International Urochordate Meeting 2003, Carry le Rouet, France, October 11 - 15th
Organized by Patrick Lemaire, Marseille (France)
Opening lecture: Let's move on ascidian biology with new ideas
Nori Satoh, Dept. of Zool.,
Ascidians begin shining as an experimental system to understand various biological phenomena. Regardless of reasons why you choose ascidians as materials, it is certain that ascidians provide an extremely convenient model for every study of cellular and molecular biology. For example, the tadpole larva of ascidians possess the most simplified chordate body plan. Analysis of the Ciona intestinalis draft genome indicates that the ~117 Mbp euchromatic genome contains ~16,000 protein-coding genes. Among these is a fundamental set of conserved chordate proteins involved in cell signaling and development. A thorough examination of Ciona gene expression, (the transcriptome) is ongoing, including large scale EST analyses, cDNA sequencing, and in situ hybridization screens. Ciona savignyi genome is also being read, leading comparative genomics of the basal chordates. In addition, recent advances in the methodology used to investigate gene regulation and function are tremendous. Mutagenesis and mutant screening is now unveiling novel genes with developmental function. Furthermore, research fields are expanding. Now, somebody is thinking apoptosis while others dramatic changes in morphology and gene expression during metamorphosis. I believe ascidians provide a very powerful experimental system to understand biological phenomena at every level. So, we need new ideas to attract general biologists with research using ascidians.
Fossil evidence on the systematic position of the tunicates and the other major deuterostome groups.
Richard Jefferies and Patricio
Dominguez, The Natural History Museum, Cromwell Rd. London, SW7 5BD,
The Palaeozoic fossils known as carpoids have a skeleton of echinoderm type, with each plate a single crystal of calcite, but lack the five-fold symmetry of extant echinoderms and have gill slits and a post-anal locomotory tail. Within the carpoids, accepting the DNA-based cladogram ((hemichordates + echinoderms) chordates), the stem groups can be recognised of tunicates, acraniates, craniates, chordates, hemichordates, echinoderms and ambulacrarians. The origin of the crown groups out of these stem groups can be reconstructed. The traditional group Cornuta, which are stem-group chordates, have external left gill slits only, whereas the traditional group Mitrata, which resemble over-sized, calcite-plated tunicate tadpoles, had internal gill slits on right and left. The mitrates were crown-group chordates and include the stem groups of tunicates, craniates and acraniates. The stem-group tunicate mitrates, in crownward order, include Peltocystis, Jaekelocarpus, Balanocystites and Anatifopsis. In this stem group the skeleton becomes progressively thinner, the left and right atrial openings at first lengthen, then migrate onto the dorsal surface, then meet mid-dorsally. A computer-tomographic study of Jaekelocarpus gives direct evidence of gill slits on right as well as left and of paired atrial openings anteriorly. The phylogenetic origin of right gill slits at the origin of mitrates is homologous with the sudden origin of right gill slits at metamorphosis in amphioxus from a larva with left gill slits only. The cranial nerves and brain of tunicate mitrates can be reconstructed and prosencephalon, deuterencephalon, trigeminal ganglia, acoustic nerves, optic system and olfactory system can be recognised. The mitrates suggest that the correct cladogram for chordates is (acraniates (tunicates + craniates)).
and Richard Jefferies, The Natural History Museum, Cromwell
Rd., London SW7 5BE, UK
The tadpole-shaped Cambrian fossils known as vetulicolids were originally described as limbless arthropods but were transferred to the Deuterostomia by Shu et al. (2001) on finding finding five pairs of gill slits in them. We have studied Chinese material of the genus Vetulicola and conclude that it was almost certainly a tunicate and probably a stem-group appendicularian. Synapomorphies with recent tunicates present in Vetulicola include: 1) mantle; 2) atria extending on right and left the entire length of the pharynx; 3) intrabranchial grill with dorso-ventral vessels arranged in primary, secondary, tertiary etc. orders and longitudinal vessels not arranged in orders; 3) brain (ganglion) at dorsal anterior in the head rather than in the primitive position at the posterior end of the head; and 4) a tail adapted for swimming forwards, rather than crawling rearwards as was the primitive habit. Probable synapomorphies with the appendicularians include: 1) reduction of the volume of the atria (as compared with the tunicate mitrate Jaekelocarpus); 2) Reduction of the gill slits to only one on either side for each transverse section, as compared with many gill slits per transverse section in in extant ascidians, three or four in Jaekelocarpus and only one in appendicularians; 3) Torsion of the tail through 90° anticlockwise seen from behind (this takes place in the three most anterior segments in Vetulicola but at the anterior end of the tail in extant appendicularians. Beside appendicularians, it occurs also in extant aplousobranchs); 4) median position of anus (though this is at the end of the tail, not at median posterior in the head). The thin skeleton was probably of calcite as in an echinoderm or mitrate.
Molecular and morphological analyses of tunicate phylogeny
Thomas Stach, Royal Swedish Academy of Sciences, Kristinebergs Marina Forskningsstation, 45034 Fiskebäckskil, Sverige email@example.com
The phylogenetic information content in molecular data sets was comparatively and consistently evaluated. To this end several methods were applied, amongst others bayesian likelihood and spectral analysis. It is concluded that the phylogenetic signal to elucidate the relationship of higher tunicate taxa in molecular data is weak. The conflict within the molecular data is strong, although several clades are repeatedly recovered. Monophyletic groups suggested by the molecular data, in decreasing order of support, are the Appendicularia; the Aplousobranchiata; the Molgulidae; a clade consisting of Pyuridae+Styelidae; the Tunicata; a clade consisting of Aplousobranchiata + Appendicularia; the Pleurogona; the Phlebobranchiata; a clade consisting of Phlebobranchiata+Thaliacea; the Thaliacea. Analysis of a data matrix of traditional, mainly morphological characters results in a highly unresolved tree topology. New morphological characters (immunoreactivity against serotonin in the central nervous system, ultrastructure of muscle cells) were investigated and their phylogenetic potential explored. Although preliminary, these data lend some support to the hypothesis proposing a sister group relationship between Appendicularia and Aplousobranchiata. A corollary of this hypothesis is the proposition that the last common ancestor of Tunicata possessed a sessile adult.
Phylogenetic analysis of Ciona intestinalis gene superfamilies supports the hypothesis of a gene expansion at the base of vertebrate ancestry
Magalie Leveugle, Karine Prat, Cornel
Popovici, Daniel Birnbaum and François Coulier Département d'Oncologie Moléculaire,
Unité 119 INSERM, IFR57, Marseille,
Understanding the formation of metazoan multigene families is a good approach to reconstitute the evolution of the vertebrate genome. In this attempt, the analysis of the genome of selected species provides valuable information. Ciona intestinalis belongs to the urochordates, whose lineage separated from the chordate lineage that later gave birth to vertebrates. We have searched available sequences from the small marine ascidian C. intestinalis for orthologs of members of five vertebrate superfamilies, including tyrosine kinase receptors, ETS, FOX and SOX transcription factors, and WNT secreted regulatory factors, and conducted phylogenetic analyses. We have found that most vertebrate subfamilies have a single C. intestinalis ortholog. Our results support the hypothesis of a gene expansion at the base of vertebrate ancestry. They also indicate that Ciona intestinalis genome will be a very valuable tool for evolutionary analysis.
