyrosine kinase is reduced in the myoplasm during oogenesis in the tailless ascidian, Molgula occulta . JM Ross, KM Suling, and BJ Swalla. Vanderbilt Univ., Nashville, TN 37235.
The recently-described SHARK family of non-receptor tyrosine kinases includes at least three genes: hydra HTK 16, Drosophila shark, and ascidian cymric. A polyclonal antibody to Drosophila shark recognizes a single protein of 68kD on western blots of gonad proteins from the ascidian Molgula oculata , which has a tailed tadpole larva, and its sister species Molgula occulta , which has a tailless larva. The protein was reduced in M. occulta gonad proteins compared to M. oculata gonad proteins. The localization of the 68kD protein was then analyzed by immunofluorescence and imunohistocytochemistry. The putative shark tyrosine kinase is present in previtellogenic oocytes of both species, but was only localized to the cortical myoplasm in M. oculata , the tailed species. The protein was abundant in presumptive muscle cells through gastrulation and neurulation, but was reduced before overt muscle differentiation in M. oculata . We suggest that the shark TK may function in signal transduction during assembly of the myoplasm and/or later during muscle specification. The evolutionary modification of the localization of this tyrosine kinase in the myoplasm may mediate the tailless M. occulta larval phenotype. Supported by NSF grant IBN-9304958.

Posterior end mark, a novel maternal gene for localized signals in the ascidian embryo. S. Yoshida, Y. Marikawa & N. Satoh. Dept. Zool., Grad. Sch. Sci., Kyoto Univ., Sakyo-ku, Kyoto 606-01, Japan.
Maternal factors are implicated in the establishment of animal body plan. The ascidian embryogenesis is regarded as a typical "mosaic" type, and recent studies have provided convincing evidence that the posterior-vegetal cytoplasm of the fertilized egg is associated with the determinant and the establishment of antero-posterior axis of the embryo. We report here an identification and characterization of a novel maternal gene, posterior end mark (pem); the transcript is initially concentrated in the posterior-vegetal cytoplasm of the fertilized egg, and later marks the posterior end of developing ascidian embryos. The predicted PEM protein showed no significant homology to known proteins. Overexpression of this gene by microinjection of synthesized pem mRNA into fertilized eggs resulted in development of tadpole larvae with deficiency of the anterior-most adhesive organ, dorsal brain and sensory pigment-cells. These data suggest that pem encodes a component of the posterior-vegetal cytoplasm and plays a role in patterning of the ascidian embryo.

Cloning and characterization of BMP-related genes of the ascidian,Halocynthia roretzi . T. Miya, K. Morita A. Suzuki, N. Ueno & N. Satoh. Address same as above.
The ascidian tadpole larva is thought to be the prototype for the ancestral chordate. Although ascidians show a highly determinate mode of development, recent studies suggest significant roles of cell-cell interaction in embryogenesis. To elucidate the signaling molecules responsible for the cellular interaction, we investigated ascidian homologues of the TGF-beta superfamily. We isolated two cDNA clones with sequence similarity to vertebrate BMPs (bone morphogenetic proteins). HrBMPa is an ascidian member of the 60A subclass of the BMP family and resembles vertebrate BMPs 5-8. The other, HrBMPb, belongs to the DPP subclass and resembles vertebrate BMPs 2-4. Both HrBMPa and HrBMPb transcripts were seen in the midline of ectoderm during embryogenesis. Ectopic expression of HrBMPb resulted in development of larvae lacking some neural tissues, suggesting its role in determination of neural/epidermal fate of ectodermal cells.

