ASCIDIAN NEWS*
Gretchen and Charles Lambert
206-365-3734
glambert@fullerton.edu or clambert@fullerton.edu
home page: http://depts.washington.edu/ascidian/
Number 60 April 2007
Thanks to all of you who sent in contributions; this is the largest issue of AN we have ever put together! There are 114 new publications listed at the end of this issue! Keep up the good work and to assure that your publications will appear in AN please don’t forget to send us hard copy or PDFs; thank you.
We heartily congratulate Dr. Xavier Turon who was chosen this year’s Paul Illg Distinguished Lecturer for the Friday Harbor Labs; we look forward to hearing about his latest findings.
In December we spent
a few days at the Friday Harbor Labs for the 90th Birthday of
Charley’s Ph.D. advisor at the
*Ascidian
News is not part of the scientific literature and should not be cited as such.
1. Upcoming 4th Intl. Tunicata Conference June 23-27, 2007, Villefranche-sur-Mer, France, organized by Christian Sardet, Station Zoologique, Observatoire, CNRS, Univ PM Curie, Villefranche. christian.sardet@obs-vlfr.fr The meeting will be held in Residence Delcloy situated on St. Jean Cap Ferrat about a mile from the Marine Station. To see the conference site: http://www.vacancesbleues.com/destinationRechercheMono.do, click on France then Mediterranee then St. Jean Cap Ferrat and click on the 360 panoramics to see the facilities. For meeting details: https://biodev.obs-vlfr.fr/tunicatemeeting/ We have already more than 100 participants and have made the call for abstracts. The meeting will be limited to 120-130 people.
We have assembled a new archive of 29 ascidian films and embryos
http://biodev.obs-vlfr.fr/recherche/biomarcell/films%2Bimages/filmarchive.htm
It goes with a review "From oocytes to 16 cell stage: the cytoplasmic and cortical reorganization which pattern the ascidian embryo" by our group which will be posted on-line in Developmental Dynamics.
2. The Biology, Biogeography, and Ecology of
Invasive Ascidians.
3. From Dr. Ivan Goodbody ivan.goodbody@uwimona.edu.jm :
The Mona Institute of Applied Sciences (MIAS)
at the University of the West Indies, Mona Campus, Jamaica has released
a CD-ROM featuring ‘An Introduction to Caribbean Sea Squirts (Ascidiacea)’
compiled by Emeritus Professor Ivan Goodbody and illustrated by photographs of
most common species taken while alive in their natural environment. Each
photograph is accompanied by both a vocal and a textual description to guide
the viewer. The work is designed for use by students and researchers working in
the
Also from Ivan
The Pelican Cays in
Recently,
disturbing reports have been received that developers have moved into the area
with the intention of creating a tourist resort. Ivan Goodbody considers that
the Pelican Cays is one of the most important sites for ascidians in the entire
4. From Lucia Manni, Dept. Biology,
More than 50 years ago, Prof. Armando Sabbadin, now professor emeritus at the University of
Padova (87 and still working in the lab almost every day rearing his selected
genotype), introduced Botryllus
schlosseri in the lab as a model organism in various field of biology,
beginning a series of researches which are still going on in our and in other
Universities. The importance of B.
schlosseri as a reference organism in the study of asexual and sexual
reproduction, developmental biology, regeneration, allorecognition,
immunobiology, stem cell biology, apoptosis, is now well established.
As pupils of Prof. Sabbadin, in order
to remember his lucky choice and to acknowledge him for his great passion for
ascidian biology and scientific rigour, we wish to dedicate a one-day meeting
to discuss, together with Prof. Sabbadin, the most recent results and views in
compound ascidian biology. The meeting will be entitled:
Fifty years of Botryllus
schlosseri as a model organism in biology: results and perspectives
The meeting will take place at the
dept. of Biology, University of Padova, Friday, June 29, 2007, a couple of days after the end of the international
Tunicate Meeting at Villefranche. It will have an informal character and will
offer the occasion to meet and exchange opinions about botryllus with prof. Sabbadin
(now 87 but always active in research).
Please, contact the organizers (paolo.burighel@unipd.it
; loriano.ballarin@unipd.it ; lucia.manni@unipd.it ; giovanna.zaniolo@unipd.it ; francesca.cima@unipd.it ) if
interested.
5. 'It is really difficult to get some drama out of a sea squirt,'
said Sir David Attenborough, in an
interview by Jonathan Dimbleby at the Natural History Museum in
6. You will find
lots of drama in the following: J. of
Experimental Mar. Biol. and Ecology special issue vol. 342 (1), 2007: Proceedings of the 1st
Introduction to the
Proceedings Pp. 1-2. Robert B. Whitlatch and Stephan G. Bullard
Invasive sea squirts: A
growing global problem. Pp. 3-4.