Gene duplication and functional adaptation: the Amt/Rh protein family
Giorgio Matassi, Rita Marino,
Raffaella Tarallo, Nietta Spagnuolo, Miriam Di Filippo, Jong-Tai Chun, Rosaria
De Santis, Paolo Sordino, Euan R. Brown Stazione Zoologica
“Anton Dohrn” Naples, Italy
We are interested in studying the relationships between gene duplication and evolution of protein function. By showing that human Rh50 proteins promote ammonium transport in yeast we took the first step to unravel the molecular basis of ammonium transport in vertebrates (Marini et al. Nature Genet. 2000; 26, 341-344). RH50 genes probably arose by duplication from an ammonium transporter (Amt) ancestor. Though with different copy numbers, RH50 and Amt genes are both present in green algae, slime-mold, nematode, fruit-fly and ascidian, but not in vertebrates (in which only RH50 paralogs are found). In the framework of the evolution of the Amt/Rh protein family, we are currently carrying out structural and functional analyses, to understand how molecular changes leading to functional adaptation correlate with different morphological, physiological and developmental complexities in both invertebrate (Ciona intestinalis) and vertebrate (zebrafish) model systems.
1.2: Evolution of developmental patterns
Session 1.2: Evolution of developmental patterns
The giant Ecteinascidia tadpole: Evidence for a migratory neural crest and induced tail musculature.
W. R. Jeffery, A. G. Strickler,
and Y. Yamamoto Department of Biology, University
of Maryland, College Park, MD, 20742 USA, and Bermuda Biological Station,
St. George’s, GE 01, Bermuda. firstname.lastname@example.org
The model ascidians have small, structurally simple, and rapidly developing tadpole larvae. In contrast, many other ascidians have larger, more complex, and slowly developing tadpoles. The development of highly differentiated tadpoles has been virtually ignored and is ripe for analysis using modern methods. Accordingly, here we introduce the ascidian Ecteinascidia turbinata, which has a giant tadpole. The Ecteinascidia tadpole has an elaborate trunk with two-pigmented siphon rudiments and a robust tail with 1134 muscle cells. The large size of the Ecteinascidia embryo allows a combination of embryological and molecular approaches to study the diversity of urochordate development. We will describe two current studies of Ecteinascidia development. First, we report DiI cell labeling results identifying a population of migratory neural crest-like cells. The dorsal neural area of developing larvae was injected with DiI and labeled cells were followed through metamorphosis. DiI labeled cells were found to delaminate from the dorsal neural region and migrate into the siphon primordia and body wall, where they differentiated into pigment cells. These results suggest that Ecteinascidia body pigmentation may be derived from a population of migratory neural crest-like cells. Second, we report antibody staining, gene expression, and embryo manipulation studies suggesting that Ecteinascidia embryos do not contain a myoplasm or use cytoplasmic determinants to generate their 1134 tail muscle cells. Instead, Ecteinascidia muscle cells appear to be specified entirely by inductive events. Our results suggest that characters attributed to vertebrate embryos, such as a migratory neural crest and induced musculature, may also be found in developing urochordates. Supported by NSF grant IBN-0212110
The complex Pitx transcriptional unit : how ancestral is it ?
Christiaen L., Bourrat F., Joly
junior group, UPR2197 DEPSN, INAF, CNRS, 1 av de la terrasse, 91198 Gif-sur-Yvette,
The Pitx gene in ascidians has been used to track the ancestry of left-right asymmetry and adenohypophyseal anlage back to the origin of Chordates. Indeed, the Ciona intestinalis Pitx exhibits an expression pattern highly reminiscent of that observed in the handfull of Vertebrate and Cephalochordate species where it has been caracterized. To go one step further in our understanding of the evolutionary history of pituitary developmental genetics, we searched for the cis-regulatory elements responsible for stomodeal expression of Ci-Pitx. Combining sequence comparison among Ciona species, functional testing by eggs electroporation and close examination of the Ciona intestinalis draft genome sequence, we caracterized the 16kb Ci-Pitx locus. Here we present evidence for the existence of two Ci-Pitx isoforms generated by alternate splicing and the use of two distinct promoters. Only the Ci-Pitxa isoform is expressed in the stomodeum in a monophasic fashion from mid-taibud to juvenile stages. We found two separate elements responsible for this stomodeal expression. They belong to separate promoter regions, and exhibit partially overlapping early and late activity respectively. The time by which the relay between the two elements occurs coincides with the end of embryogenesis and might thus reflect a switch from an embryonic to a metamorphic developmental program. In light of these data, we discuss the evolutionary relevance of the apparently conserved genomic organization of the Pitx locus in Chordates, with special emphasis on pituitary and stomodeal development and evolution.
Retinoic acid (RA) is a signaling molecule required for the formation of a number of chordate-specific and vertebrate-specific characteristics. These include the anteroposterior pattern of the dorsally located central nervous system, pharynx with gill slits, neural crest cells and limbs. The necessity of endogenous RA and the RA receptor (RAR) has been demonstrated in vertebrates by mutant analyses, vitamin A-deficient animals and various other methods. We identified cDNA clones encoding RAR, retinaldehyde dehydrogenase (RALDH), and RA-degrading enzyme (CYP26) in the ascidian Ciona intestinalis. Since RAR has been identified only in chordates, the acquisition of the RAR-mediated RA signaling pathway is thought to be an important event for the innovation of the chordate body plan. Raldh and Cyp26 also seem to be chordate specific. The expression pattern of these genes in the Ciona embryo is essentially similar to that in vertebrate embryos. A microarray analysis including 9287 non-redundant cDNA clones was carried out to screen RA target genes in the Ciona embryo. The spatial expression pattern of candidate RA target genes was examined by in situ hybridization. We identified various novel RA target genes as well as well-known vertebrate RA target genes including Hox-1 and Cyp26. RA also regulates morphogenesis during the asexual reproduction of ascidians, suggesting that RA may also have played a part in producing diversity within the chordate groups. We would like to report the results suggesting that RA signaling cascade had already been well established in the common chordate ancestor.
Evolutionary analysis of Hox1 regulation in the neural tube and neural crest
The vertebrate characteristic of finely patterned craniofacial structure is intimately related to neural crest. Anteroposterior positional information carried by the neural crest is important for development of the vertebrate craniofacial region, and this information is encoded as the pattern of Hox gene expression. I challenge this molecular evolutionary background for new expression of Hox genes in the neural crest. We have already tested putative enhancers of amphioxus Hox genes in mice and chickens by performing transgenic assays, where we found that the element designated as element 1A, which sits 3' downstream of AmphiHox1, drives expression in the neural tube with the anterior limit in posterior hindbrain and the neural crest cells. In order to tell whether cis elements for neural crest expression are separable from those for the neural tube, I have performed the deletion assays for the element 1A. The deletion assays showed that a DR5 type retinoic acid responsive element is indispensable for both the neural tube and the neural crest expression. I also found that DR5 alone is enough for the expressions. Therefore, I propose that the neural crest expression of the Hox genes is evolved by co-opting the retinoic acid dependent regulatory machinery, which is originally functioning in the protochordate neural tube, for the vertebrate neural crest. So far it has been believed that the neural crest expression of the Hox genes is regulated independently from that in the neural tube. This idea is mainly based on the two observations; 1) the anterior limit of expression for Hox genes is not the same in the neural tube and the neural crest. 2) The neural crest specific cis element is present in Hoxa2, which is regulated by AP-2. These observations will be discussed based on some our data and recently reported evidences.
