Number 43 May 1998
Charles is retiring at the end of this semester,
after 28 years at Calif. State Univ. Fullerton. On June 4 we are
moving to Seattle, Washington. Our new address will be 12001 11th Ave.
NW, Seattle, Wa 98177, telephone number 206-365-3734. We will continue
to use our same email addresses temporarily. We will both be doing research
in association with the Univ. of Washington. We will continue our studies
on the introduction of non-indigenous ascidians to the U.S. Pacific Coast,
but move our sampling from southern California to the Pacific Northwest.
We also plan to do some travelling; we will be spending the month of October
in Honolulu, with Charles doing research on ovulation in Herdmania momus
and Gretchen working at the Bishop Museum identifying some of their ascidians.
Gretchen will continue to produce Ascidian News twice a year
on our web site. We have compiled this newsletter since 1975, and we hope
you continue to find it useful. Please keep in touch, and please
continue to send us a copy of your new publications, which will assure
that they are included in the Recent Publications section at the end of
each AN. There are 60 new publications in this issue; if you have not yet
sent us a copy of your latest papers, please send them to our new Seattle
address.
*Ascidian News is not part of the scientific literature and should
not be cited as such.
NEWS AND VIEWS
Dr. Paul Illg: We are very sorry to report that
our long term friend and author of books and numerous monographs on copepods
that inhabit ascidian hosts passed away on May 10th after a long illness.
His knowledge of invertebrate
biology was truly amazing. Paul was influential to both of our careers
and we learned a great deal from him. We greatly enjoyed talking
to him on a daily basis when we were graduate students. He helped to generate
our interest in systematics and we have counted upon him as a friend throughout
our subsequent lives. He will be greatly missed by all who were fortunate
enough to know him during his many years at the Friday Harbor Laboratories
and in the Zoology department of the University of Washington.
WORK IN PROGRESS
Drs. V.K. Meenakshi, Dept. of Zool, APC Mahalaxmi college for Women,
Tuticorin 628 002 and T.K. Renganathan, Dept. of Zool. VOC College, Tuticorin
628 008, India.
We are working on the ascidians collected from the
SE coast of India along the Gulf of Mannar from Tuticorin to Rameswaram.
A preliminary search has yielded many new records to Indian waters and
a few species which are new to science. Our papers on the new reports
are under preparation. (We congratulate Dr. Meenakshi on receiving her
Ph.D. in Dec. 1997; her thesis was "Biology of a few chosen ascidians"
with a chapter on taxonomy which included 8 new species.)
ABSTRACTS FROM RECENT MEETINGS
1. Zool. Soc. of Japan 68th annual meeting, 1997. (Publ.
in Zool. Sci. vol. 14. supplement, December 1997).
EXAMINATION OF RETINAL BINDING PROTEINS IN THE EGGS OF
AMPHIBIAN, TELEOST AND UROCHORDATE.
T. Irie1, T. Seki2, S. Kajiwara3 and M. Iwamoto4.
1Osaka Meijo Women's Coll., Osaka; 2Dept. Health Sci., Osaka Kyoiku Univ.,
Osaka; 3Dept. Biol., Fac. Educ., Iwate Univ., Iwate and 4Dept. Biophys.
Engn., Fac. Engn. Sci., Osaka Univ. Osaka.
Lipovitellin 1 (LV1), a yolk protein,
was shown to be the retinal binding protein in Xenopus laevis eggs.
In the eggs of amphibian, teleost and urochordate, corresponding proteins
to LV1 were found by SDS-PAGE. For the confirmation of retinal binding
to a specific yolk protein, rat antisera against LV1 of X. laevis, E1a
of chum salmon (Oncorhynchus keta) and flatfish (Limanda yokohamae)
eggs, and against retinal were obtained. By western blotting analysis,
antiserum against X. laevis stained only one band of LV1.
Using the anti-retinal antiserum, positive bands have not been observed,
but retinyl product of X. laevis LV1 was found to be fluorescent
on the blotted membrane. Among the egg proteins, two bands were stained
with anti-O. keta E1a antiserum, while many protein bands of L.
yokohamae were stained with anti-L. yokohamae E1a antiserum.
Identification of the retinal binding protein with one or some stained
bands is a problem for further study. In the ascidian eggs, four
bands corresponding to LV1 were observed, but none reacted with anti-retinal
antiserum.
HYPERPOLARIZATION OF SPERM PLASMA MEMBRANE INDUCES SYNTHESIS
OF CYCLIC AMP AND ACTIVATION OF SPERM MOTILITY IN THE ASCIDIANS
Ciona intestinalis and C. savignyi.
H. Izumi1, T. Marian2, K. Inaba1, Y. Oka1, M. Morisawa1.
1 Misaki Mar. Biol. Sta., Graduate Sch. of Sci., Univ. of Tokyo; 2 Positron
Emission Tomograph Center, Univ. Med. Sch. of Debrecen, Hungary.
Sperm-activating and -attracting factor
(SAAF) released from egg at fertilization activates sperm motility in the
ascidians Ciona savignyi or C. intestinalis through synthesis
of cAMP and influx of Ca2+ (Yoshida et al. 1994). We show here that
the activation of Ciona sperm motility was suppressed by extracellular
K+. A K+ ionophore, valinomycin, substitutes the SAAF effect; it activated
sperm motility in the absence of SAAF. The effect of the ionophore
is reduced by higher concentration of external K+ . On the other hand.
measurement of membrane potential with a slow voltage-sensitive carbocyanine
dye, DisC3(5) showed that both valinomysin and SAAF induce hyperpolarization
of the sperm plasma membrane in the presence or absence of extracellular
Ca2+ and the amplitude of the potential was dependent on external K+ concentration.
Valinomycin as well as SAAF caused an increase in intracellular cAMP.
A K+ channel blocker, MCD-peptide, suppressed the activation of sperm motility
and membrane hyperpolarization. MCD-peptide also inhibited
cAMP elevation by SAAF but did not inhibit valinomycin-induced cAMP
synthesis. These results suggest that hyperpolarization of the plasma membrane
through an increase in K+ permeability causes the synthesis of cAMP, resulting
in the activation of sperm motility. It is also suggested that Ca2+ influx
is necessary for cAMP synthesis and sperm motility activation, but is unnecessary
for the membrane potential changes.
PHOSPHOPROTEINS PARTICIPATE IN THE SAAF-DEPENDENT ACTIVATION
OF Ciona SPERM.
M. Nomura, K. Inaba, M. Morisawa. Misaki Mar. Biol.
Station, Graduate Sch.of Sci., Univ. of Tokyo.
Sperm of the ascidian Ciona
intestinalis initiates motility by unfertilized egg-derived factor,
SAAF (Sperm Activating and Attracting Factor) which -raises intracellular
cyclic-AMP (cAMP) level dependent on extracellular Ca2+ (1). On the other
hand, demembranated sperm of C. intestinalis require cAMP to reactivate
(2,3,4). In order to elucidate a relation between SAAF-dependent motility
initiation and sperm protein phosphorylation, we used demembranated Ciona
sperm with SAAF-dependent in vivo activation. From experiment using
demembranated sperm model, SAAF activated sperm model have already been
prepared to motile with ATP alone, furthermore extracellular Ca2+ was required
to initiate the motility in intact sperm prior to cAMP-dependent activation
pathway. Intact sperm motility initiation in SAAF-dependent manner was
inhibited by W-7 and ML-7. Remarkable sperm protein phosphorylation corresponding
to SAAF-dependent sperm motility initiation, was seen in 21kDa and 26kDa
proteins. These two proteins were phosphorylated in intact sperm which
had been activated by SAAF. These protein phosphorylations may be regulated
by CaM-like protein. Furthermore the 32P-labeled 21kDa protein was identified
as one having a dynein light chain. We conclude that SAAF activates Ciona
sperm via the dynein light chain, which is upregulated by Ca2+ efflux into
sperm prior to regulation by calcium binding protein. These support
a previous report related to Ciona sperm activation by theophylline
(4). (1) Yoshida, M. et al. 1994. Develop. Growth & Differ.
36: 589-595. (2) Brokaw, CJ 1985. Ann. N. Y. Acad. Sci. 438, 132-141. (3)
Dey, CS & Brokaw, CJ 1991. J. Cell Sci. 100, 815-824. (4) Chaudhry,
PS et al.1994. Cell Motil. Cytoskeleton 32, 65-79.
ISOLATlON AND EXPRESSlOIN ANALYSlS OF RXR FROM Polyandrocarpa
misakiensis.
M. Kamiimura, K. Hisata, S. Fujiwara, K. Kawamura and
T. Yubisui. Dept. of Biol., Fac.of Sci., Univ. of Kochi.
It has been suggested that in the
budding tunicate Polyandorocarpa misakiensis, retinoic acid (RA)
is an endogenous regulator of bud development (Kawamura et al.).