Gretchen Lambert
The non-native solitary
ascidian Ciona intestinalis (L.)
depresses species richness. Pp. 5-14. Julia C. Blum, Andrew L. Chang,
Marcela Liljesthröm, Michelle E. Schenk, Mia K. Steinberg and Gregory M. Ruiz
Effects of small-scale
disturbance on invasion success in marine communities. Pp. 15-29. Safra
Altman and Robert B. Whitlatch
Spatial and temporal
effects of pre-seeding plates with invasive ascidians: Growth, recruitment and
community composition. Pp. 30-39. Brad P. Agius
Variation in the ability
of Didemnum sp. to invade established
communities. Pp. 40-53. Richard W. Osman and Robert B. Whitlatch
Do artificial substrates
favor nonindigenous fouling species over native species? Pp. 54-60. Megan
C. Tyrrell and James E. Byers
Distribution and
long-term temporal patterns of four invasive colonial ascidians in the
Invasion of the southern
Reproduction and early
life history of an invasive ascidian Styela
clava Herdman in
Spatial and temporal
patterns of recruitment of the tunicate Ciona
intestinalis on a mussel farm in
Feeding preference of Strongylocentrotus droebachiensis
(Echinoidea) for a dominant native ascidian, Aplidium glabrum, relative to the invasive ascidian Botrylloides violaceus. Pp.
93-98.Melissa Simoncini and Robert J. Miller
The colonial ascidian Didemnum sp. A: Current distribution,
basic biology and potential threat to marine communities of the northeast and
west coasts of
Ecological observations
on the colonial ascidian Didemnum sp.
in a
Recent population
expansions of non-native ascidians in The Netherlands. Pp. 122-126. Adriaan
Gittenberger
Ascidian suspension
feeding. Pp. 127-137. Jens Kjerulf Petersen
Phylogenetic diversity
of bacteria associated with ascidians in Eel Pond (Woods Hole,
Benthic foraminifera
associated with the invasive ascidian, Didemnum
sp. A. Pp. 147-153. Mary R. Carman
Development and
application of tools for incursion response: Lessons learned from the
management of the fouling pest Didemnum
vexillum. Pp. 154-162. Ashley D.M. Coutts and Barrie M. Forrest
Competition among
invading ascidians and a native mussel. Pp. 163-165. Rubi Rajbanshi and Judith
Pederson
Fragmentation of
colonial ascidians: Differences in reattachment capability among species.
Pp. 166-168. Stephan G. Bullard, Betsy Sedlack, James F. Reinhardt, Carolyn
Litty, Keith Gareau and Robert B. Whitlatch
The role of colonial
ascidians in altering biodiversity in marine fouling communities. Pp. 169-171.
Jennifer Dijkstra, Hillary Sherman and Larry G. Harris
Effects of temperature
on growth rates of colonial ascidians: A comparison of Didemnum sp. to Botryllus
schlosseri and Botrylloides violaceus.
Pp. 172-174. Anna McCarthy, Richard W. Osman and Robert B. Whitlatch
Water quality, nitrogen
pollution, and ascidian diversity in coastal waters of southern
The occurrence of the
colonial ascidian Didemnum sp. on
The distribution of Styela clava (Tunicata, Ascidiacea) in
European waters. Pp. 182-184. Martin H. Davis and Mary E. Davis
Spread of Microcosmus squamiger (Ascidiacea:
Pyuridae) in the
Occurrence and
distribution of ascidians in
7. From Erin Newman-Smith, Director, Ascidian Stock Center, MCD Biology, University of California, Santa Barbara, CA 93106-9610.
The
1. Anna Epelbaum (NSERC Visiting Fellow, EpelbaumA@pac.dfo-mpo.gc.ca), Chris Pearce, and Tom Therriault (Pacific Biological Station, Nanaimo, British Columbia, Canada) have a project in progress regarding abiotic and biotic factors affecting the growth, survivorship, and reproduction of non-native tunicate species in British Columbia (BC), Canada. Currently, there are at least four species of tunicates in BC which are non-native and potentially invasive: the solitary tunicate Styela clava and the colonial tunicates Botrylloides violaceus, Botryllus schlosseri, and Didemnum sp. While these species are present at many shellfish culture leases around Vancouver Island, they do not appear to have proliferated to the extent that they have in Atlantic Canada and the BC shellfish culture industry would like to minimize the potential impact of invasive tunicate species by better understanding the factors affecting their invasion success. We have conducted preliminary lab experiments examining survivorship and growth of juvenile colonies of B. schlosseri and B. violaceus under 36 combinations of a wide range of temperatures and salinities (0-25ºC, 8-38 ppt). Lab experiments aimed at assessing predation effects on survivorship and growth of B. schlosseri, B. violaceus, and S. clava are currently underway. We are testing predators which can potentially be used to control tunicate fouling on aquaculture gear (e.g. sea urchins, skeleton shrimps), as well as those which would potentially control tunicate spread on natural substrates and limit their ability to invade new habitats (e.g. various species of crabs and sea stars). We are also planning to perform a series of trials to assess the feasibility of various physical and chemical treatments to eradicate these non-native tunicate species.
2. From Dan Minchin, 3
Marina Village, Ballina, Killaloe, Co Clare,
New tunicate records
in
A rapid assessment
survey in
In an earlier study
the Asian tunicate was found on the south-west coast of
The researchers in
Minchin D, Davis MH,
Minchin D, Sides E 2006. Appearance of a cryptogenic
tunicate, a Didemnum sp. fouling
marina pontoons and leisure craft in Ireland.
Aquatic Invasions 1(3): 143-147.
Minchin D 2007. Rapid coastal survey for targeted alien species associated with floating pontoons in Ireland Aquatic Invasions 2(1): 63-70.