Oikopleura dioica HOX genes are not clustered
Seo HC1, Maeland AD1, Edvardsen RB1, Bjordal M1, Hansen A1, Flaat M1, Jensen MF1, Lehrach H2, Reinhardt R2, Chourrout D1
1Sars International Centre for Marine
The Oikopleura dioica genome, represented by a 41 Mb shotgun dataset, has been examined for the presence of HOX and HOX-related genes. Independent cloning has been performed and essentially provided the same set of genes. In total we isolated genes and cDNAs, including splicing variants, encoding nine HOX genes, named HOX 1, 2, 4, 9, 9B,10, 11, 12,13, EVX, GSX and CDX. Phylogenetic analysis confirmed the identity of these genes. The upstream sequences of two HOX genes (HOX1 and HOX10) contain retinoic acid response elements. Expression patterns were studied at three development stages through RNA-RNA in situ hybridizations. The patterns of HOX genes showed a general tendency for tissue-specific expression and poorly evoked the classical expression colinearity. To search for the HOX cluster, nine distinct gene probes were hybridized with a BAC library covering the genome 15-20X and none of the positive BAC clones hybridized with more than one gene probe. A BAC walk, conducted on both sides of each HOX gene over 300 kb, did not permit to join distinct HOX genes. Nine HOX-containing BACs were sequenced, revealing a large number of surrounding genes. For each HOX gene, cDNAs of five surrounding genes were recloned and their expression patterns were examined. These did not show similarity with the HOX gene expression patterns. The absence of HOX gene clustering in Oikopleura, and the possible loss of HOX function will be discussed with reference to other imperfect HOX clusters (C. elegans, Ciona), low level of body plan complexity and lineage vs position dependent cell fate.
The ins and outs of retrotransposable elements in the miniature genome of Oikopleura dioica
Volff JN1, Lehrach H2, Reinhardt R2, Chourrout D3 1Biofuture Research Group, Biozentrum, University of Würzburg, Germany 2Max-Planck Institute for Molecular Genetics, Berlin, Germany 3Sars International Centre for Marine Molecular Biology, Bergen, Norway
Retrotransposable elements have certainly played an important role in shaping eukaryotic genomes, and their levels of activity and elimination are major parameters influencing the size of a genome. Interactions between retroelements and host genomes are extremely complex and still poorly understood. With about 15,000 genes within 65 megabases of DNA, the marine non-vertebrate tunicate Oikopleura dioica has the smallest genome ever found in a chordate, and provides an excellent model to study retroelement behavior in a very compact genome. Consistently with a massive elimination of retroelements, none of 14 major clades of non-long-terminal-repeat (non-LTR) retrotransposons was detected in 41 Mb of non-redundant genomic sequences from O. dioica, while at least six clades were identified in the 2-3 times less compact genome of the ascidian Ciona intestinalis. Only one, apparently novel family of non-LTR retrotransposons was found in O. dioica. In contrast, Ty3/gypsy LTR retrotransposons presented an astonishing level of diversity. They were generally poorly corrupted and frequently bore an envelope-like open reading frame, suggesting that they might have been acquired through infection. These elements potentially represent, besides vertebrate retroviruses, the second family of infectious retroelements identified in chordates.
The non-LTR retrotansposons in Ciona intestinalis.
Ricard Albalat, Jon Permanyer and Roser Gonzàlez-Duarte email@example.com
Departament de Genètica. Facultat de Biologia. Universitat
de Barcelona, Av. Diagonal, 645. 08028
An analysis of the Ciona intestinalis non-LTR retrotransposon families will be presented. The Ciona genome database has been used to identify representatives of the eukaryotic reported clades. Experimental and in silico approaches have been combined to assess the overall features of each family: structural organization, copy number, genome distribution and methylation status. The Ciona genome harbors a low copy-number of representatives of the I, L1, L2, LOA and R2 clades. The elements are not randomly distributed but associated to genomic regions whose gene-density is lower than average. Moreover, Southern analyses reveal that the non-LTR elements are contained in the unmethylated fraction of the ascidian genome.
Department of Zoology,
We sequenced 11 sets of several thousands expressed sequence tags (ESTs) derived from various staged embryos and tissues of Ciona intestinalis (Satou et al. 2002). In general, sequence similarity of each 3-prime tags was used for characterizing ESTs into independent clusters. We attempt to modify the EST-cluster using full-length cDNAs and draft genome DNA sequence data in addition to EST sequence. Comparing ESTs to genome-DNA sequences was effective for dividing paralogue genes. For example, muscle actin genes in which 6 paralogues exist (Chiba et al, 2003) were distinguishable by the compareing EST-genome strategy. For more complicated gene structures, however these simple sequence comparing was inadequate (e.g. two paralogues exist in close distances, or two different genes coded overlap domain of genome). Several numbers of tandem duplicate genes are coded in Ciona genome (Dehal et al. 2002), so that we try to divide these type paralogous genes. In this report, we present a distinct number of genes and unsolved problems of EST clustering.
Hypervariable and highly divergent intron/exon organizations in the chordate Oikopleura dioica
Rolf B. Edvardsen1, Emmanuelle Lerat2, Anne Dorthea Maeland1, Mette Flåt1, Rita Tewari1, Marit F. Jensen1, Hans Lehrach3, Richard Reinhardt3, Hee-Chan Seo1 & Daniel Chourrout 1 1Sars Centre for Marine Molecular Biology, Bergen, Norway, 2Laboratoire de Biométrie et Biologie Evolutive, Université Lyon1, France 3Max-Planck Institute for Molecular Genetics, Berlin, Germany.
We have isolated and characterized the organization of thirty-four genes (12 ribosomal protein genes, 2 EF1-a genes, 8 Hox genes and 12 alpha-tubulin genes) of Oikopleura dioica. Their intron positions have been compared with those of the same genes from various invertebrates and vertebrates, including four species with entirely sequenced genomes. Oikopleura genes, like Caenorhabditis genes, have introns at a large number of non-conserved positions, which must originate from late insertions or intron sliding of ancient insertions. In these two species, distinct genes of the alpha-tubulin family have markedly different intron-exon organizations because most non-conserved positions are located in a single gene. The hypervariability and divergence of intron positions in Oikopleura and Caenorhabditis may be related to the predominance of short introns, the processing of which is little dependent upon the exonic environment compared to large introns. Also, both species have an undermethylated genome, and the control of methylation-induced point mutations imposes a control on exon size, at least in vertebrate genes. That introns placed at so variable positions in Oikopleura or C. elegans may serve a specific purpose is not easy to infer from our current knowledge and hypotheses on intron functions. We propose that new introns are retained in species with very short life cycle, because illegitimate exchanges including gene conversion are strongly repressed. We also speculate that introns placed at gene-specific positions may contribute to suppress these exchanges and thereby favorize their own persistence.