In mammals, RA exerts a wide variety of activities via its nuclear receptor,
retinoic acid recepter (RAR) that can form a heterodimer. We have
recently isolated almost the full length of RAR cDNA from Polyandrocarpa
PmRAR (Hisata et al. 1995). The aim of this study was to isolate
Polyandrocarpa homologs of retinoid X receptor (PmRAR) that would
be a partner for heterodimerization. In order to amplify cDNA fragments
by PCR, degenerate primers were designed encoding conserved amino acid
sequences of the known RXRs. The strands were extended by 3' RACE, and
eventually a total of 2132 bp was sequenced. Deduced amino acid sequence
was 80% identical with that of vertebrate RXRs. Both DNA-binding
domain and ligand binding domain were highly conserved RTPCR and Southern
hybridization analyses showed that PmRXR was expressed in developing buds
in the same developmental time course as PmRAR. Our results indicate that
there is a good chance of PmRXR forming heterodimer together with PmRAR.
PURIFICATION AND PROPERTIES OF NDADH-CYTOCHROME B5 REDUCTASE
FROM Styela plicata.
K. Kurita, K. Kawamura and T. Yubisui, Dept. Biol., Fac.
Sci., Kochi Univ.
NDADH-cytochrome b5 reductase is considered to
play an important role in the development of ascidians, based on the studies
of the mammalian enzymes. Therefore, we studied the properties of
NDADH-cytochrome b5 reductase of S. plicata. In this
study, the enzymes of S. plicata were partially purified by
an affinity chromatography on a 5'-AMP-S column, and we investigated some
kinetic properties. Optimal pH of the enzyme was determined to be
6.2, and apparent Km values for NADH and cytochrome b5 were determined
to be 1.4, and 3.6 uM, respectively. An apparent Vmax value was 3.0
umol/min/mg. An immunoblot study with an antibody against human enzyme
showed a distinct band at around 67kD, which is about double the size of
mammalian enzymes.
AN ASCIDIAN DEAD-BOX GENE LOCALIZES MOST POSTERIOR BLASTOMERES
DURING EMBRYOGENESIS.
M.Fujimura and K.Takamura, Div. of Biotech., Fac. of
Engineering, Fukuyama Univ., Fukuyama.
We isolated a DEAD-box gene from Ciona
ovary cDNA library by PCR method with degenerate primers and RACE method.
The protein deduced from this cDNA sequences showed good homology to vasa
homolog of other animals. The mRNA for this cDNA was about 2.8 kb by nothern
blot analysis, and was detected through embryogenesis by RT-PCR. In situ
hybridization showed that this mRNA existed in the inner region of eggs
and was segregated into most posterior region during the second cleavage
(2 to 4 cell stages). These results suggest that the segregation mechanism
of this mRNA is different from that of ooplasm which occurs immediately
after fertilization. As a result, this mRNA was inherited by most posterior
blastomeres through succeeding cleavages and, finally, was detected in
a part of the caudal endodermal strand at the early tailbud stage.
ASCIDIAN NETRIN HOMOLOG EXPRESSES IN NOTOCHORD AND BRAIN
LINEAGES DURING EMBRYOGENESIS.
K.Takamura and S. Kawano, Div. of Biotech., Fac.
of Engineering, Fukuyama Univ., Fukuyama.
We isolated two types of cDNA for an ascidian
netrin homolog by PCR methods. Each cDNA was designated CINETA and CINETB,
respectively. Their sequences were determined and compared with known netrin
homologs of C.elegans, D.melanogaster and G. gallus.
CINETA had all conserved domeins, while CINETB lacked last laminin-like
domein. RT-PCR showed that both mRNAs for these cDNAs were detected from
the middle gastrula stage and decreased from the late tailbud stage.
In situ hybridization with RNA probes for CINETA showed that this mRNA
expressed in the primary notochord lineage blastomeres (A-line) at the
gastrula stage and in the notochord at the early tailbud stage. Additional
expression was detected in the brain lineage at the neurula stage and in
the ventral region of brain at the early tailbud stage. These results show
that the expression pattern of netrin in ascidians is different from that
in other vertebrates whose netrin expresses in the ventral spinal cord.
IDENTIFICATION OF THE BLASTOMERES INVOLVED IN THE INDUCTION
OF EPIDERMAL SENSORY NEURONS DURING ASCIDIAN DEVELOPMENT.
Y. Ohtsuka1, Y. Okamura2 and T. Obinata1. 1Dept.
of Biol., Fac. of Sci., Chiba Univ., Yayoi-cho, Chiba; 2Natl. Inst. of
Biosci. & Human Technology Agency of Industrial Science and Technology
M.I.T.I.,Tsukuba .
Previously, we found that gelsolin, an actin
filament severing and capping protein, is present abundantly in epidermal
sensory neurons of the ascidian larva. In this study, we examined how vegetal
blastomeres are involved in sensory neuron differentiation during ascidian
larval development by using gelsolin as a molecular maker. When ascidian
embryos at the 8-cell stage were manipulated to remove vegetal cells, either
pairs of A4.1 cells or pairs of B4.1 cells, and then cultured until reaching
the late tailbud stage, gelsolin-positive cells were detectable by anti-gelsolin
antibody (AS23) in each case. The anterior half (a4.2+A4.1) and posterior
half (b4.2+B4.1) embryos cleaved at the 8-cell stage produced gelsolin
positive cells, whereas isolated (b4.2+A4.1), vegetal half (A4.1+B4.1)
and animal half (a4.2+b4.2) embryos did not produce the neurons. These
results indicate that signals from A4.1 and B4.1 are necessary for gelsolin
expression in a-line and b-line blastomeres, respectively. At the16-cell
stage, all of progenies derived from the A4.1 and B4.1 showed the ability
to induce the neurons in animal blastomer when cultured in combination
with them.
EXPRESSION OF EMBRYONIC AND ADULT-TYPE TROPONIN T ISOFORMS
DURING ASCIDIAN EMBRYOGENESIS AND METAMORPHOSIS.
T. Kawakami, Y. Ohtsuka, T. Endo & T. Obinata. Dept.
of Biol., Fac. of Sci., Chiba Univ., Yayoi-cho, Chiba.
Troponin T (TnT) is a component of troponin,
a Ca2+-dependent regulator for striated muscle contraction in a variety
of vertebrates and invertebrates. TnT is not detectable in vertebrate
smooth muscle, but we previously demonstrated that TnT exists in adult
body wall smooth muscle of ascidian, Halocynthia roretzi, as well
as in the larval striated muscle, and in addition, that the embryonic TnT
(eTnT) and adult TnT (aTnT) are present in this animal, which are encoded
by distinct genes (Endo et al.1996. J. Biol. Chem. 271: 27855-27862).
In the present study, we examined the expression of the two types of TnT
during ascidian embryogenesis and metamorphosis using specific cDNA probes
and an anti-TnT monoclonal antibody (NT302). The results show that
mRNA for eTnT is detectable in the nuclei of several blastomeres at the
110-cell stage or a little earlier, while TnT protein is detectable only
after the neurula stage. The switching from eTnT to aTnT occurs about
7 days after tail absorption (onset of metamorphosis). Co-expression of
eTnT and aTnT was not observed at any stage, suggesting that expression
of the two TnT isoforms is precisely regulated.
CLONING AND SEQUENCE OF ASCIDIAN TROPONIN I ISOFORMS.
H. J. Yuasa and T. Takagi, Biol. Inst., Graduate Sch.
of Sci., Tohoku Univ., Sendai.
Previously we identified two distinct cDNAs
of troponin C (TnC) isoforms from the ascidian, Halocynthia roretzi.
These two TnCs (larval TnC and adult TnC) were encoded by a single gene,
and expressed through alternative selection of a third exon (1).
This time, we determined cDNA sequences of three TnI isoformes from H.
roretzi. One of the three isoforms (adult TnI) was composed of
173 amino acids, and expressed in body-wall smooth muscle and heart muscle.
The other two isoforms (larval TnIƒ¿ and ƒÀ) were isolated
from a cDNA library of larvae. Both larval TnIs are composed of only
142 amino acids, and are the smallest of all known TnIs. They are
ca. 30 amino acid residues shorter at the C-termini than other TnIs.
The position of the intron of these TnIs C-terminal regions was also determined.
When compared with vertebrate TnI genes, the intron of the ascidian adult
TnI gene is located at 6 nucleotides downstream, and the introns of the
two larval TnIs are positioned at 9 nucleotides upstream. These results
suggest that H. roretzi TnI is encoded by at least three genes (2).
(1) Yuasa et al. 1997. J. Biochem. 121, 671-676. (2) Yuasa et al. 1997.
J. Biochem. 122, 374-380.
REGULATION OF THE TYROSINASE GENE EXPRESSION IN THE ASCIDIAN
EMBRYO.
R. Toyoda1, S. Sato1,T. Numakunai2, T. Gojobori3, K.
Ikeo3 and H.Yamamoto1.