3. Patrick Frank,
Dept. of Chemistry,
We are in the final presubmission stages of work that will include a hypothesis describing the biological reduction of inorganic vanadate to vanadyl and V(III) in ascidians. This work was done with Bob and Elaine Carlson, Britt Hedman, and Keith Hodgson. We used x-ray absorption spectroscopy to follow the fate and uptake of vanadyl ion in the blood cells of A. ceratodes. The results are fully consistent with previous work on Phallusia nigra with Bill Robinson and Ken Kustin (2001 J. Inorg. Biochem. 86, 635-648). All the detected intracellular forms of vanadyl ion varied in linear concert, implying biological regulation with active feedback. The enzymatic hypothesis, which proposes the existence of a vanadium reductase in ascidians, follows directly from the results of our study, and includes details of an active site mechanism that accomodates the varied structural requirements of all three vanadium oxidation states. We will also report new inorganic chemistry that validates this proposed mechanism. The brand-new and undoubtedly rich field of vanadium metalloenzyme redox biochemistry is indicated.
4.Maximilian Haeussler, Equipe INRA U1126 "Morphogenèse du système
nerveux des Chordés", UPR 2197 DEPSN, CNRS, Institut de Neurosciences A.
Fessard, Gif-sur –
In a side-activity from
my thesis on Ciona intestinalis, I
have built a local website that my group leader Jean-Stéphane Joly and I would like to make available to the Ciona community. At present, it contains
a home page, a simple genome browser customized for ascidian studies (see
below), a series of images of Ciona
intestinalis tailbud stage embryos obtained by confocal microscopy that were
used to refine the definition of stages, and links to the site of the Gif
transgenesis platform (under construction)
and to other ascidian websites. Here is the rational for adding one more
genome browser to the already excellent existing ones: I first spent a lot of
time using them. What bugged me was that I had to switch all the time between
the browsers made by Ensembl, UCSC,
The idea is that you can type in any kind of gene number (even human symbols) and see where there is information in one of the various databases about it, to compare their intron/exon structure and 5' / 3' ends. As the browser is in a very preliminary stage (e.g. many gene models don't distinguish between UTR and coding sequence yet), I'm happy for any feedback on it or on the developmental table to haeussle@iaf.cnrs-gif.fr or joly@iaf.cnrs-gif.fr . http://www.ciona.cnrs-gif.fr/ The site will be online by about mid-April.
1. Intl.
Council for the Exploration of the Sea (ICES) Symposium,
Reducing the Impact
of the Invasive Ascidian Didemnum sp.
on the Shellfish Industry in
Several species of
ascidians have invaded the
2. VIIIth scientific meeting of the Italian Association for
Developmental and Comparative Immunology (IADCI), 1 and 2 March 2007, Area
della Ricerca, CNR, Naples,
a. A novel rhamnose-binding lectin from the compound ascidian Botryllus
schlosseri. L Ballarin,
Animal lectins play a fundamental role in
invertebrate immunity, as they are involved in the recognition of microbial molecular
patterns which, in turn, triggers various effector responses, such as
opsonisation, encapsulation, activation of the pro-PO activating system,
phagocytosis. In a previous study, we purified by affinity chromatography and
partially characterised a soluble Ca2+-independent lectin, with
specificity for b-galactosides, from the blood of the colonial ascidian Botryllus
schlosseri. The molecule can agglutinate rabbit erythrocytes, is secreted
by haemocytes upon the recognition of foreign particles and behaves as an opsonins (Ballarin et al., 1999, 2000).
Recently, we purified further this protein by RP-HPLC, obtaining 4 lightly
different peaks, likely isoforms of the same molecule. The MWs estimated using
mass spectrometry ranged between 10.7 and 11.1 Kda. The lectin was digested
with trypsin and tryptic fragments were sequenced by mass spectrometry. Blast
analysis of the main sequences obtained indicated a high degree of homology
with rhamnose-binding proteins, a family of S-type lectins described in sea urchin
and teleosts. The specificity for rhamnose (and the similar melibiose) was
successively demonstrated in haemoagglutinating inhibition assays. We prepared
a full length cDNA library from Botryllus colonies from which we
obtained three full sequences of transcripts which, after BLAST analysis,
resulted highly homologous to known genes for rhamnose-binding lectins. Their
putative aminoacid sequences contained our tryptic peptide sequences.
b. Undifferenteed cells in the blood of the colonial
ascidian Botryllus schlosseri: a morpho-functional charaterisation. F Cima, L Ballarin. Dept.
of Biology,
Colonies of the ascidian Botryllus
schlosseri undergo a periodic tissue renewal in the take-over stage of the colonial
blastogenetic cycle, during which an extensive apoptosis occurs in the adult
zooid tissues and the senescent cells are progressively removed by circulating
phagocytes. The haemocytes which circulate in the common vascular system also
die partly by apoptosis during this stage. These cells are replaced by new
haemocytes, likely differentiating from stem cells. Up to now, haemopoiesis was
observed only in solitary ascidians in which haematopoietic noduli were
described in the branchial wall. Nothing is known on haemopoiesis in colonial
species, in the blood circulation of which two cell types with the morphology
of undifferentiated cells are recognizable: haemoblast and lymphocyte. We have
studied the cytochemical and immunocytochemical properties of these haemocytes:
results indicate the haemoblast as a pluripotent stem cell since it shows a
basophilic nucleus labeled either with Hoechst 33342 for euchromatin or
anti-Ki-67 and anti-PCNA antibodies specific markers of nuclear proteins
involved in cell proliferation and its plasma membrane is labeled by anti-CD34
and anti-CD100 antibodies, specific for haemopoietic cells in vertebrates. Commercial antibodies for cytokine receptors, like interleukin 1
receptor I (IL-1RI) and stem cell factor receptor (SCF-R) label haemoblast
plasma membrane, suggesting the presence of growth factor receptors.