Organization of spliced-leader RNA (SL RNA) genes in Ciona
Kenneth E.M. Hastings, Montreal Neurological Institute and Department of Biology, McGill University, 3801 University St., Montreal, Quebec, Canada H3A 2B4 (firstname.lastname@example.org)
mRNA 5'-leader trans-splicing (SL trans-splicing), originally discovered in protostome invertebrates and protists, was recently found to occur in the deuterostomes, in ascidians. In Ciona intestinalis a 16-nucleotide leader sequence, representing the 5'-segment of a 46-nucleotide spliced leader donor RNA (SL RNA), is transferred to a splice acceptor site upstream of the protein-coding sequences of >7 different mRNA species. I have characterized the genes encoding SL RNAs of Ciona intestinalis and Ciona savignyi (JGI and Whitehead databases). Both species contain multiple SL RNA-encoding DNA sequences, including tandemly repeated head-to-tail arrays. In Ciona intestinalis the SL RNA gene repeat is 264 bp - the shortest yet reported. In Ciona savignyi the SL RNA gene repeat is 575 bp and shows little similarity to the Ciona intestinalis SL RNA repeat outside of the SL RNA sequence. However, within the SL RNA sequence 35 of the 46 nucleotides are conserved, including all the elements thought to be functionally important, i.e., the 16-nt leader sequence itself, the splice donor site, and a putative binding site for Sm proteins within the “intron”moiety. Thus although there has clearly been dynamic evolution of the repeat within the genus Ciona, sequences associated with SL RNA function have been strongly conserved. I will discuss evolutionary implications of the SL RNA gene organization in the Cionidae, evidence for trans-splicing in more distantly-related ascidians, and the concept that ascidian SL trans-splicing may represent an ancestral chordate character that has apparently been lost in the vertebrate lineage. I will also discuss implications of SL trans-splicing for experimental studies involving expression of vertebrate genes in ascidian cells and vice versa.
Trans-splicing in Oikopleura dioica
P.Ganot, T.Kallosoe, D.Chourrout
& E.M.Thompson, Sars centre
SL-RNA trans-splicing is a bimolecular reaction where the donor splice site (5’SS) of the SL-RNA single exon is spliced in trans onto specific acceptor splice sites (3’SS) of a subset of pre-mRNAs. SL-RNA trans-splicing is observed in the kinetoplastid, cnidarians and nematode subphyla and in ascidians. Trans-splicing requires part of the cis-splicing machinery and the SL-RNA as part of the spliceosome. Remarkably, in trypanosome and nematodes, SL-RNA trans-splicing allows processing of polycistronic pre-mRNA into monocistronic mRNAs. There are two SL-RNAs in C.elegans: SL1 trans-splices the first cistron whereas SL2 is specific of downstream cistrons on the operon. The intergenic (outron) distance is 500bp and 100bp in trypanosomes and nematodes respectively; both utilise cognate polyadenylation signals for the 3’end processing of individual cistrons. 5’end investigation of mRNAs in Oikopleura dioica reveals RNA trans-splicing for a subset of genes (15-25%). The likely single SL-RNA is 93nt long and adopts a secondary structure similar to the non-chordate SL-RNAs. The exon-intron boundary is defined by a consensus 5’SS, the intron being associated with the spliceosomal SM proteins. Like in other species, the SL-RNA is encoded nearby the 5S rRNA gene. Genomic analysis of trans-spliced genes reveals unusual compact gene organisation. Indeed, we have characterised the transcription of one operon containing at least 5 genes. The subsequently processed mRNAs were trans-spliced and polyadenylated. Also, trans-splicing can occur before or after cis-splicing. Outrons have unexpectedly small size, up to 10bp shorter than the characteristic 35-40bp short introns of Oikopleura. Outrons harbour strong 3’SS but lack 5’SS. Importantly, the first 4 cistrons are devoid of cognate polyadenylation signals, contrary to the last cistron of the operon. A model where the 3’end processing machinery is brought in trans with the SL-RNA is very likely.
Session 3: Functional analysis of the ascidian genomes: Tools and approches
Ciona heart development: a role for the pole plasm?
Brad Davidson and Michael Levine,
We are investigating heart development in Ciona intestinalis. Although the Ciona heart differentiates after metamorphosis, gene expression analysis indicates that heart lineage specification is initiated during embryogenesis. By the tailbud stage, we have characterized a distinctive set of overlapping expression domains within this lineage, including the specific expression of Ci-Nkx (the single Ciona Nkx 2.5 ortholog). We also elucidated the initial fusion of the bilateral heart field, which occurs just prior to larval hatching. A review of published gene expression data has led us to generate two hypothesis. First , that specification of the heart field may be initiated as early as the 110 cell stage. Secondly, maternally loaded germ cell determinants may influence early heart specification. Currently, we are defining an expanding set of enhancers which regulate Ciona heart gene expression. By identifying clusters of conserved motifs within these enhancers, we hope to delineate additional factors involved in Ciona heart specification, while simultaneously searching the Ciona genome for novel heart genes which are also regulated by these motifs.
Subcellular localization and functional analyses of notochord gene products in the ascidian embryos of Ciona intestinalis
Hiroki Takahashi1,2, Kohji Hotta3.
Shigehiro Yamada1, Noriyuki Satoh4,2,
and Naoto Ueno1 1Dept. of developmental Biology, National Inst. for Basic Biology
2CREST, JST 3National Institute of Genetics 4Department
The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tubes and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the notochord itself. In the urochordate ascidian Ciona intestinalis, Brchyury (Ci-Bra) plays a key role in notochord differentiation. In previous study, we isolated cDNA clones for nearly 40 potential target genes that are expressed in notochord cells. Here we characterized of subcellular-localizations of the 20 GFP fusion gene products in the notochord cell. These products observed after electroporating the embryos at one-cell stage with GFP fusion gene containing notochord specific promoter. They showed various subcellular localizations in the notochord cells of the tadpole tails. In addition to investigate the actual functions of the genes during the notochord formation, functional analyses were performed by injecting of antisence morphollino oligos.
Computational and functional analyses of coregulated muscle genes in Ciona savignyi
DS Johnson & A Sidow. Department of Genetics,
We are using computational and experimental approaches to analyze regulation of suites of coregulated genes in the solitary ascidian, Ciona savignyi. The assumption is that noncoding sequences adjacent to coregulated genes share elements that drive their common expression. Comparative analysis of these adjacent sequences should reveal the essential elements necessary for expression. One current effort focuses on muscle terminal differentiation genes. We use a reporter assay approach (Corbo et al., 1997) to test sequences upstream of the transcriptional start site for activity. Construct design is guided by analysis of M-LAGAN (Brudno et al., 2003) alignments between C. intestinalis and the C. savignyi. This technique has been used to generate functional minimal promoter constructs for a variety of genes strongly expressed in the tadpole muscle. Another effort in our lab explores regulation of muscle actins in the C. savignyi embryo. Comparative sequence analyses, coupled with functional analyses, may provide insight into the regulation of both the actin family and the regulation of muscle genes generally. Meanwhile, we are developing other computational methods to discover functionally important noncoding sequences. Eventually, we hope to expand this type of analysis to other tissues (and other closely coregulated gene families) in the ascidian embryo. Brudno M, Do CB, Cooper GM, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Research 13(4): 721-31. Corbo, JC, M Levine, & RW Zeller. (1997). Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124: 589-602.