1Biol. Inst. Tohoku Univ., Sendai; 2Mar. Biol. Stn. Tohoku
Univ., Asamushi, 3Natl. Inst. Genet. Mishima.
Tadpole larvae of the Japanese ascidian
Halocynthia roretzi deve1op two sensory pigment cells in the brain,
the otolith and ocellus. These melanin -containing cells specificaIly
express tyrosinase activity and play an important role in detection of
gravity and light. Genes encoding tyrosinase, a key enzyme in melanin biosynthesis,
have been isolated from various vetebrate species. The 5' flanking sequences
of the vertebrate genes have several conserved sequences, but their function
as cis-elements remains to be solved. To carry out a phylogenetic study
of the regulation of tyrosinase gene expression, we cloned a putative tyrosinase
gene and a gene encoding tyrosinase c-related protein TRP from Halocynthia
roretzi. To localize the sequences regulating expression of these genes,
each 5' flanking sequence was ligated to a lacZ reporter gene and used
for microinjection into fertilized ascidian eggs. 1.8 kb of the upstream
sequence of the ascidian tyrosinase gene drives expression of the reporter
gene in pigment precursor cells at the tailbud stage. On the other hand,
573 bp of the ascidian TRP gene were found to be sufficient to express
in pigment precursor cells.
A MATERNAL WNT-RELATED GENE OF THE ASCIDIAN Halocynthia
roretzi IS STRONGLY LOCALIZED IN AN EMBRYO.
Y. Sasakura, M. Ogasawara, K.W. Makabe, Dept. of Zool.,
Grad. Sch. of Sci., Kyoto Univ., Kyoto.
To understand the roles of maternal
mRNAs in early development, we have started a mass screening of cDNAs of
the fertilized egg of Halocynthia roretzi. Here we report
the cloning and characterization of a member of the Wnt family. This
Wnt-related gene shows the highest homology with Wnt-5, thus we named it
HrWnt-5. The maternal transcripts of HrWnt-5 are distributed at the cortical
region of the unfertilized egg. After secondary ooplasmic segregation,
the transcripts are strongly localized at the posterior region of the egg.
This localization pattern continues to the tailbud stage, and at that stage
maternal transcripts are seen only in the endodermal strand. Zygotic transcripts
are detected at the 64-cell stage in various regions of the embryo.
At the tailbud stage, zygotic transcripts are seen only in the notochord
cells.
ANALYSIS FOR THE ROLE OF POSTERIOR END MARK, A LOCALIZED
MATERNAL FACTOR
IN THE PATTERNING OF THE ASCIDIAN EMBRYO.
S. Yoshida and N. Satoh, Dept. of Zool., Graduate
School of Science, Kyoto Univ., Sakyo-ku, Kyoto.
Maternal factors are implicated
in the establishment of the animal body plan. Ascidian embryogenesis
is regarded as the 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 the antero-posterior
axis of the embryo. We report here an analysis for the role 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 lacking the anterior-most
adhesive organ, dorsal brain and sensory pigment-cells. Lineage tracing
analysis revealed that the anterior dorsal neuronal cells were translocated
posteriorly into the tail region, and this translocation could be rescued
by LiCl treatment. These data suggest that pem plays a role in patterning
of the ascidian embryo.
EXPRESSION OF ENDOSTYLE-SPECIFIC GENES IN THE ASCIDIAN
Ciona intestinalis.
M. Ogasawara & N. Satoh, Dept. of Zool., Grad.
Sch. of Sci., Kyoto Univ., Kyoto.
The endostyle is an organ key
to an understanding of the molecular mechanism underlying the origin and
evolution of chordates. We reported previously an isolation
of two cDNA clones of the Halocynthia roretzi endostyle-specific
genes, HrEnds1 and HrEnds2. In this study, we isolated and examined novel
endostyle-specific genes of Ciona intestinalis, which belongs to
another order of ascidians. Transcripts of the CiEnds1 and CiEnds3
were detected in the protein secreting element of zone 6, and CiEnds2 transcripts
were detected in other protein secreting element of zone 2 under our in
situ hybridization. These genes may serve as probes for further analysis
of molecular mechanisms involved in the formation and evolution of the
endostyle.
ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR TISSUE-SPECIFIC
GENES IN
Ciona savignyi EMBRYOS.
S. Chiba1,2 , Y. Satou2, T. Nishikata1 and N.
Satoh2. 1Dept. of Biol., Konan Univ., Kobe; 2Dept. of Zool.,
Grad. Sch. of Sci., Kyoto Univ., Kyoto.
We prefer Ciona eggs and embryos
as an experimental system for future studies, in particular with respect
to those with genetic approaches, because their spawning season is basically
all year round and because their generation time is about 3 months. But,
at present, we have isolated few tissue-specific genes in Ciona
species that are useful items for the marker. Therefore we tried to isolate
cDNA clones for tissue-specific genes from a library of tailbud-embryo
mRNAs subtracted with fertilized-egg mRNAs of Ciona savignyi. Thus
far we were able to isolate several cDNA clones for tissue-specific genes.
We characterized two cDNA clones for muscle-specific genes and two
clones for epidermis-specific genes. All of the genes are useful for the
marker. One of the muscle-specific genes is identified to be a muscle actin
gene of Ciona savignyi.
FULL-LENGTH cDNA CLONING OF NOTCH HOMOLOGUE FROM THE ASCIDIAN
Halocynthia roretzi.
S. Hori, T. Saitoh,& H. Nishida. Dept. of Life
Sci., Tokyo Inst. of Tech., Yokohama.
Ascidian embryogenesis involves the various cell-cell
interactions to specify the particular cell fate. To know the molecular
basis of such cell interactions, we isolated the full-length cDNA of the
ascidian homologue of the Drosophila neurogenic gene Notch (HrNotch).
HrNotch transcripts encode a protein of 2352 amino acids and share the
principal features of the Notch gene family; extracellular EGF-like repeats,
three Notch/Lin-12 repeats, six intracellular ankyrin repeats and RAM domain.
Yet ascidian Notch contains only 33 EGF-repeats in the putative extramembrane
domain and specifically lacks the three EGF-like repeats. We have previously
reported the zygotic expression of HrNotch in sensory pigment cell precursors,
which suggests its roles in the determinative events in the sensory pigment
cell equivalence group. In this report, the zygotic expression of HrNotch
is also detected in the nerve cord lineage (b-line cells) and the presumptive
larval adhesive organ (a-line cells).
THREE Y-BOX PROTEINS IN THE ASCIDIAN (Ciona intestinalis)
EGG.
M. R. Wada, Y. Ohtani, Y. Shibata, K. J. Tanaka, &
T. Nishikata, Fac. of Sci., Konan Univ., Kobe.
The localization of maternal mRNAs
and their segregation to different embryonic cells has been proposed to
mediate cell fate determination during early development. Recently,
Y-box protein family which is known as the nucleic acid-binding protein
has been suggested to have an ability to repress the translation
of mRNAs. In this paper, we isolated three cDNA clones for Y-box
protein mRNAs (CiYB1, CiYB2 and CiYB3) from eggs and embryos of the ascidian,
Ciona intestinalis. The genomic sequence analysis of CiYB
gene revealed that the three mRNAs were produced by the alternative splicing.
CiYB1 and CiYB2 were expressed in the very early period of oogenesis and
suggested to be recruited into maternal ribonucleoprotein particles (mRNPs).
CiYB1, CiYB2 and CiYB3 expressed zygotically in tissue restricted manners.
Using gel-mobility shift assays, we found that CiYB1 protein have an RNA-binding
ability. This could suggest that the ascidian Y-box proteins play an important
role for the storage and/or repression of the translation of the maternal
informations.
CELL LINEAGE ANALYSIS OF THE ENDODERMAL CELLS OF ASCIDIAN
LARVAE DURING METAMORPHOSIS.
T. Hirano & H. Nishida, Dept. of Life Sci., Tokyo
Inst. of Technology, Yokohama.
To study the origin of adult
tissues of the ascidian Halocynthia roretzi, we traced the cell
fate by intracellular injection of horseradis peroxidase (HRP) into identical
blastomeres at 110-cell stage. Descendants of the injected cells
were histochemically detected at juvenile stage. In the previous
meeting, we reported the results of the cell lineage analysis of mesodermal
cells of ascidian larvae and suggested that the cell lineages during metamorphosis
were also invariant. In this meeting, we present the results
about endodermal cells of larvae. Endodermal cells of the larvae
were indicated to give rise to adult endodermal tissues, that is endostyles,
peribranchial epithelium, and digestive organs and so on. Furthermore
these results suggested that the boundaries of adult-endoderm precursors
do not correspond to the clonal boundaries of larval endodermal cells.
GENETICAL VARIETY OF rRNA GENE IN Ciona.
M. Matsumoto and M. Hoshi, Dept. of Life Science, Tokyo
Inst.of Tech., Kanagawa.