Both lymphocytes and haemoblasts during the colonial cycle show a significant
increase in concentration during the blastogenetic replacement. However,
mitosis figures were rarely observed in circulating haemocytes. In vitro assays
of haemocyte exposure to colchicine showed the presence of mitosis figures,
which significantly increase after exposure to bacteria indicating a
proliferating capability in blood circulation mainly as an immune response as
observed in other invertebrates like molluscs.
c. Toll-like receptors in haemocytes of the colonial ascidian Botryllus
schlosseri: preliminary results. A
Menin, L Ballarin, Dept. of Biology,
Toll-like receptors (TLRs) represent a
well-known family of pattern recognition receptors, expressed by immunocytes,
the importance of which in non-self recognition was demonstrated in both
Vertebrates and invertebrates. In the colonial ascidian Botryllus schlosseri,
we used commercial anti-TLR2 and anti-TLR4 antibodies to inquire into the
presence of Toll-like receptors (TLR) in haemocytes lysates. After SDS-PAGE,
the immunoblot analysis revealed single protein bands recognised by the two
antibodies, of 34 kDa and 32 kDa for TLR2 and TLR4, respectively.
Immunocytochemical investigation on monolayers
of fixed haemocytes, previously exposed to E. coli LPS and yeast cells,
revealed the expression of molecules recognised by TLR2 on activated phagocytes,
whereas no labelling was observed with TLR4. We also studied the role of NF-kB
in the signal transduction pathway related to phagocytosis. Immunocytochemical
analysis with anti-NF-kBp65 antibody revealed the labelling of the cytoplasm of
untreated cells, whereas haemocytes exposed to yeast cells or Bacillus
clausii spores showed a marked staining of the phagocyte nucleus. The NF-kB
inhibitors Na-pyrrolidinedithiocarbamate and parthenolide, at sublethal
concentrations, significantly inhibits both the ingestion of yeast cells by Botryllus
phagocytes and the nuclear translocation of the activated factor. The same
molecules have no effects on the morphology of haemocytes. On the whole, our
data suggest that, in our species, TLR are involved in phagocytosis and act
through the activation of NF-kB.
d. Signal transduction in phagocytosis of the colonial ascidian Botryllus
schlosseri: a preliminary approach. A Menin,
In the course of our study on the role of
immunocytes of the colonial ascidian Botryllus schlosseri in immune
responses, we began to investigate the signal transduction pathways involved in
yeast cell phagocytosis. Both calphostin C, a specific inhibitor of protein
kinase C (PKC), and H-89, a specific inhibitor of protein kinase A (PKA)
significantly inhibit the increase in the phagocytic index. This indicates that
both cyclic AMP, which activates PKA, and phospholipase C, which results in the
production of IP3 and DAG (the former mobilising Ca2+ from
intracellular stores, the latter activating PKC), are routinely required for
phagocytosis. In addition, manumycin A, inhibiting Ras activation, PD98059,
inhibitor of ERK activation, SP600125, preventing JNK activation, SB202190,
inhibiting p38 kinase, significantly inhibit yeast phagocytosis by Botryllus
phagocytes. This suggest that the main MAP kinase
pathways are involved in the ingestion of foreign cells. The frequency of
phagocytes expressing molecules recognised by anti-pan Ras antibody increase
significantly when haemocytes were pre-incubated in the presence of foreign
cells. Activated haemocytes also express molecules recognised by anti-p-ERK and
anti-p38. Therefore, a complex network of intersecting pathways is emerging and
future research will aim to a better clarification of the main steps of signal
transduction in ascidian phagocytosis.
3. 48th Symp. of the Soc. for Histochemistry, Stresa,
a. Recognition and clearance of apoptotic
cells in colonial ascidians. L. Ballarin, Dipt. di Biol.,
Univ.
The colonial ascidian Botryllus
schlosseri forms new zooids by blastogenesis, through the formation of
palleal buds which progressively grow and mature until an adult is formed. At a
temperature of 19°C, adult zooids remain active for about one week; then they
contract, close their siphons and are gradually resorbed, being replaced by
buds which reach functional maturity, open their siphons and begin their
filtering activity as adult zooids. This recurrent generation change, known as
regression or take-over, is characterised by the occurrence of diffuse
programmed cell death by apoptosis. During the take-over, circulating phagocytes
infiltrate in zooid tissues and engulf apoptotic cells; in addition, the
frequency of haemocytes showing nuclear condensation and annexin-V labelling
significantly increases. Moreover, the number of circulating phagocytes showing
a globular morphology and containing ingested cells or cell debris
significantly rises whereas the frequency of hyaline amoebocytes, which
represent mobile, active phagocytes decreases. Phagocytes, both professional
and occasional, actively recognise senescent cells and ingest them. As regards
the eat-me signals, PS seems to be involved in the recognition of effete cells,
as the addition of phospho-L-serine, a soluble analogue of PS, inhibits in
vitro phagocytosis of apoptotic cells. CD36, a part of the receptorial complex
binding thrombospondin which acts as a bridging molecule between phagocyte
surface and apoptotic cells, is expressed on Botryllus phagocytes: the
frequency of cells recognised by anti-CD36 antibodies significantly increases
during the take- over and the expression pattern changes from a patchy
distribution to a uniform staining of the phagocyte surface during the
take-over. Anti-CD36 antibodies significantly decreases the phagocytosis of
effete cells suggesting that similarly to that described in Vertebrates the
thrombospondin receptor play a role in apoptotic cell removal by phagocytes.