High throughput screens for cis-regulatory DNA in Ciona intestinalis
David Keys Joint Genome Institute,
The number of cis-regulatory DNAs that have been characterized in transgenic animals remains relatively small. This is largely due to the difficulty of producing transgenic embryos in traditional model systems. Because of the relative ease of transient transformation using Ciona intestinalis, it has become practical to use this organism to perform large scale experiments to identify and characterize DNA with cis-regulatory activity. Here we describe several high throughput strategies to screening for cis-regulatory activity using randomly generated and targeted genomic domains. These strategies have been used to identify over 300 individual DNA fragments with the ability to drive tissue specific expression. The identification of potential functional domains within this data set using computational methods is discussed.
Germline transgenesis of Ciona intestinalis using a Tc1/mariner superfamily transposon Minos
Yasunori Sasakura, Satoko Awazu, Terumi Matsuoka and Nori Satoh email@example.com
Because Ciona intestinalis has a compact genome with a complete draft sequence, a large quantity of EST/cDNA information, and a short generation time, it is a suitable model for future genetics. Among several techniques of genetics, transgenic technology, especially using transposable elements, is useful. To establish this technique in Ciona, we searched for a transposon that is active in Ciona. By the excision and interplasmid transposition assay, we found that Minos, one of the Tc1/mariner superfamily transposable elements, remains high activity when expressed in Ciona. When Minos containing a reporter gene was introduced into Ciona with transposase mRNA, one third of these founder animals transmitted reporter gene to the next generation. By Southern blot and PCR analyses, we showed that Minos was integrated into genomic DNA by transposase. In addition, up to four independent insertions were obtained from one founder, suggesting the high efficiency of Minos. One of the transgenic line showed irregular expression pattern of GFP. This line has an insertion that targeted Ciona Musashi-2 orthologous gene (CiMsi-2). In situ hybridization of CiMsi-2 showed almost the same expression pattern as the GFP, suggesting that the insertion is responsible for the altered expression pattern of GFP. This is a first evidence of enhancer trap in Ciona. These results indicate that Minos will provide powerful tool for creation of transgenic animals, insertional mutagenesis, and other useful transgeneic technology that is represented by the enhancer trap technique.
Mutational analysis of ascidians: advances in positional cloning
William C. Smith, Carolyn Hendrickson, and Yuki Nakatani. MCD Biology, University of California,
Santa Barbara, CA 93106,
Several approaches for isolating developmental mutations in Ciona, including screening wild populations for pre-existing mutations, and the use of chemical mutagens, have been explored. Among the mutations isolated are three non-complementing mutations, chongmague, chobiesque, and chobi, that each disrupt notochord development at a different stage. Several other mutations profoundly disrupt the nervous system, including the mutant frimouuse (frm), in which there is a complete loss of anterior ectoderm derivatives including the sensory vesicle and palps. Finally, the tadpoles for the draemong (dra) mutation have a abnormally rounded trunks and are lacking the atrial siphon primordia (ASP). After metamorphosis, dra juveniles lack gill slits and the atrial siphons. Both the lack of the ASP and the malformed trunks appear to be the result of a disruption in ectoderm morphogenesis. We speculate that in the absence of the ASP, an inductive signal that is required for the gill slits to form in the pharyngeal endoderm is lost. A major goal of our research is to identify the mutated genes responsible for the phenotypes. For mutations with no obvious candidate, positional cloning is the alternative. Preliminary results for positional cloning of two recessive mutants, chongmague and draemong, are very promising. We have been using the bulked segregant analysis (BSA) approach to identify linked markers. The nucleotide sequence of the linked AFLP markers has allowed us to identify candidate regions of the sequenced ascidian genomes. New genetic markers within the genomic region are then developed to test for linkage in single tadpoles. In a pilot study, we have identified a 300,000 bp region containing one excellent candidate gene that shows very tight linkage to chongmague.
Natural mutations in Ciona savignyi in
Di Jiang*, Edwin M. Munro#,
and William C. Smith*. * Molecular,
Cellular, and Developmental Biology Department,
We have isolated natural recessive mutations affecting embryonic development of Ciona savignyi from the Santa Barbara harbor. Our aims were: 1) to obtain developmental mutants for embryological studies, and 2) to examine the frequency, distribution, and spectrum of natural mutations in a wild population. Several mutants have been isolated from a preliminary screen: Chobiesque affects the notochord development. As the result, the tail of mutant tadpole is half of the size of the wild type. Other tissues in the larva develop normally. Homozygous tadpoles in our laboratory can settle, undergo metamorphosis, grow to sexual maturity, and the adults are fertile. Our analyses suggest that the establishment of cell polarity, the initial stage of convergence-extension movement in ascidian notochord morphogenesis, is deficient in Chobiesque. Immaculate has pigmentation and behavioral defects. The pigmentation of the otolith and ocellus are absent or greatly reduced, although all sensory cells appear to be present. As the result, the swimming behavior of the larvae is greatly compromised. Homozygous tadpoles fail to swim against the gravity and away from the light. Immaculate tadpoles can also grow into fertile adults. A recent screen identified 24 individuals, out of 104, that carry obvious embryonic mutations at the larval stage with phenotypes ranging from early arrest, anterior morphological defects, pigmentation defects, tail morphogenesis defects, and behavior defects. We are in the process of confirming the inheritability of these mutants, and are planning a new screen to measure the total mutation load (affecting all stages of development) in our local population.
Classical genetics in Ciona intestinalis in the genome era: polymorphisms, strains, and mapping
1. Department of Zoology,
Little classical genetics in Ciona has
been studied although it is essential. To establish
genetic basis, we have approached the following issues: 1) investigation
of polymorphisms in C. intestinalis, 2) establishment of
a few breeding lines which are distinguishable, and 3) a genetic mapping
using polymorphisms between populations. We have confirmed
that each population is distinguishable among several populations collected
from different sites worldwide using RAPD and microsatellite markers that
could be non-coding regions, whereas there was little polymorphism in several
coding regions. A population from South British, especially,
was unique in morphological features, genetic fingerprints, and length polymorphisms
in some genes. Having focused on these, we crossed
the British individuals (Br) with the Neapolitan individuals (Fu; Fusaro),
and cultured in the breeding facility of SZN. We observed
in the F1 progeny that the British genome decreased fitness in the facility. The F1 progeny inherited some phenotypes concerned with
red pigments in siphon edge, top of endostyle, and end of gonoduct according
to dominant manner. We keep other breeding lines derived
Functional diversification of duplicates during evolution as implied via a comparative insitu screen
Georgia Panopoulou, Vesna Radosavljevic, Heike Kröger, Steffen Hennig, Detlef Groth and Hans Lehrach. Evolution and Development Group, Dept. Prof. H.Lehrach, Max-Planck Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany.