Genetic
variety of rRNA gene (rDNA) repeated units was studied in Ciona savignyi
and Ciona intstinalis. We previously reported that the internal
spacer region between 18S rDNA and 5.8S rDNA had a wide variety in length.
For further investigation, we analyzed animals from three bays where both
species live alongside (Onagawa, Honmoku and Qingdao). Contrary to
the previous data, all animals so far studied gave very similar PCR products
of the spacer region regardless of species and of geography, at least
within these three bays.
CONTROL OF CELL DIVISION NUMBER DURING ASCIDIAN EMBRYOGENESIS.
A. Yamada and H. Nishida, Dept. of Life Sci., Tokyo Inst.
of Technology, Yokohama.
Cell number during ascidian
embryogenesis were counted every hour. Number of nuclei of the squashed
embryos and larvae were counted on photographs. Cell number of a
whole larva was about 2700 just after hatching. Next, animal and vegetal
egg fragments were made by bisecting unfertilized eggs to alter the egg
volume and the nucleocytoplasmic ratio. Egg fragments were fertilized and
cell number of the resultant larvae were estimated. The results suggest
that three kinds of mechanisms are involved in control of cell division
number during embryogenesis: (1) Mechanism that depends on the nucleocytoplasmic
ratio. (2) Mechanism that depends on the absolute volume of cytoplasm.
(3) Mechanism that is not affected by changes of both factors.
MODE OF ACTION OF THE SPERM PROTEASOMES ON FERTILIZATION
OF THE ASCIDIAN, Halocynthia roretzi.
H. Sawada, J. Fujino, Y. Takahashi, & H. Yokosawa,
Dept. Biochem., Fac.Pharm. Sci., Hokkaido Univ., Sapporo.
We have previously reported that the sperm
20S proteasome and 930-kDa prot easome are involved in the sperm binding
to and in the sperm penetration through the vitelline coat of the eggs
of the ascidian, Halocynthia roretzi, respectively. In the present
study, we investigated the mode of action of the proteasomes in fertilization.
MG115 (an inhibitor for proteasome and cathepsin B/L) inhibited the fertilization
of intact eggs, while E-64-d (an inhibitor for cathepsin B/L and calpain)
showed no inhibition. This potent inhibitory ability of MG115 against fertilization
appears to be diminished by using the eggs treated with acidic seawater
(pH 3.0), which elicits the release of 70-kDa component of the vitelline
coat. In addition, MG115 inhibited the sperm binding to the vitelline
coat of the glycerinated eggs. The anti-proteasome antibody also inhibited
the fertilization. These results suggest that the proteasome is involved
in the sperm binding to and penetrating through the vitelline coat, and
that the 70-kDa component of the vitelline coat may be a candidate substrate
for the proteasome. It was also revealed that the proteasome functions
extracellularly during fertilization.
EVIDENCE FOR THE INVOLVEMENT OF PROTEIN KINASES AND MICROTUBULES
IN REGULATION OF PROTEASOME ACTIVATION DURING ASCIDIAN EGG ACTIVATION.
Y. Aburatani & H. Yokosawa, Dept. of Biochem., Fac.
of Pharm. Sci., Hokkaido Univ., Sapporo.
The ubiquitin-dependent proteolysis is required
for the progression of cell cycle. We previously reported that the proteasome
is activated in response to intracellular calcium increase during activation
of ascidian and Xenopus eggs. To define factors functioning in calcium-induced
proteasome activation, we investigated effects of three protein
kinase inhibitors and an anti-microtubule compound on proteasome activation
in ascidian eggs, which was triggered by calcium ionophore. Protein kinase
inhibitors (olomoucine, curcumin, and erbstatin analog) and an inhibitor
of microtubule polymerization (nocodazole) inhibited the proteasome activation,
which is due to the assembly of the 26S proteasome from the 20S proteasome.
This result indicates that protein kinases play important roles in
the calcium-induced proteasome activation and that the presence of intact
microtubules is indispensable for this event.
PREPARATION AND LOCALIZATION OF A MONOCLONAL ANTIBODY
AGAINST A VANADIUM-ASSOCIATED PROTEIN EXTRACTED FROM THE BLOOD CELLS OF
THE VANADIUM-RICH ASCIDIAN, Ascidia sydneiensis samea.
H. Michibata, J. Wuchiyama, Y. Nose, T. Uyama. Mukaishima
Mar. Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch.
Sci., Hiroshima Univ., Hiroshima.
Recently we reported that a vanadium-associated
protein (VAP) was extracted from the blood cells of an ascidian, Ascidia
sydneiensis samea. VAP was estimated to associate with vanadium
at an approximate ratio of 1 mol : 16 mols. In the present experiment,
we raised a monoclonal antibody, designated F8DH, against VAP in blood
cells of a vanadium-rich ascidian, A. sydneiensis samea. Immunoblot
analysis showed that F8DH recognized 2 related peptides of 15 kDa and 16
kDa of VAP. Using F8DH, VAP was shown to be in the cytoplasm of vanadocytes
and compartment cells, both of which cells were reported to contain vanadium.
F8DH also stained the vanadocytes distributed in the connective tissues
around the alimentary canal, suggesting that vanadocytes in the connective
tissue contained VAP. Furthermore, blood cells of 3 different species of
ascidian having high levels of vanadium, A. sydneiensis samea, A.
ahodori, and Ciona intestinalis, showed reactivity of F8DH but
little reactivity was observed in 2 species having less vanadium, Halocynthia
roretzi and Pyura michaelseni, suggesting that VAP recognized
by F8DH is a common protein in vanadium-rich ascidians.
CDNA SEQUENCE FOR A 45 KDA-ANTIGEN RECOGNIZED BY THE MONOCLONAL
ANTIBODY S4D5 SPECIFIC TO VANADOCYTES IN THE VANADIUM-RICH ASCIDIAN, Ascidia
sydneiensis samea.
T. Uyama1, H. Takahashi2, N. Satoh2, and H. Michibata1.
1 Mukaishima Marine Biol. Lab., Fac. Sci. and Lab. Marine
Molec. Biol., Graduate Sch. Sci., Hiroshima Univ., Hiroshima and 2Dept.
Zool., Graduate Sch.Sci., Kyoto Univ., Kyoto
To characterize a 45 kDa-antigen recognized
by the monoclonal antibody S4D5, specific to vanadocytes in vanadium-rich
ascidian Ascidia sydneiensis samea, the gene encoding the antigen
was screened using S4D5 monoclonal antibody as a probe from a cDNA library
prepared from the blood cells. Three positive clones were isolated.
The longest insert was found to cover other two clones by nucleotide sequencing.
A full length cDNA designated sd45 for the newly cloned molecule was subcloned
into the expression vector according to the sequencing strategy of the
cDNA. Consequently, sd45 encompassed 92-bp of the 5' untranslated
region, a 1542-bp open reading frame, and 449 bp of the 3' untranslated
sequence. A search of the SwissProt sequence data base for similarities
with sd45 detected a match with 6-PGDH both in levels of nucleotide and
amino acid sequences. The open reading frame encoded a protein of
483 amino acids with 74.6% identity and 87.1% similarity to 6-PGDH of sheep.
A 45 kDa-antigen recognized by the monoclonal antibody S4D5 is, therefore,
shown to be 6-PGDH which is an enzyme of the pentose phosphate pathway.
FINDING OF ENZYMATIC ACTIVITY OF 6-PHOSPHOGLUCONATE DEHYDROGENASE
IN VANADOCYTES OF THE VANADIUM-RICH ASCIDIAN, Ascidia sydneiensis samea.
T. Kinoshita, T. Uyama, and H. Michibata, Mukaishima
Marine Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch.
Sci., Hiroshima Univ., Hiroshima.
A 45 kDa-antigen recognized by the monoclonal
antibody S4D5, specific to vanadocytes in vanadium-rich ascidian Ascidia
sydneiensis samea, was found to be 6-phosphogluconate dehydrogenase
(6-PGDH) which is an enzyme of the pentose phosphate pathway, based on
cDNA isolation of RNA samples from blood cells of the ascidian. Western
blot analysis confirmed an abundance of 6-PGDH protein in the vanadocytes
and localization of 6-PGDH in soluble extract in the blood cells.
Soluble extract further exhibited a correspondingly high level of 6-PGDH
enzymatic activity. Km for the substrate and Vmax were calculated to be
62.5 micromol/l and 129 nmol/min at pH 8.9. Almost all vanadium ions
are reduced to the +3 oxidation state via the +4 oxidation state in vanadocytes,
although vanadium ions are dissolved in the +5 oxidation state in sea water.
Some reducing agents must, therefore, participate in the accumulation process.
These observations suggest that NADPH produced in the pentose phosphate
pathway conjugates reduction of vanadium from the +5 oxidation state through
the + 4 oxidation state in vanadocytes of ascidians.
GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN THE PENTOSE PHOSPHATE
PATHWAY IS LOCALIZED IN VANADOCYTES OF VANADIUM-RICH ASCIDIAN, Ascidia
sydneiensis samea.
K. Yamamoto, T. Uyama, and H. Michibata. Mukaishima Marine
Biol. Lab., Fac. Sci. and Lab. Marine Molec. Biol., Graduate Sch. Sci.,
Hiroshima Univ., Hiroshima.
Ascidians are sessile marine animals known
to accumulate high levels of vanadium selectively in vanadium-containing
blood cells (vanadocytes). Almost all the vanadium accumulated in
the vacuoles of vanadocytes is reduced to the +3 oxidation state via the
+4 oxidation state, although vanadium is dissolved in the +5 oxidation
state in sea water. Some of the reducing agents that participate
in the reduction have been found. By chemical study, vanadium in
the +5 oxidation state was reported to be reduced to the +4 oxidation state
in the presence of NADPH. The present study revealed the existence
of glucose-6-phosphodehydrogenase (G6PDH), the first enzyme to produce
NADPH in the pentose phosphate pathway, in vanadocytes of a vanadium-rich
ascidian. Western blot analysis with anti-G6PDH antibody demonstrated
that a positive band of 58 kDa exhibited in soluble proteins. The
soluble proteins were confirmed to exhibit a high level of G6PDH enzymatic
activity. Km for the substrate and Vmax were 78.6 micromol/l and
220 nmol/min, respectively, at pH 8.0. Taken together, it is suggested
that G6PDH conjugates the reduction of vanadium from the +5 through to
the +4 oxidation state in vanadocytes of ascidians.
ELECTRONMICROSCOPIC OBSERVATION OF CELLS THAT FIRST APPEAR
IN THE TUNIC OF Halocynthia roretzi AFTER HATCHING.
S. Ohtake, T. Abe, F. Shishikura, and K. Tanaka, Dept.
of Biol., Nihon Univ. Sch. of Med., Tokyo.
In order to clarify the nature of the tunic
cells of H. roretzi, we examined by transmission electron microscopy
their fine structure after hatching out, and compared their morphological
characteristics to those of the hemocytes in juvenile individuals. The
cells were observed first in tunic immediately after the beginning of tail
absorption distributed at the frontal part of the tadpole larva. The cells
were characterized by their electron-dense cytoplasm and large nucleus.
The cytoplasm wa packed with ribosome-like granules and well-developed
endoplasmic reticulum. Mitochondria were relatively rich and Golgi were
found occasionally. The number of cells, here called larval tunic cells,
increased in tunic, and granules and vacuoles progressively appeared in
their cytoplasm during metamorphosis. 14 days after tail absorption,
we could distinguish in the tunic of juveniles two types of tunic cells.
One was probably the final stage of the larval tunic cell and the other,
characterized by numerous dense granules, corresponded to the dense-granular
cell in adult tunic. The larval tunic cells probably stay in the
tunic from metamorphosis to juvenile stage. However, the fine structure
of these cells differs from hemocytes of the juveniles.
AUTOFLUORESCENCE AND ACIDIC VACUOLES IN TUNIC LUMINOCYTES
OF A LUMINESCENT ASCIDIAN.
E. Hirose1 and K. Chiba2. 1Dpt. Chem. Biol. &
Mar. Sci., Univ. of the Ryukyus, Nishihara, and 2Dpt. Biol., Ochanomizu
Univ., Tokyo.
In the colonial ascidian Clavelina
miniata, physical stimulation induces strong luminescence of yellow-green
in the tunic. The light source is a particular type of tunic cells
(free cells in the tunic), and we call them tunic luminocyte. Under
blue light excitation, tunic luminocytes showed a weak yellow-green autofluorescence
that is similar in color to the luminescence. It is possible that
this fluorescent molecule is concerned with the luminescence system in
the cell. Staining with acridine orange or LysoSensor (Molecular
Probes Inc.) demonstrated the presence of acidic vacuoles in the tunic
luminocytes. The pair images of Nomarski DIC suggested that the acidic
vacuoles seem to correspond to the clear vacuoles that are characteristic
to the luminocytes. Luminescence occurs when water is added to the
freeze dried zooids. The amount of luminescence is much smaller in
acidic condition, e.g., pH 3, than that in neutral condition. This
might suggest that the acidic vacuole contributes to keeping the luminescent
system silent within the cell.
WHICH CELLS CARRY THE INFORMATION OF VISCERAL PATTERN
IN Polyandrocarpa misakiensis?
T. Ishii1, Y. Saito2 and Y. Taneda3. 1Biol. lab.,
Coll. of Edu., Akita Univ., Akita; 2Shimoda Mar. Res. Center, Univ. of
Tsukuba, Shimoda, Shizuoka; 3Dept. of Biol., Fac. of Edu., Yokohama National
Univ., Yokohama.
In the compound
ascidian P. misakiensis, there are two different strains of body
color pattern, white-spot and spotless. In this species, situs inversus
or reversed zooids are easily induced experimentally from the normal zooids.
We created chimeric zooids between body fragments with reversed white-spot
and ones with normal spotless. Then we analyzed the visceral pattern of
asexual offsprings from the chimeric zooids. The blastozooids with normal
white-spot and reversed spotless appeared from the fused area of the chimeric
zooids. These blastozooids were new combination of body color pattern and
visceral pattern. A bud consists of two cell layers, epidermal layer and
peribranchial wall. We already had revealed that the epidermal layer is
the carrier of color pattern. Therefore, our results suggest that the information
of visceral pattern exists in the peribranchial wall.
ABILITY OF VASCULAR BUDDING IN Botryllus schlosseri.
Y.Taneda, M. Ochmai, and Y. Arimoto. Dept.
of Biol., Fac. of Edu., Yokohama Natl. Univ., Yokohama.
In botryllid ascidians a unique budding
called vascular budding is well known. In Botryllus primigenus
it occurs in intact colony. However in Botryllus schlosseri
it does not occur in intact colony, but it occurs when all zooids
and buds are removed from the co1ony. Why does it occur in B.
schlosseri only when all zooids are removed from the colony?
First of all, we investigated the condition under which vascular budding
could occur. Not only in the case without any zooids and any buds
but also in that with some zooids and no buds, vascular budding occurred.
In the latter case vascular budding occurred on the process of physiological
regression of functional zooids. Next we investigated the ability
of vascular budding of the colony. Colony with one functional zooid
(oozooid or first blastozooid) did not have an ability of vascular budding,
while that with two or more functional zooids produced vascular buds.
Ability of vascular budding may be acquired between the colony with functional
first blastozooid and that with functional second blastozooids.
EXPRESSION OF A CALCIUM CHANNEL ALPHA1-SUBUNIT IN ASCIDIAN
EMBRYO.
T.Okada 1, K.Nakajo 2, R.Okagaki 3,
L.Chen 1, Y.Okamura 1,2,4.
1Lab of Cell Biochem., NIBH.,Tsukuba, 2Univ.of Tokyo.,Tokyo,
3Kasumigaura Hospital, Tsuchiura, 4 PRESTO, Japan Science and Technology
Corp., "Intelligence and Synthesis".
Recently we cloned a cDNA putatively
coding for voltage-gated Ca2+ channel, TuCa1, from young tadpole larvae
of the ascidian, Halocynthia roretzi. The amino acid sequence of
TuCa1 was 50% identical to that of the rabbit skeletal muscle Ca2+ channel
alpha1-subunit. Here we studied expression of TuCa1 by whole mount in situ
hybridization. TuCa1 mRNA was expressed in neurons, some epidermal cells,
and all of muscle cells. We also compared development of calcium channel
currents in cleavage-arrested neuronal and muscular blastomeres with the
two-electrode voltage clamp method. Kinetic properties were similar between
Ba2+ currents through Ca2+ channels from neuronal and muscular blastomeres.
These results indicate that the single Ca2+ channel ƒ1-subunit gene encodes
Ca2+ currents in ascidian neurons and muscle cells.This is compatible with
the idea that the EC-coupling of ascidian striated muscle cell is similar
to vertebrate "cardiac" type rather than to "skeletal" type.
INTRACELLULAR CALCIUM OSCILLATIONS AND MEIOSIS RESUMPTION
FROM METAPHASE I INDUCED BY INJECTION OF THE SPERM EXTRACT IN THE ASCIDIAN
Ciona savignyi.
K. Kyozuka1, R. Deguchi2, T. Mohri2 and S. Miyazaki2.
1Asamushi Mar. Biol. Stn., Tohoku Univ., Aomori and 2Natl.
Inst. for Phys. Sci., Okazaki.