The cellular basis of ascidian notochord formation. E.M. Munro & G.M. Odell. Dept. Zool., Univ. of Washington, Seattle WA 98192.
Timelapse observations of live ascidian embryos and confocal analysis of fixed, whole-mounted phalloidin-stained embryos reveal a highly stereotyped pattern of 3D cell rearrangements and shape changes that accompanies the transformation of the notochord from a monolayer epithelial disk of 40 cells into a single stack of coin-shaped cells. Globally, the notochord folds ventrally and its lateral edges fuse at the midline to form a tube. Locally, notochord cells retain their monolayer organization, elongate and intercalate perpendicular to the AP axis - first as columnar epithelial cells, then as pizza slices stacked around the tube. Direct observations of live embryos combined with SEM and confocal microscopy show that notochord cells extend their edges across the interior faces of adjacent notochord cell neighbors by means of F-actin rich protrusions, while exterior notochord boundaries are stabilized by interdigitating lamelliform protrusions. We find that notochord cell shape changes and rearrangements are patterned with respect to both the AP axis and to cell movements and tissue deformations taking place within surrounding tissue (e.g. mesoderm, endoderm, neural plate). Moreover, notochord primordia isolated at early gastrula stage complete their final divisions and become motile with normal timing, but fail to form notochords. On the basis of tissue ablation and recombination experiments, we find that no individual bounding tissue is required for notochord formation after the early gastrula stage but each tissue plays a distinct role in determining the normal pattern of cell shape changes and rearrangements. We interpret these results in the context of several possible models for notochord formation.

Abas, S.A., M.B. Hossain, D. vanderHelm & F.J. Schmitz 1996. Alkaloids from the tunicate Polycarpa aurata from Chuuk atoll. J. Org. Chem. 61:2709-2712.

Araki, I. & N. Satoh 1996. cis-regulatory elements conserved in the proximal promoter region of an ascidian embryonic muscle myosin heavy-chain gene. Dev. Genes & Evol. 206:54-63.

Araki, I., K. Tagawa, T. Kusakabe & N. Satoh 1996. Predominant expression of a cytoskeletal actin gene in mesenchyme cells during embryogenesis of the ascidian Halocynthia roretzi . Dev., Growth & Differ. 38:401-412.

Arizza, V., M. Cammarata, M.C. Tomasino & N. Parrinello 1995. Phenoloxidase characterization in vacuolar hemocytes from the solitary ascidian Styela plicata. J. Invert. Pathol. 66:297-302.

Azumi, K., H. Takahashi, R. Ishimoto & H. Yokosawa 1996. Effects of plant lectins on cellular defense reactions of ascidian hemocytes. Experientia 52:839.

Azumi, K. & H. Yokosawa 1996. Characterization of novel metallo-proteases released from ascidian hemocytes by treatment with calcium ionophore. Zool. Sci. 13:365-370.

Bak, R.P.M., D.Y.M. Lambrechts, M. Joenje, G. Nieuwland & M.L.J. Van Veghel 1996. Long-term changes on coral reefs in booming populations of a competitive colonial ascidian. Mar. Ecol. Prog. Ser. 133:303-306.

Bates, W.R. & C. Bishop 1996. Localization of constitutive heat shock proteins in developing ascidians. Dev., Growth & Differ. 38:307-314.

Bishop, J.D.D. 1996. Female control of paternity in the internally fertilizing compound ascidian Diplosoma listerianum. 1. Autoradiographic investigation of sperm movements in the female reproductive tract. Proc. Roy. Soc. Lond. ser. B 263:369.

Bishop, J.D.D., C.S. Jones & L.R. Noble 1996. Female control of paternity in the internally fertilizing compound ascidian Diplosoma listerianum. 2. Investigation of male mating success using RAPD markers. Proc. Roy. Soc. Lond. ser. B 263:401-408.

Bishop, J.D.D. & A.D. Sommerfeldt 1996. Autoradiographic investigation of uptake and storage of exogenous sperm by the ovary of the compound ascidian Diplosoma listerianum. Mar. Biol. 125:663-670.

Bolton, T.F. & J.N. Havenhand 1996. Chemical mediation of sperm activity and longevity in the solitary ascidians Ciona intestinalis and Ascidiella aspersa. Biol. Bull. 190:329-335.

Bruno, J.G., S.B. Collard, D.J. Kuch & J.C. Cornette 1996. Electrochemiluminescence from tunicate, tunichrome-metal complexes and other biological samples. J. Biolumin. & Chemilumin. 11:193-206.

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