The high rate of gene duplication along evolution is accompanied by a high rate of gene loss in a relatively short period after gene duplication. Duplicated genes often undergo a period of relaxed selection that enables for potential functional diversification. The dependence of gene preservation on functional diversification as well as the mechanism that defines the function of the new duplicates are unknown. We have started to systematically catalogue via a comparative whole mount insitu hybridisation screen of amphioxus (Branchiostoma floridae) and zebrafish orthologs, the change in a gene's role (as far as this can be implied through its expression domain) after it is duplicated. A zebrafish and an amphioxus set of 49,888 and 14,000 consensus ESTs respectively that have been classified into orthologous groups are used as the basis for the selection of genes to be studied. 650 groups that include orthologs from both organisms are defined, 293 of these groups include a single amphioxus and multiple zebrafish orthologs. Details on the system that we use for defining gene orthology will be presented. We intend to include Ciona intestinalis genes in the above screen as this will enable the comparison of orthologs on the functional level while it will also allow to determine the contribution of regulatory elements in the functional diversification of duplicates. 597 Ciona intestinalis predicted proteins from the recently completely sequenced genome have orthologs in both the amphioxus and zebrafish gene sets that we are using. A few examples of insitu hybridisations of Ciona orthologs included in the above screen will be shown.
Modelisation and representation of expression data in ascidians: the virtual ascidian embryo project
Patrick Lemaire LGPD, Campus de Luminy case 907 F-13288
Marseille Cedex 09 France
With more and more data available for Ascidian systems, the need of an informatics system able to correlate different kinds of information with an efficient knowledge representation system becomes essential. The ANISEED (Ascidian Network for In Situ Expression and Embryological Data) system has been designed to offer an advanced database able to relate expression data with other knowledge fields such as lineage, induction, cell fates, genomic information, or blastomere position and morphology in the developing embryo. Each in situ picture entered in the database is described in term of stained molecules, experimental conditions, and expression profiles using a hierarchical anatomical dictionary. The dictionary is then used to clusterise genes sharing similar expression profiles. These clusters constitute the preliminary step to identify conserved regulatory sequences, motifs and regulons in the vicinity of co-regulated genes. A web portal is available to access these data, and provides a way to compare different species and the effect of varying experimental conditions (deregulated molecules, removed anatomy parts…). A schematic view allows to graphically display gene expression through space and time, in the whole developing embryo, or in a specific lineage or tissue precursor. We also provide 3D virtual embryos, reconstructed from confocal microscopy optical sections. This tool allows to display gene expression territories, lineages and precursors in an interactive manner. A clipping tool allows to see the embryo internal organisation, while biometry tools allow to compute surfaces, volumes and surfaces of contact between neighbouring cells. These tools allow for instance to search for asymmetric cell divisions, to correlate the extent of blastomere contacts with the reception of inductive signals, or to study mechanical constraints driving gastrulation.
An Ets transcription factor, HrEts, is target of FGF signaling and involved in induction of notochord, mesenchyme, and brain in ascidian embryos
Hiroki Nishida and Takahito Miya Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta,
In ascidian embryos, a fibroblast growth factor (FGF) signal induces notochord, mesenchyme, and brain formation. Although a conserved Ras/MAPK pathway is known to be involved in this signaling, the target transcription factor of this signaling cascade has remained unknown. We have isolated HrEts, an ascidian homolog of vertebrate Ets1 and Ets2, to elucidate the transcription factor involved in the FGF signaling pathway in embryos of the ascidian Halocynthia roretzi. Maternal mRNA of HrEts was detected throughout the entire egg cytoplasm and early embryos. Its zygotic expression started in several tissues, including the notochord and neural plate. Overexpression of HrEts mRNA did not affect the general organization of the tadpoles, but resulted in formation of excess sensory pigment cells. In contrast, suppression of HrEts function by morpholino antisense oligonucleotide resulted in severe abnormalities, similar to those of embryos in which the FGF signaling pathway was inhibited. Notochord-specific Brachyury expression at cleavage stage and notochord differentiation at the tailbud stage were abrogated. Formation of mesenchyme cells was also suppressed, and the mesenchyme precursors assumed muscle fate. In addition, expression of Otx in brain-lineage blastomeres was specifically suppressed. These results suggest that an Ets transcription factor, HrEts, is involved in signal transduction of FGF commonly in notochord, mesenchyme, and brain induction in ascidian embryos.
Polarisation of the egg cortex, cortical ER and mRNAs during early development in 3 ascidians (Phallusia, Ciona, Halocynthia)
Francois Prodon1 (firstname.lastname@example.org),
Christian Sardet1, Philippe Dru1, Kaichiro Sawada2,
Hiroki Nishida2 1BioMarCell, UMR7009, CNRS/UPMC,
Station Zoologique, Villefranche-sur-mer, 06230,
In ascidians as in Drosophila and Xenopus mRNA determinants for embryonic polarity and differenciation are located in the egg cortex. What exactly the cortex is remains a matter of debate ( Sardet et al.,2002, Dev. Biol 241, 1-23). The submembranous region of the ascidian egg is a basket made of 2 juxtaposed domains: a mitochondria-rich sub-cortical domain (myoplasm) and an endoplasmic reticulum-rich cortical domain (cER). Myoplasm and cER domains are polarized in the egg and relocate after fertilization. A class of cortical maternal mRNAs (postplasmic RNAs) including PEMs and the muscle determinant macho-1 are initially polarized along the A-V axis and relocate like the cER domain after fertilization. Since the network of cER is retained in isolated cortices prepared from eggs and embryos of Phallusia, Ciona and Halocynthia we tested whether some postplasmic RNAs were associated with cER in these isolated cortices. Using high resolution in situ hybridization under conditions which preserve membranes we show that: 1) macho-1 and HrPEM are retained with the isolated cortex and bind to the polarized cortical rough ER network, 2) macho1 and HrPEM mRNAs concentrate in the vegetal cortex with the cER after fertilization and move posteriorly with it after meiosis completion, 3) the maternal mRNAs accumulate as patches in the CAB at the 8 cell stage, together with the cER network. We propose that the polarized rough cER network in the egg is a major precursor of the CAB and plays an important role in the localization and segregation of maternal mRNAs including determinants such as macho-1 mRNA.
The Cititf1 endoderm enhancer.
Albertina Fanelli, Gabriella Lania, Antonietta Spagnuolo, and Roberto Di Lauro Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy
Cititf1 is an early and specific marker of endoderm development in Ciona intestinalis. We examined Cititf1 transcriptional regulation focusing in particular on its endodermal restricted expression. Through the analysis of Ciona embryos, electroporated with different portions of Cititf1 5'-flanking region fused to lacZ, we characterized a minimal 300 bp cis-regulatory sequence able to closely reproduce the spatial and temporal expression pattern of the endogenous gene. This enhancer contains at least three distinct regulatory regions, two of which are responsible for activation of transcription in the endoderm and in the mesenchyme, respectively, while the third is a negative control element that represses mesenchyme transcription. We have further defined the sequences responsible for transcriptional activation in the endoderm by clustered point mutations and DNA-binding assays.