We examined effects of sperm extract (SE)
of Ciona savignyi, a supernatant of the centrifuged sperm homogenates,
on changes in intracellular free Ca2+ concentration ([Ca2+]i) and the associated
meiotic process in the oocyte. Microinjection of SE into the unfertilized
oocyte caused two sets of [Ca2+]i spikes, mimicking the [Ca2+]i pattern
at normal fertilization. When SE was microinjected into the cortical region
of the oocyte, the [Ca2+]i rise started from the nearest cortex and propagated
across the oocyte. A similar Ca2+ wave from the cortex was detected
even when SE was injected into the central region. SE injection also
triggered meiosis resumption from metaphase I, resulting in emission of
both first and second polar bodies. SE was heat-unstable and was
speculated to contain a molecule(s) between 30 kDa and 100 kDa. These
results support the ''sperm content model'' that sperm introduce soluble
substances inside the egg following gamete fusion to activate the egg.
DISTRIBUTION ON GONADOTROPIN-RELEASING HORMONE (GNRH)
IMMUNOREACTIVE NEURONS IN AND AROUND THE NEURAL COMPLEX OF THE ASCIDIAN
Halocynthia roretzi.
K. Terakado, Dept. Regul. Biol., Fac. of Sci., Saitama
Univ., Urawa.
Using antiserum against to human or salmon
GnRH, we stained immunohistochemically the neural complex and its neighboring
regions by streptavidin-biotin-peroxidase complex (ABC) method in adults
and juveniles of the ascidian Halocynthia roretzi. Adult neural
complex were also examined with immunoelectron microscopy. There
were numerous GnRH immunoreactive neurons in the cerebral ganglion and
along the dorsal strand. In addition, many immunoreactive axons ran
in the connective tissue matrix to pharyngeal epithelium, epithelium of
dorsal tubercle, ciliated epithelium of gill baskets, etc. These axons
originated from distributed in the connective tissue matrix. GnRH
neurons along the dorsal strand seemed not to extend to these regions and
remained within the blood sinus. In small juveniles (about 2mm in
length), there were already a number of GnRH immunoreactive neurons arranged
longitudinally in the cerebral ganglion and some cell bodies arranged in
lines, presumably along the dorsal strand. However, juveniles reared
for 30 days in refrigerator were GnRH negative in whole body. Immunoelectron
microscopy revealed that GnRH in localized in the 100-250nm granules in
the GnRH neurons associated with the dorsal strand. These results
suggest that GnRH functions as a neurotransmitter or a neuromodulator as
well as a hormone.
PRESENCE OF A PROLACTIN-LIKE IMMUNOREACTIVE SUBSTANCE
IN THE ASCIDIAN NEURAL COMPLEX.
G. Kawahara1, K. Terakado2, K. Yamamoto1, S. Kikuyama1
1Dept. Biol., Schl. Education, Waseda Univ., Tokyo, and
2Dept. Regul. Biol., Fac. of Sci., Saitama Univ., Urawa.
The neural complex (dorsal strand,
cerebral ganglion and neural gland) of Halocynthia roretzi
was examined using light and electron microscopy. Cells scattered
along the dorsal strand and in the cerebral ganglion contained electron-dense
secretory granules with diameters of 100-250nm. Immunoelectron-microscopic
studies with an antiserum against bullfrog prolactin (PRL) revealed that
the secretory granules of some of the granulated cells contained a PRL-like
immunoreactive substance. Gel chromatography and western blot analysis
revealed that the PRL-like substance is a protein with an apparent molecular
mass of 75kDa. The cells of the neural gland and the dorsal
strand neither contained the dense secretory granules nor showed PRL immunoreactivity.
These findings raise the possibility that the granulated cells along the
dorsal strand and those in the cerebral ganglion are phylogenetic progenitors
of vertebrate adenohypophyseal cells.
G-PROTEINS EXPRESSED IN ASCIDIAN LARVA.
T.Iwasa, T.Tajika, K.Kanehara, M.Ohkuma, M.Tsuda. Dept.
of Life Sci., Fac. of Sci., Himeji Inst. of Technol., Hyogo.
The small number of neural cells
in the head and the well-characterized cell lineage based on the stereotyped
cell cleavage pattern make the ascidian larva a simple system for examining
neural differentiation and neural signal transduction. We have isolated
a novel G protein subunit, Gn, from the larvae of Halocynthia
roretzi . Gn has the consensus sequence of N-terminal myristylation,
which is characteristic for various Gi class a subunits. Gn has a
unique amino acid sequence in its C-terminal and lacks a cystein residue
for ADP-ribosylation by pertussis toxin. These data suggest that Gn defines
a novel subclass within the Gi family of a subunits. This is also
supported by a phylogenetic tree of Ga subunits made by the NJ-method.
In situ hybridization and northern analysis of the Gn message revealed
that a small amount of the message is maternal, evenly distributed within
the blastomere, and the expression of the message is first detected at
the 64-cell stage in eight blastomeres containing neural lineage cells.
At the early tailbud stage, it is expressed in several cells of brain and
mesenchyme. Four more cDNA clones were isolated from H. roretzi larvae.
They were Gs, Gi, Gq and one similar to Gq. The class of the last
one is not clear yet.
LIGHT INDUCED SPAWNING AND PHOTORECEPTOR IN THE CEREBRAL
GANGLION OF THE ASCIDIAN Halocynthia roretzi.
M. Ohkuma and M. Tsuda. Dept. of Life Sci., Fac.
of Sci., Himeii lnst. of Tech., Hyogo.
Light signals can shift the phase
of the circadian oscillator of the biological clock for most animals.
In the avian pinealocyte, it was shown that the retinal protein pinopsin
is a photoreceptor that resets the phase of the circadian pacemaker controlling
the rhythmic production of melatonin D. Spawning of ascidians has
been reported to be triggered by exposure to light following a period of
darkness. It was suggested that the cerebral ganglion is a possible
photoreceptor that is related to gamete release in the adult ascidian.
ln order to determine the localization of retinal protein in the cerebral
ganglion of ascidians, the fluorescence of retinal protein were used for
histochemical studies. As in many other ascidian species, the thickest
nerves arise as a pair from the anterior and posterior end of the ganglion.
Ye11ow- green fluorescence due to retinal protein appeared on both sides
of the crossing point of the branch, indicating the presence of retinal
protein.
RELATIONSHIP BETWEEN METABOLIC RATE AND COLONY SIZE OF
THE ASCIDIAN Botryllus schlosseri.
F. Nakaya1, Y. Saito2 and T. Motokawa1. 1Basic
Biol., Fac. of Biosci. & Biotechnol., Tokyo Inst. of Technol.; 2Shimoda
Mar. Res. Center, Univ. of Tsukuba.
The allometric relationship
between metabolic rate and body size is well established in organisms with
individuality. In colonial organisms, however, there is little research
on the relationship between metabolic rate and body size. We measured the
metabolic rate of the colonial ascidian Botryllus schlosseri to
investigate the relationship between metabolic rate and colony size. A
small colony was put on a slide glass which was set in a container submerged
in the open sea water. The colony spread on a slide glass as it grew to
be a sheet. The zooid size did not change with colony growth. We measured
oxygen consumption of these reared colonies of various sizes with 30-800
zooids by oxygen electrodes. The allometric relationship obtained is:
VO2 = 0.0643 W 0.674 (VO2: oxygen consumption
rate, ml/h; W: wet weight, g; 20)
A clear allometric relationship exists between the metabolic
rate and colony size of this colonial ascidian.
GENETIC DIFFERENTIATION IN THE PYURID ASCIDIAN Halocynthia
hispida (Herdman).
T. Kakuda, Dept. of Biol. Sci., Kanagawa Univ., Hiratsuka.
Genetic differences among two morphological
forms of the ascidian Halocynthia hispida - igaboja and ritteri
forms, sympatric at Shimokita Peninsula - were investigated by restriction
fragment analysis of mitochondrial DNA (mtDNA) and allozyme analysis.
Based on restriction patterns of eight restriction enzymes, there were
no shared restriction patterns between two forms. The nucleotide
divergence d which is estimated by the proportion of shared restriction
fragments (Nei & Li '79) were 4.246x10^-2. In allozyme analysis,
a remarkably different electrophoretic pattern was observed in SOD, between
the two forms. These differences are far larger within reproductive
isolated three types of H. roretzi, and equal to the rate between
different species, H. roretzi and H. aurantium. In
addition, by some morphological characters, H. hispida were definable
to the two forms. At the end of September, the igaboja form
was observed spawning, but ritteri form did not. These results
suggest that the two forms of H. hispida may represent separate
species.
SEASONAL VARIATlON IN EMBRYONlC THERMOTOLERANCE OF THE
ASCIDIAN Ciona savignyi.
S. Minowa1, C.Nishijima1,C. Haraguchi1, M. Hashimoto1,H.
Fujisawal and T. A. Nomaguchi2.
1Fac. of Educ., Saitama Univ., Urawa and 2Dept. of Cell
Biol., Tokyo Metropolitan lnst. of Gerontol., Tokyo.