Tissue degeneration which occurs during
development of organisms is often of morphogenetic importance as well as
proliferation and differentiation. Cyclic apoptosis of organs was progressively
lost in Chordates. Botryllus schlosseri is a colonial ascidian
continuously forming new zooids by blastogenesis, through the recurrent
formation of palleal buds, which grow and mature until an adult is formed.
Three blastogenic generations are commonly co-present:
adult, filtering zooids, their buds and budlets on buds. At a temperature of
19°C, adult zooids remain active for about one week (mid-cycle stages); then
they contract, close their siphons and are gradually resorbed, being replaced
by a new generation of adult zooids, represented by buds which reach functional
maturity, open their siphons and begin their filtering activity (regression or
take-over stage). This stage is characterised by the occurrence of diffuse
programmed cell death by apoptosis in zooid tissues, as evidenced by TUNEL
reaction for chromatin fragmentation and annexin V labelling for detection of
exposed phosphatidylserine, whereas infiltration of circulating phagocytes,
which appear engulfed with apoptotic cells, is observed. With these
characteristics, colonial tunicates are suitable subjects for studies on
cyclical involution and resorption of tissues. In residual zooids remaining for
a long time in the centre of each colony, melanin and lipofuscins accumulate as
detected with Masson-Fontana, Ziehl-Nielsen and H2O2
bleaching methods. Immunocytochemical assays to detect pro- and antiapoptotic
factors reveal an opposite expression which progressively extends in tissues of
adult zooids with an organ gradient starting from the branchial basket. Results
support the idea that fundamental mechanisms for the induction of apoptosis are
well conserved throughout chordate evolution.
4. Marine Genomics
International Conference. Sorrento (Naples)
28 Oct.- 1 Nov., 2006.
a. Regulation of the meiotic cell cycle in ascidian oocytes: a comparison with vertebrate models
Russo, G.L.,
Bilotto, S., Cuomo, A., Silvestre, F., and Tosti, E., Stazione Zoologica
‘Anton Dohrn’,
In all vertebrate, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase I, others at pronucleus (G1) stage. Fertilization induces completion of meiosis and entry into the first mitotic division. How the different mechanisms underlying meiotic regulation evolved is very far from being clarified. In the past decades, several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the molecular mechanisms responsible for lack of S phase between the two meiotic divisions, and the regulation of the cytostatic factor (CSF) in metaphase I or II. Some of these questions remain elusive and could be approached by the introduction of new experimental models. For these reasons, few years ago, we proposed ascidian Ciona intestinalis oocytes as a new model to study the meiotic division both at the physiological and molecular level. Recently, this model has been strengthened by the publication of the draft copy of C. intestinalis genome. In the present communication, we are reporting data on the role of protein kinase CK2 and MAP kinase in regulating meiosis completion in C. intestinalis oocytes. These two kinases are physically interacting in arrested oocytes. After fertilization, degradation of CK2 regulatory subunits and enzymatic inactivation of MAP kinase drive fertilized oocytes to metaphase-anaphase I transition. We believe that this mechanism might be responsible for CSF inactivation. The results obtained indicate that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus, more than that of other invertebrates. Finally, CK2 catalytic subunits (CK2-alpha) has been expressed in E. coli and purified, in order to study its biochemical and functional properties. In fact, we previously demonstrated that, in fertilized oocytes, C. intestinalis CK2-alpha is enzymatically active independently from its regulatory subunit (CK2-beta). A comparative biochemical analysis of CK2 enzyme from different sources will be presented.
b. HAP/APEX/Ref-1, apurinic/apyrimidic
endonuclease mRNA is expressed in the oocytes of ascidian Ciona intestinalis. Elisabetta Tosti1,
Stefania Bilotto2, Said El-Mouatassim3,
Yves Menezo3, Gian Luigi Russo1,4 1Lab Biologia Cellulare, Stazione Zoologica “Anton
Dohrn”, Napoli; 2Dipartimento delle Scienze Biologiche, Università
degli Studi di Napoli Federico II; 3Lab.
Marcel Mérieux, Ave. Tony Garnier LYON, France; 4Ist. di
Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche,
DNA repair is probably one of the most
important processes to be performed in the oocytes and zygote, at the time of
fertilization and immediately after, in order to allow complete embryonic
development. APEX/Ref1, Apurinic/apyrimidinic endonuclease-Red-Ox
factor1 is capable of initiating the repair of apurinic/apyrimidic
(AP) sites, the most common decay, in damaged DNA. In vertebrate models, the
enzyme is also supposed to play an important role in response to oxidative
stress. The DNA-binding activity of APEX is modulated by a post translational
mechanism involving reduction oxidation and so at least partly mediated by ROS.