All vertebrates develop asymmetric viscera, and this is under the control of a conserved genetic pathway centred on the TGFbeta family member nodal and the homeobox gene Pitx2. Upstream of these factors, however, there is less indication of conservation, and evidence for similarity of mechanisms in different vertebrate groups is poor. One potential unifying theory involves the asymmetric localisation of ions, generating a gradient that is read to produce asymmetric gene expression. Ascidians are also asymmetric, and both nodal and Pitx are asymmetrically expressed in embryonic epidermis. I have used the asymmetry of CiPitx as a read-out in a screen of ion channel and ion pump activity using pharmacological antagonists. My results show BaCl has a predictable effect of asymmetry. BaCl is a K+ channel antagonist. Correspondingly, I have examined the expression of H+/K+ ATPase subunit genes in Ciona. One gene is expressed in dorsal and ventral midline epidermal cells at the tailbud stage, a pattern compatible with a role in the regulation of asymmetry. These results provide preliminary evidence for a role for active transport of K+ ions in the regulation of Ciona asymmetry.
Formation of the anterior nervous system is impaired in the Ciona intestinalis frimousse mutant
Deschet and William C. Smith, MCDB, University of California, Santa Barbara,
CA 93106 USA
Intrinsic features of ascidians, such as invariant lineage, cellular simplicity and capacity for self-fertilization, make them ideal model organisms for the genetic analysis of neural development. In the course of a screen conducted in Ciona intestinalis, we isolated a spontaneous and lethal recessive zygotic mutation, frimousse (frm). This mutation specifically affects the morphogenesis of the anterior nervous system. The frm larva fails to form the pigment organs of the sensory vesicle, the ocellus and the otolith, and the adhesive organs (or palps). Analysis of the frm axon tracts shows a disruption of the anterior sensory nervous network including the rostral trunk epidermal neurons (RTEN). However, the projections of the motor neurons of the visceral ganglion and caudal epidermal sensory neurons are properly differentiated in the tail. The mutant phenotype first becomes evident in early-tailbud stage embryos and is characterized by a round head and a dorsal protrusion of the brain, suggesting a severe truncation and altered regulation of proliferation of the anterior region. Using anterior-specific markers, we confirmed that the precursors of the rostral nervous system, including the palps (Ci-islet), RTEN (ETR-1), pharynx primordium (Ci-Pitx, Ci-Six3) and anterior sensory vesicle (Ci-Otx, Ci-Not) were absent in the frm tailbud embryos. Interestingly, the neural tissues that are missing in frm all derive from the a-line anterior and animal blastomeres. The formation of the a-lineage descendants requires inductive interactions with vegetal cells during the cleavage stages. The frimousse phenotype could result from an early induction or specification defect in this lineage. The characterization of this mutant should provide insight into the ancestral molecular mechanisms of Chordate forebrain patterning.
Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors.
V. Bertrand1*, C. Hudson1,2, D. Caillol1, C. Popovici3, and P. Lemaire1* email@example.com
1: Laboratoire de Génétique et Physiologie
du Développement. IBDM. CNRS/INSERM/Université
de la Méditerranée/AP de Marseille. Parc Scientifique
de Luminy, Case 907. F-13288, Marseille Cedex
In chordates, formation of neural tissue from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in vertebrates. Here, using the early neural marker Otx as an entry point, we dissected the neural induction pathway in the simple Ciona intestinalis embryos. We first isolated the regulatory element driving Otx expression in the prospective neural tissue, showed that this element directly responds to FGF signalling and that FGF9/16/20 acts as an endogenous neural inducer. Binding site analysis and gene loss of function established that FGF9/16/20 induces neural tissue in the ectoderm via a synergy between two maternal response factors. Ets1/2 mediates general FGF-responsiveness, while the restricted activity of GATAa targets the neural programme to the ectoderm. Thus, our study identifies, for the first time, an endogenous FGF neural inducer and its early downstream gene cascade. It also reveals a novel function for GATA factors in FGF signalling.
Session 5.1: Neural development and chordate evolution
Development of the ascidian CNS: early neural specification and lineages
Vincent Bertrand, Danielle Caillol, Sébastien Darras, Vanessa Fox, Eric Frangulian, Clément Lamy, Patrick Lemaire, Andrea Pasini, Ute Rothbächer, Olivier Tassy, The Ciona group, LGPD/IBDM, Campus de Luminy, F-13288 Marseille Cedex 9, France Lemaire@ibdm.univ-mrs.fr
The aim of our group is to generate a precise description of the ontogeny and lineage of the larval central nervous system of the ascidian Ciona intestinalis. Because this CNS contains less than 100 neurones, it constitutes the only chordate system in which a complete description of the formation of the CNS and its connectivity can be achieved with a single-cell resolution level. We focus our attention on the formation of the anterior central nervous system, which forms from the a-line. I will first report on the projects that we have achieved in the understanding of the mechanisms that restrict the anterior neural programme to the a-line blastomeres, in the identification of the neural inducer and its transcriptional logic, and in the characterisation of novel transcriptional targets of the inducer. Downstream of the induction process, I will present our efforts to describe the precise fate and cell behaviour of the progeny of each neural plate cell by combining advanced live imaging with high-resolution gene expression studies.
The molecular function of RNA-binding proteins, musashi and Hu, in the ascidian neural tube formation
Takahito Nishikata (
A neural tube is one of the distinguishing characteristics of chordate. In order to analyze the molecular mechanism which underlying neural tube formation in the ascidian embryo, we focused on the RNA-binding protein genes, CiMsi and CiHu. CiMsi is expressed in the presumptive neural tube and mesenchyme cells, while CiHu is expressed in the presumptive mesenchyme cells. Overexpression of CiMsi caused ectopic neural differentiation. On the other hand, disruption of CiMsi function by the morpholino antisense oligo nucleotides (MO) resulted in the disordered cell-movement of the neural tube precursor cells. CiHu gene function was analyzed in the PC-12, rat pheochromocytoma cell line. It can differentiate into neurons by the NGF stimulation and the transformation with mouse HuD gene. When the full-length CiHu construct was introduced into PC-12, CiHu protein was localized in the nucleus and the cells did not differentiate into neurons. When the deletion CiHu construct, which lacked nuclear localization signal, was transformed, deletion CiHu protein was localized to the cytoplasm, and the PC-12 showed the neurite outgrowth. This result revealed the conserved neurite-inducing activity of ascidian CiHu and mouse HuD. While the protein structure responsible for the subcellular localization was different among them. This is the important clue to understanding what kind of change in the amino acid sequence caused the alteration of the developmental system and brought the evolutional change from urochordates to vertebrates. More precise understanding of the CiMsi and CiHu gene functions in the ascidian neural tube formation will provide us an insight how chordates obtained the neural tube, and how vertebrates evolved the tubular brain and dorsal hollow nerve chord.