Embryonic thermotolerance of the ascidian
Ciona savignyi was examined with special reference to acclimatization
to seasonal changes in seawater temperature. This ascidian spawns throughout
the year. Its life span is completed within six months and depends on the
cumulative environmental temperature. The optimal temperature range for
development from early cleavage stage to metamorphosis in embryos produced
by individuals raised in warmer seasons differed significantly from that
of individuals raised in colder seasons. Within the common optimal
temperature range, developmental times at any given temperature were the
same for both groups of embryos. The thermal acclimation of ascidian embryos
is discussed in comparison with that of echinoid embryos.
IMMUNOHISTOCHEMISTRY OF HATSCHEK'S PIT DURING POST-BREEDING
PERIODS IN AMPHIOXUS, Branchiostoma belcheri.
K. Kuobkawa1 and M. Nozaki2. 1Ocean Research Inst., Univ.
of Tokyo, Tokyo, 2 Marine Biol. St., Niigata Univ., Niigata.
Amphioxus were collected
in the coast of the Atsumi peninsula, Aichi prefecture during the
summer and early autumn of 1995, 1996 and 1997. They were fixed in
a 10% formaldehyde solution except for individuals collected in September,
1995. The latter individuals were fixed in a Bouin-Hollande sublimate fixative
solution for immunohistochemistry. When an anti-human LH antibody
was used, a strong immunoreactivity was detected in the apical area of
Hatschek's pit, but no immunoreactions were detected against antibodies
of Salmon GTH I and II, and sturgeon GTH. The positive reaction was observed
in all individuals examined which possessed different stages of the gonad,
matured, regressed and developing. Interestingly, small spots showing
a strong immunoreactivity were found in the abdominal part of the neural
tube extending to Hatschek's pit in individuals possessing the developing
gonad. This suggests that the immunopositive substance to anti-human
LH antigen exists not only in Hatschek's pit but also in the neural tube,
although the relation between these two regions is still to be elucidated.
Judging from the gonadal condition, this species breeds once a year between
July and August. In September, gonads are completely regressed and only
early stage germ cells were observed . Mature gonads were usually observed
in individuals with longer body length than 30 mm. This species seems
to become sexually mature within a short time, about 6 weeks after metamorphosis
and settlement.
A CONNECTIN-LIKE PROTEIN IN Amphioxus STRIATED
MUSCLE.
S. Kimura1, Y. Kawamura1, A. Watanabe1, K. Kubokawa2,
and K. Maruyama1. 1Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba,
and 2Ocean Res. Inst., Univ. of Tokyo.
Connectin/Titin is a 3000 kDa
elastic protein that positions the myosin filament at the center of a sarcomere
in vertebrate striated muscle myofibrils. It has been shown that
connectin is present in all the vertebrate skeletal muscles examined from
mammals to fishes including lamprey (Cyclostomata). In the present study,
it was demonstrated that the 3000 kDa band of Amphioxus striated
muscle in an SDS gel electrophoresis reacted with polyclonal antibodies
to chicken breast muscle connectin (Pc1200). Although other kinds
of antibodies to connectin did not give positive reactions, it is regarded
that a connectin-like 3000kDa protein is present in protochordate skeletal
muscle. In addition, it is observed that a 750 kDa band of Amphioxus
striated muscle crossreacted with polyclonal and monoclonal antibodies
to skeletal muscle nebulin. This information is of particular interest
because nebulin is present in invertebrate skeletal muscle as well as in
vertebrate skeletal muscle.
2. Italian Embryol. Group 43rd annual meeting, Grottammare, Italy June 8-11, 1997. Publ. in Anim. Biol. 6(3).
HEMOCYTES AND PHAGOCYTOSIS DURING THE COLONIAL LIFE CYCLE
OF THE ASCIDIAN Botryllus schlosseri.
L. Ballarin, F. Cima and A. Sabbadin. Dpto. di
Biol., Univ. di Padova, Padova, Italy.
Even if ascidian hemocytes are the
subject of a vast scientific literature, controversial aspects still exist
mainly regarding their differentiation pathways and their biological role.
For instance phagocytosis raises interesting questions about the analogies,
homologies of this process with its counterpart in vertebrates. We focussed
our attention to phagocytosis in
the colonial ascidian B. schlosseri, both in
vitro and in vivo. In vitro investigations led us to
identify the phagocytes (hyaline amoebocytes and macrophage-like cells)
and at least two receptors involved in the recognition and adhesion of
foreign/non-self particles to the phagocyte surface. One is the mannose
receptor, required to bind yeast in the absence of any opsonizing molecule
and playing a similar role in mammals. The other is the antigen CD36 involved
in the recognition of apoptotic cells by mammalian macrophages and probably
required for the recognition of apoptotic cells of degenerating zooids
ingested during the takeover phase of the colonial life cycle. The study
of phagocytosis in vivo took advantage of the peculiar life cycle
of Botryllus colonies, which is the sum of several blastogenetic
cycles: at 19° C old zooids are weekly resorbed and replaced by their
palleal buds reaching maturity and opening the siphons. During the takeover
we observed a significant increase in the frequency of circulating macrophage-like
cells containing digested material inside their vacuoles and a parallel
decrease in the frequency of hyaline amoebocytes. This fact supports our
hypothesis of a single phagocyte-type in Botryllus, with two functional
stages: the hyaline amoebocyte, moving actively and ingesting foreign particles,
which changes its morphology to the macrophage-like one upon ingestion.
These changes were coupled with a cytoskeletal reorganization, both in
the actin and tubulin components. The takeover phase is characterized by
a massive infiltration of phagocytes inside the tissues of the old zooid
(not into the bud tissues) and by an intense phagocytosis: we observed,
analogously to that reported for the same process in mammals, a significant
increase in the concentration of reactive oxygen metabolites, such as peroxides,
and of hydrolytic lysosomal enzymes, such as acid phosphatase. We
also observed a significant increase in the frequency of circulating macrophage-like
cells containing apoptotic cells inside their vacuoles, as revealed with
the TUNEL reaction, thus confirming the morphological data indicating the
occurrence of apoptosis associated with zooid regression. At the beginning
labeling was limited to the nucleus of the ingested cells whereas, as regression
went on, it diffused inside the vacuoles, due to the release of small DNA
fragments from the nuclear envelope. We are now interested in defining
the morphological changes on the surface of senescent cells responsible
for their recognition by phagocytes.
EFFECTS OF RETINOIC ACID ON ASCIDIAN EMBRYOS.
C. Sotgia, U. Fascio, S. Groppelli,* M. De Vincentiis
and F. De Bernardi.
Dept. of Biol., Univ.of Milan; *Dept. of Genetic, General
& Molec. Biol., Univ. of Naples.
Ascidian embryos showed malformations
similar to those described in many vertebrate embryos. Previous researches
on amphibian embryos (Xenopus laevis) have shown that treatment
with RA at early gastrula stages affects the development of head structures
leading to dose-dependent progressive truncation of the antero-posterior
axis, preventing the formation of forebrain, cement gland and hatching
glands (Durston et al., 1989. Nature 340:140-144). The results of RA treatment
are fully described in two species of solitary ascidian: Phallusia mammillata
(De Bernardi et al.1994. Anim. Biol. 3:75-81) and Halocynthia
roretzi (Katsuyama et al.1995. Development 121:3197-3205). In
both species malformations were detectable in the larvae, the anterior
part of which were truncated, and adhesive papillae failed to develop;
the posterior part of the larvae was not affected. Phallusia
embryos at the two blastomere stage treated with 10-7 M RA developed larvae
that were unable to hatch. On the contrary, Halocynthia embryos
treated with higher RA concentration developed larvae that normally hatched.
We tried to understand the reason for the different effect of RA on the
two species and we performed RA experiments on Ciona intestinalis,
a solitary ascidian belonging to a different family. C. intestinalis
embryos were treated at early cleavage stage with 10-6 M RA for at least
four hours in the dark. We obtained hatched swimming larvae with rather
normal tail. but the cephalenteron was small and rounded. Papillae were
absent and papillary nerves were damaged. The brain vesicle was reduced,
lacking sensory organs whose pigment was extruded. The mesenchymal cells
beneath the papillae were absent and the other mesenchymal cells were kept
close to the epithelium of the pharynx. The malformations obtained confirmed
that the effect of RA is always the same in ascidian embryos and it is
comparable to that in vertebrate embryos. We used a polyclonal antibody
raised against Ciona intestinalis hatching enzyme to perform whole
mount immunofluorescence experiments. By confocal microscopy bright fluorescence
was localized in the peripheral secreting cells of the adhesive papillae
and in the cells of the epidermis of the tip of the tail of newly hatched
C. intestinalis larvae. The same antibody recognized hatching enzyme
in Phallusia mammillata newly hatched larvae, in which only the
peripheral secreting cells of the adhesive papillae react. Since RA causes
truncation of the anterior region of the larvae, papillae do not develop,
and consequently Phallusia larvae cannot hatch. Ciona intestinalis
larvae lacking the papillae after RA treatment do hatch by hatching enzyme
secreted by the tail epidermal cells, which are not affected by RA treatment.