Preliminary data indicate that APEX
mRNA is expressed in human spermatozoa and oocytes and in pre-implantation
embryos. In order to investigate if the level of expression of APEX was evolutionarily conserved, we
extended our study to oocytes ad spermatozoa of ascidian Ciona intestinalis (sea squirt), an organism intensively
studied in developmental biology and, more recently, proposed as a model to
study meiotic regulation. From an evolutionary point of view, tunicates
(appendicularians, salps and sea squirts) have very recently been re-evaluated
as the closest relatives of vertebrates, more than cephalochordates, like
amphioxus. This important discovery has been made possible since the advent of
genomics that actually provide the opportunity for phylogenetics to resolve a
number of outstanding evolutionary questions. In this respect, the draft copy
of the C. intestinalis genome became publicly available providing new
insights into origin and evolution of chordates. Based on this evidence, we
used C. intestinalis gametes as
comparative model to study the conservativity of APEX function. Our data
indicate that APEX transcripts were detected in oocytes and embryos, but not in
spermatozoa, of C. intestinalis. Of
philogenetic significance is also the observation that ascidian APEX lacks
redox transcriptional activity.
5. NW Developmental
Biol. Conference at
a. Whole body regeneration in a colonial ascidian, Botrylloides violaceus. Anna D. Le, Federico D. Brown and Billie J. Swalla, Dept. of Biology, Univ. of Washington, Seattle, WA and Friday Harbor Laboratories, Univ. of Washington, Friday Harbor, WA.
Most organisms have the capacity to regenerate organs or tissues through the proliferation and differentiation of stem cells, but the phenomenon of whole body regeneration id s rare event in highly
complex animals. Stem cells are self-renewing and unspecialized, but their daughter cells develop into specialized cells. There have been many studies documenting regeneration by stem cells throughout
metazoans. Some classically described stem cell lineages are the hematopoietic blood cell lineage and primary germ cells (PGCs) that populate the gonads. However, there is little known about the other
stem cell lineages that are not as easily localized and characterized. Colonial ascidians offer a remarkable advantage to studying these stem cell lineages in chordates. These animals are
capable of budding, or growing an entire individual from the interaction of the epithelial bud with a pluripotent stem cell circulating in the blood. Thus these complex colonial ascidians offer
the possibility of studying cell fate, maintenance, and proliferation in an invertebrate chordate, with a less complex genome than the vertebrates. Here we study the phenomenon of whole body regeneration
in a colonial ascidian, revealing the capability of regenerating the entire body from stem cells circulating throughout the vasculature.
b. The evolution of cartilage and the
deuterostome body plan. Amanda L. Rychel and Billie J. Swalla. Dept. of
Biology, Univ. of Washington, Seattle, WA and Friday Harbor Laboratories, Univ.
of Washington, Friday Harbor, WA.
Deuterostomes are a monophyletic group of animals from which the vertebrates evolved. The invertebrate deuterostomes are Cephalochordata, Tunicata, Hemichordata, Echinodermata, and
Xenoturbellida. Hemichordates and cephalochordates have pharyngeal skeletons morphologically similar to vertebrates, despite not having neural crest. Presently, only vertebrates are known to have neural crest cells, and they give rise to multiple cell types including the chondrocytes of the pharyngeal skeleton, although tunicates have placodes, and may have neural crest cells. We have shown that
invertebrate pharyngeal cartilages are acellular and appear to be secreted by the pharyngeal endoderm in hemichordates and a combination of pharyngeal endoderm, ectoderm, and mesoderm in cephalochordates. We also examined the expression of soxE in hemichordates and found it expressed in the pharyngeal endoderm, suggesting that this vertebrate developmental program may be traced back to the deuterostome ancestor. Preliminary results suggest that ptychoderid hemichordates have cellular cartilages in their proboscis skeleton, so we are now conducting experiments to examine the source of this cartilage. We are particularly interested in the development of the proboscis skeleton, stomochord, and heart/kidney complex as possible homologues to vertebrate structures.
c. An in-depth look at
invagination mechanisms in ascidian gastrulation. Kristin M.
Sherrard , Ed Munro, Center for Cell Dynamics, Univ. of
Dr.
Sherrard won the second prize for
best talk by a post-doc.
Invagination is a fundamental mode of morphogenesis whose mechanical
basis remains poorly understood. We have
been studying the process in gastrulating ascidians, whose ten endodermal cells
(each the size of a C. elegans egg) invaginate in a simplified cellular
context of a hundred-cell, monolayer blastula lacking extracellular
structures. Time-lapse videomicroscopy,
immunofluorescence of mono- and di-phosphoyrlated myosin II regulatory light
chain, explants, and experiments with inhibitors to myosin phosphorylation
support a novel, 2-step mechanism of invagination whereby cells first
columnarize and subsequently actively shorten and round up while maintaining
tight apical contacts. Apical
constriction of the endoderm cells appears to drive the columnarization but
does not operate during invagination proper, which is instead driven by
contraction of the basolateral surfaces. Mono-phosphorylated MRLC is implicated
in both apical constriction and shortening, but di-phosphorylated MRLC may be
instrumental in maintaining tight apical collars during the second step. Preliminary modeling simulations of
invagination in two-dimensional cross-section demonstrate that such “collared
rounding” of columnar cells can indeed drive an invagination.