LOCALIZATION OF HATCHING ENZYME FROM THE SEA-SQUIRT Ciona
intestinalis.
U. Fascio, S. Groppelli,**A. D’Aniello, *M. De Vincentiis,
*S. Scippa and F. De Bernardi.
Dept. of Biol., Univ. of Milan;*Dept. of Genetic, Gen.
& Molec. Biol., Univ. of Naples; **Dept. Biochem. & Molec. Biol.,
Staz. Zoologica “Anton Dohrn”,Naples.
A polyclonal antibody raised against
the hatching enzyme of Ciona intestinalis (D’Aniello et al.,
1997 Biochim. Biophys. Acta, 1339:155-163) was used on larvae of different
ages in whole mount immunofluorescence experiments to localize the
cells secreting the enzyme. After staining with FITC-conjugated second
antibody, the larvae were observed by confocal microscopy. The larvae just
before hatching (9-10 hours after fertilization) showed the presence of
the enzyme in the peripheral cells of the adhesive papillae. These cells
secrete and produce the hatching enzyme which digests the egg chorion.
The newly hatched larvae showed fluorescence both on the adhesive papillae
and in the epidermal cells of the tip of the tail. Higher magnification
confocal images of the papillae revealed bright fluorescence both in peripheral
cells of the papillae and in the cavity between the tunic and the apex
of the papillae (hyaline cap). The secreting cells were still fluorescent.
The swimming larvae maintain the fluorescence in the peripheral cells and
in the hyaline cap for some hours until the beginning of metamorphosis,
when the fluorescence of the tip of the tail disappears. We also used the
same antibody on larvae of another ascidian, Phallusia mammillata.
The peripheral cells of the papillae of the newly hatched larvae were fluorescent,
and a bright fluorescence was also present between the two layers of the
tunic above the papillae and the anterior part of the cephalenteron. We
never observed fluorescence in the cells of the tail epidermis. When the
larvae start the tail resorption, just before settlement and the beginning
of metamorphosis, some vesicles with a positive fluorescent reaction appeared
at the tip of the adhesive papillae. These tunic vesicles, already observed
by SEM, soon break and some microvilli protrude from the central sensorial
cells (De Bernardi et al., 1996 Anim. Biol. 5:83). We hypothesize
that at this stage the hatching enzyme, or a modification of it, could
help the next protrusion of the microvilli interacting with protein components
of the tunic.
PERIPHERAL NERVOUS SYSTEM DEVELOPMENT IN Botryllus
schlosseri (ASCIDIACEA).
L. Manni, G. Zaniolo & P. Burighel. Dpto. di
Biol., Univ. di Padova, Italy.
The brain of adult ascidians is composed
of an oval cerebral ganglion lying within the connective tissue of the
mantle between the two siphons. Some nerves, directed to the periphery,
emerge from its ends. Knowledge of the peripheral nervous system (PNS)
and, in particular, of its development in ascidians, is incomplete. Detailed
information on branchial basket innervation was recently obtained (Arket
et al.1989, Cell Tiss. Res. 257:285-294) by staining nerves for acetylcholinesterase
(AChase), which is not restricted to synaptic sites but occurs throughout
the neurons. Using the same method, we studied the complete organization
of the cholinergic PNS and its development in relation to forming organs
in blastozooids of the colonial ascidian Botrylllus schlosseri.
B. schlosseri forms colonies of numerous zooids, each possessing
its own nervous system. Colonies cultured in the laboratory were followed
daily under the stereomicroscope, and buds and adults were dissected and
processed at appropriate stages of development to reveal AChase activity.
Whole mount preparations and thick sections of embedded specimens were
observed.
Achase activity appears in buds at
the stage at which all organs are present in rudimentary form, before the
heart begins to beat. In these young buds, 4 or 5 bundles of neurites emerge
from each side of the developing ganglion, running in the mantle toward
the latero-ventral surface of the bud. During development, the number of
nerve roots increases to 10-15 in both sides of the ganglion, in addition
to some anterior, posterior and ventral nerves. Nerves split repeatedly
into thinner branches penetrating bud organs. In particular, nerves from
the anterior end of the ganglion innervate the most anterior region of
the animal (especially the oral siphon) and give rise to two main parallel
nerves, directed ventrally to the endostyle. Nerves from the posterior
end run in the dorsal-posterior mantle to the developing atrial siphon,
and a bundle of neurites (the visceral nerve) passes from the ventral surface
of the ganglion to the viscera, running along the roof of the branchia.
The viscera are also innervated by branches of nerves coming from the mantle
and passing through the trabeculae, and from the endostyle region. Nerves
display numerous swellings along their length, thus looking like a string
of pearls, sometimes very large and regularly arranged. Nerve development
usually parallels that of the organs they accompany. In the adult, local
nerve plexa may be recognized innervating branchia, gut (stomach, intestine
and pyloric gland), heart and the two siphons. Activity-dependent selection
of neurites probably occurs during development, so that adults possess
fewer nerves than buds. In fact, only two pairs of anterior and posterior
nerves, one visceral nerve, and 4 or 5 minor lateral nerves lead
from the brain in adults.
3. RETINOIDS ’97 European Retinoid Research Group, Nice (France) Sept. 28- Oct. 1, 1997.
RETINOIC ACID TREATMENT OF EARLY ASCIDIAN EMBRYOS PRODUCES
TRUNCATION OF ANTERIOR STRUCTURES OF THE LARVAE.
F. De Bernardi, S. Groppelli and C. Sotgia. Dept.
of Biol., Univ. of Milan, Milan, Italy.
Embryos of Phallusia mamillata
and Ciona intestinalis were treated at two-cell or at four-cell
stage with different concentrations of all-trans retinoic acid (RA) for
two hours, rinsed with sea water and allowed to develop at 20°C to
the swimming larva stage. The RA-treated embryos give rise to hatching
or non-hatching larvae that show dose-dependent reduction of anterior adhesive
papillae, alterations of the brain vesicle and of sensory organs and extrusion
of pigment. Pharynx structures appeared to be unaffected, but the button
cells of P. mammillata larvae appeared unable to move and remained
close to the endodermal cells. The larvae, still inside the chorion, retracted
the tail and carried out metamorphosis quite regularly, but without settlement.
The Ciona intestinalis larvae hatched but failed to develop adhesive
papillae. The effect of RA on both ascidian species was to truncate the
anterior structures, as it does in amphibian and in other vertebrate embryos,
probably acting on migrating cells.
4. Gordon Conf. on natural products research, Ventura, Calif. Feb. 22-27, 1998.
OCCURRENCE OF AN OVERSULFATED DERMATAN SULFATE WITH A
HIGH HCII-MEDIATED THROMBIN INHIBITORY ACTIVITY IN ASCIDIANS (CHORDATE-TUNICATE).
M.S.G. Pavao1,2, K. Rebeca1, L.C.F.
da Silva1, P.A.S. Mourao1, N.S. Colwell2
& D.M. Tollefsen2.
1Dept. Biochquim., Inst. Ciencias Biomedicas, Univ. Fed.
Rio de Janeiro, C.P. 68041, Rio de Janeiro, RJ 21941-540, Brazil; 2Div.
of Hematol., Depts. Internal Med, Biochem & Mol. Biophys., Washington
Univ. Sch. Med., St. Louis, MO.
We previously isolated an oversulfated
dermatan sulfate-like glycosaminoglycan from the body of the ascidian Ascidia
nigra, composed of disaccharide units of [IdoAc(2S04) -
GalNAc (6S04)] (J. Biol. Chem. 270:31027-31036, 1995). Different
from mammalian dermatan sulfate, the ascidian glycosaminoglycan had no
HCII-mediated thrombin inhibitory activity because of the absence of sulfation
on carbon 4 of the galactosamine residues. We now describe a new oversulfated
dermatan sulfate with a high HCII activity, isolated from the body of the
ascidian Halocynthia pyriformis. The structure was determined by
analysis with specific glycosidases and sulfatases, and by 1H
Nuclear Magnetic Resonance. The polymer is composed of repeating
disaccharide units of [IdoAc(2S04) - GalNAc (4S04)].
It has a very high HCII-mediated thrombin inhibitory activity, 13 times
more potent than that of the mammalian counterpart, estimated by a chromogenic
substrate assay. The activity of the ascidian glycosaminoglycan is
specific for HCII, and experiments using native or mutated recombinant
HCII indicate that it binds to the dermatan sulfate binding site on the
inhibitor. We also describe the occurrence of a heparin-dependent thrombin
inhibitor in the ascidian hemolymph, which inhibited thrombin activity
in the presence of 1ug of heparin. The inhibitor forms a 97,000
MW covalent complex with human thrombin. No inhibition or complex
is observed in the presence of mammalian dermatan sulfate.
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