6. Third Asian Biological
Inorg. Chemistry Conference,
a. Accumulation
mechanism of vanadium by ascidians based on genes and proteins involved in
the accumulation. H. Michibata, M. Yoshinaga, M. Yoshihara, N. Kawakami,
N. Yamaguchi and T. Ueki. Mol. Physiol. Lab, Dept. of Biol. Sci.,
The intracellular vanadium concentration of some species in the family Ascidiidae can be as high as 350 mM, which is 10(^7) times the concentration in seawater. Vanadium ions, thought to be present in
the +5 oxidation state in seawater, are reduced to the +3 oxidation state via the +4 oxidation state and are stored in the vacuole of vanadocytes, the vanadium-containing cells, where high levels of
protons and sulfate are also contained. To investigate this unusual phenomenon, we have isolated many proteins and genes that might be involved in the accumulation and reduction of vanadium. To date, more than five types of vanadium-binding protein, designated as Vanabin family, have been isolated from vanadocytes. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes, and NADPH produced by the pentose phosphate pathway participates in the reduction of vanadium(V) to vanadium(IV). Vacuolar-type H(^+)-ATPase (V-ATPase) maintains the low pH in the vacuole and is thought to provide the
energy for vanadium accumulation. Using an immobilized metal-affinity chromatography (IMAC) and gene homology cloning, we have further obtained a Vanabin homologue in blood plasma, metal-ATPase, glutathione-S-transferase and SO(_4)(^-2) transporter. Now, it becomes important to elucidate not only physiological roles of these proteins but also to resolve how these proteins share their roles to accumulate vanadium in ascidians.
b. Metal ion
selectivity and affinity of wild type and mutant Vanabins. T. Ueki, H.
Michibata
Ascidians, tunicates or sea squirts, are well known to accumulate high levels of vanadium ions in the vacuole of one or more type(s) of blood cells. We previously identified five low molecular weight
vanadium-binding proteins, designated Vanabin1, Vanabin2, Vanabin3, Vanabin4 and VanabinP, from a vanadium-rich ascidian Ascidia sydneiensis samea. All of these Vanabins are about 100 amino acid in length and contain 18 cysteine residues, whose intervals were conserved very well. Among them, Vanabin2 is most predominant vanadium-binding protein expressed in the cytoplasm of vanadium- accumulating cells, or vanadocytes. EPR and NMR analyses suggested that lysines and arginines in Vanabin1 and Vanabin2 mainly contribute as coordination sites for vanadium(IV) ions. We performed in vitro mutagenesis of Vanabin2 to modify lysines, arginines and some amino acid residues in possible binding sites, and assessed the metal binding ability of mutants by immobilized metal ion affinity chromatography. Mutation of some of lysines and arginines affected the vanadium binding ability of Vanabin2. Removal of all three histidines apparently did not affect the binding of vanadium.
THESIS ABSTRACTS
1. Study on AsGSTs,
vanadium-binding glutathione transferases, identified from the
vanadium-rich ascidian, Ascidia sydneiensis
samea. M. Yoshinaga, Mol. Physiol. Lab, Dept. of Biol. Sci.,
hmichi@hiroshima-u.ac.jp [No abstract available]
2. Analysis of the
allorecognition mechanism of Halocynthia
roretzi hemocytes. Yuko Doi,
Dept. of Biosci. and
Informatics, Fac. of Sci. and Technol,.
Recognition strategies play an essential role in the immune system, which faces thousands of foreign substances. They are classified into two groups; recognition of non-self and recognition of
self. The latter has been important topic as the possible explanation for the transplantation-immunity-like reaction that can be seen throughout the animal kingdom. However, the molecular entity of self-marker is hardly elucidated with the only exception of the major histocompatibility complex in vertebrates. Ascidians belong to the phylum Chordata and hold the phylogenetic key position to discuss the evolution of the vertebrate allorecognition system. Hemocytes of the solitary ascidian, Halocynthia roretzi undergo the contact reaction (CR) against allogeneic hemocytes, indicating that they have the ability of allorecognition. Hemocytes of H. roretzi are morphologically classified into nine types and every type of hemocyte undergoes CR. The missing-self recognition model is proposed for the recognition mechanism of H. roretzi hemocytes. In this study, I analyzed the allorecognition mechanism of H. roretzi hemocytes using the moloclonal antibody ku-4-96, which inhibits the
releasing of phenoloxidase (
ku-4-96 inhibited the releasing of
measured by the releasing of the cytoplasmic fluorescent viability dye calcein AM and the releasing of calcein was suppressed in the cells treated with ku-4-96. These two results suggested that the
function of ku-4-96 antigen(s) is different for each cell type. To identify the ku-4-96 antigen(s), I purified plasma membrane fraction by chromatography on phenyl-Sepharose. Among the three
molecules recognized by ku-4-96, the molecules of 180kDa (ku496P-1) and 160 kDa (ku496P-2) were isolated and each N-terminal amino acid sequence was determined. As for ku496P-1, partial degradation by the Cleaveland method was carried out and the fragment containing N- terminal was obtained.
3. Search for self-marker molecules of ascidian hemocytes in Halocynthia roretzi. Masaki Ema, Dept. of Biosci. and Informatics, Fac. of Sci. and Technol., Keio Univ., Japan. Master’s thesis. Advisors Motonori Hoshi and Midori Matsumoto.
Many metazoan cells have a system to recognize alien substances as non-self (for example microorganisms and chemical substances etc.). And, vertebrate and their close relatives have a system to distinguish their own cells from other cells in the same species. The former system is called xenogenic recognition and the latter system is called allogenic recognition. It is thought that the difference of these recognition systems is decided with self-marker molecules, which is indispensable to identify self from non-self. It is the major histocompatibility complex (MHC) that is well known as a self-marker in the allo-recognition mechanism of vertebrates. The transplant rejection happens because the type of MHC is not suitable. The feature of MHC is having a wide variety of alleles in the locus. By this combination, MHC can invent advanced diversity between individuals. Recently, MHC was identified in Chondrichthyes, and it was suggested that all vertebrates except the cyclostomes, the most primitive vertebrates, retained MHC.
Self-marker molecules in allo-recognition mechanism of invertebrates are not well known. Recently, FuHC, which are polymorphic gene loci to determine colony fusion or rejection, was identified in one kind of ascidian, which is closely related to vertebrates, Botryllus schlosseri. But this is the only report that describes self-marker molecules in allogenic recognition mechanism of invertebrates.
In a solitary ascidian, Halocynthia roretzi, their hemocytes have a capacity for allogenic recognition. When hemocytes from two individuals are mixed in vitro, they immediately start to agglutinate
each other and emit their cellular vacuoles. This reaction was named the Contact Reaction (CR). It is thought that self-marker molecules exist in Halocynthia roretzi hemocyte, because CR never happens in the contact of their own cells. To find the origin of self-marker molecules in CR, I designed the experimental system to detect the unique hemocyte proteins in each individual. The cellular
extract from each ascidian hemocyte was labeled by fluorescents and separated by 2 D electrophoresis. By this system, a group of protein spots, which expressed differently between individuals, was
discovered. They are candidates for self-marker molecules. These spots were excised, and then PMF and MS/MS were taken with MALDI-TOF MS. Then, those candidate self-marker molecules, amino acid sequences of the fragments, were sequenced.
4. Impacts of changing water temperatures on
the life histories of two invasive ascidians in the
Temperature and
its effects on life cycles in organisms have received great attention recently
due to heightened awareness of global warming.
Relative growth and reproductive development were monitored in two
dominant species of ascidians from the
5. Phylogeny,
population genetics and biology of the genus Pycnoclavella (Ascidiacea). Rocío Pérez-Portela, Dept. of
Animal Biol. (Invertebrates), Fac. of Biology,
The taxonomy and
phylogeny of the genera Pycnoclavella
and Clavelina, and their inclusion in
one or two different families within the order Aplousobranchia, have been
controversial since the description of both genera. The genus Pycnoclavella has been widely studied in
tropical areas but its diversity was scarcely known in Atlanto-Mediterranean
waters, where the species Pycnoclavella
aurilucens had been described as featuring high morphological variability.
Moreover, its possible synonymy with another species Clavelina nana, has been
a matter of debate not yet settled. Combining morphological, molecular and
biological data we have explored the intra- and inter-specific variability of
the genus Pycnoclavella, as well as
its relationship with Clavelina, and
its abundance and distribution in Mediterranean waters. Phylogenetic analyses
were performed under different criteria (Maximum Likelihood and Bayesian
Inference) on sequences of the cytochrome c oxidase I (COI) mitochondrial gene,
of 21 species of Pycnoclavella, Clavelina and Nephtheis fascicularis
collected in the
Four new species (P. atlantica, P. brava, P.communis
and P. martae) were formally
described and holotypes erected. Three of them were described from
The genetic
variability of Pycnoclavella communis,
studied from sequences of COI of W. Mediterranean Sea and
6. Contribución al conocimiento de las ascidias coloniales
(Chordata, Tunicata) de la Antartida Occidental y Región Magallánica.
Contribution to the knowledge of colonial ascidians (Chordata, Tunicata)
from western
Research over the past few decades has established that the Antarctic benthos, especially on the shelf, is characterized by high biomass and abundance levels on both hard and soft substate (White, 1984; Clarke and Crame, 1989; Clarke, 1990; Knox, 1994). These bottoms reveal a rich and diverse fauna of suspension feeders (Dell, 1972; Gallardo, 1987; Arntz et al., 1994; Starmans et al., 1999; Gili et al., 2001). One of the most important taxa in numerical and biomass abundances is ascidians in the epibenthic communities on the Antarctic continental shelf (Monniot & Monniot, 1983; Gallardo, 1987; Kirkwood & Burton, 1988; Galéron et al., 1992; Arnaud et al., 1998). Also, about 90% of the ascidians found in antarctic and subantarctic waters have a high percentage of endemisms. (Monniot & Monniot, 1983).
The focus of this study was to enlarge our
knowledge about the ascidian fauna in Scotia Arc,
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(Crustacea : Copepoda : Cyclopoida : Notodelphyidae), a new genus and species,
living in the compound ascidian Diplosoma virens at
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