Development of the nervous system in the larvacean urochordate Oikopleura dioica
Bassham, C. Cañestro, and J.H. Postlethwait
Key steps in the evolution of the vertebrate nervous system have been thought to involve the evolution of a three-part brain, the neural crest, and placodes from early filter-feeding chordates that lacked these features. To contribute to a deeper understanding of the evolutionary origins of these features, we have begun to investigate the development of the central and peripheral nervous system in the larvacean Urochordate Oikopleura dioica. Because larvaceans maintain a chordate body plan as adults, investigation of their developmental mechanisms can complement extensive knowledge already obtained for ascidians, which undergo a dramatic metamorphosis that substantially reorganizes the animal’s nervous system. We isolated larvacean orthologs of genes shown to be necessary for the development of the three-part vertebrate brain, neural crest, and placodes, and have investigated their expression patterns and functions in developing Oikopleura embryos. These results provide an initial comparative outline of the development of the larvacean brain, and suggest precursors for vertebrate crest and placodes.
Homologues of vertebrate placodes in Ciona intestinalis
Francoise Mazet, James Hutt,
John Millard, Josselin Milloz and Sebastian Shimeld School of AMS, University of Reading, Whiteknights,
Reading RG6 6AJ,
Ectodermal sensory placodes are often considered a vertebrate character, however recent molecular studies suggest that two ascidian embryonic structures, the primordial pharynx and the atrial invaginations, could be considered homologous to placodes. Much of the molecular evidence from protochordates can, however, be challenged as the analyses focused on genes involved in relatively late differentiation of placodes rather than on a conserved, interacting ensemble of molecules involved in early steps of placode induction. To test this further, we examined a range of C. intestinalis genes that are orthologs of markers of vertebrate placode development and differentiation, including members of the Pax, Six, Eya, Dach, Fox and Sox families. Our results show that both atria and pharynx express marker genes in agreement with their status as proposed placode homologues. They also suggest a complex coordination of their expression as well as the subdivision of the ascidian primordia into discrete units. To better understand this we examine how expression of specific genes is restricted to these cells, and the similarity of the patterns and pathways to those observed in vertebrates. We will also discuss what mechanisms might have lead to the evolution of elaborated sensory organs and ganglia in vertebrates.
Morphogenesis of the neural gland in ascidians: studying a conserved genetic network leading to divergent functions
Lionel Christiaen, Carole Deyts, Franck Bourrat , Jean-Stéphane Joly UPR 2197 DEPSN IAF CNRS Av. de la Terrasse 91 198 Gif-sur-Yvette, France
Taking advantage of the almost unique wealth of cellular and molecular data available on hypophyseal development and function in mammals, we decided to analyse this system in Ciona intestinalis. Ascidians indeed exhibit a set of organs -the so-called neural gland complex- proposed to be homologous to adeno/neurohypophysis. This set derives from the anterior stomodeal cells. Our project now focuses on the morphogenetic description of these anterior cells and their derivatives, where we have the opportunity to unravel the cis-regulatory logic of Pitx regulation, focusing on the description of the modular organisation of the promoter and signaling pathways integrated by enhancer elements. By studying cis-regulatory regions of other genes acting on the proliferation or differentiation of the precursors of the neural gland complex, which we are currently isolating by various approaches, we hope to unravel conserved regulatory logics necessary for gene activation in this domain.
Characterisation of Ci-GnRHLP gene which codes GnRH-like peptide in ascidian
Katsumi Takamura, Fukuyama University,
In ascidian, neurosecration system for neurotransmitter and neurohormone has been not studied enough. We have cloned and analysed several genes coding neurotransmitter synthesis enzymes and neurohormone precursors using PCR methods and EST analysis in Ciona intestinalis. In those studies, we found a precursor gene in which GnRH-like peptides were coded. GnRH is a key peptide for the control of releasing Gonadotropin and is composed of 10 amino acids conserved in any vertebrates. This gene,Ci-GnRHLP (Ciona GnRH-like peptides) is 1082 bp long and its ORF (open reading frame) codes 729 amino acids. We can find a hydrophobic region at N-terminal end, GnRH-like amino acid sequences, followed by three amino acids GRR or GKR which seem to be a protease recognition site, and GnRH-associated peptide (GAP) at C-terminal end in protein precursor coded by this ORF, as other vertebrate GnRH precursor. Interestingly, this GnRH precursor in Ciona contains three GnRH-like amino acid sequences, while those in vertebrate contain only one GnRH peptide sequence. RT-PCR analysis showed that this RNA product existed preferentially in adult neural complex. Additionally, two cDNAs, which might play a role in GnRH peptide maturation, were isolated from neural complex cDNA library. One coded a glutaminyl-peptide cyclotransferase and another coded a peptidyl-glycine alpha-amidating monooxygenase. Although expression of these genes in adult neural complex is only indirect evidence, matured and functional GnRH peptides may be produced from Ci-GnRHLP protein by these enzymes. To confirm this possibility, we prepared antiserum against synthetic Ci-GnRH peptide. Preliminary experiment with this antiserum showed that some immunostained cells were located in papillae of tadpole larvae and in adult neural complex. Now we attempt to analyse maturation process and function of Ci-GnRH peptide by immunoblott analysis.
Comparative analysis of synaptotagmin promoter between two ascidian species
Jun Matsumoto1, You Katsuyama2, and Yasushi Okamura3 1Department of Biological Sciences Tokyo Institute of Tech-nology Nagatsuta, Midori-ku Yokohama 226-8501, 2Department of Neuroscience Kobe University Graduate School of Medicine 3Department of Developmental Neurophysiology Center for Integrative Bioscience Okazaki National Research Institutes
Synaptotagmin, a key protein for neuronal exocytosis, is expressed abundantly in all types of neurons. Thus, syt may serve as a critical molecular marker for studying neuronal gene regulatory mechanisms. In the present study, regulation of syt gene is comparatively analyzed between two distally related species of ascidians, Halocynthia roretzi (Hr) and Ciona intestinalis (Ci). Hr-syt gene is expressed in all types of neurons. A 5’ fragment of Hr-syt promoter mimics expression pattern of endogenous syt gene. Deletion experiments have shown that multiple regions of Hr-syt promoter regulate neuronal gene expression. The following three important regions for neuronal gene expression are defined: NRR1 (-558 to -246), NRR2 (-2900 to -2500) and NRR3 (+100 to +203). NRR2 construct that consists of 200bp sequence (-2900 to –2700) repeated in tandem has most highly intensified neuron-specific gene expression. The Hr-syt regulatory regions have also driven gene expression in the Ci neuronal cells. Furthermore, a 3-kb 5’ fragment of Ci-syt promoter has shown pan-neuronal promoter activity in both Ci and Hr embryos. These results suggest that the neuronal gene regulatory mechanism that involves syt is conserved in both Hr and Ci promoters. It is possible that other critical binding sites for neuronal transcription factors may be conserved. Some candidate binding sites, which includes zinc finger (MyT1), basic Helix-Loop-Helix (E-box) and homeodomain (TAAT-core), are found at high frequency within the critical regulatory regions of Hr-syt promoter. Comparison of restricted regulatory regions across species may lead to further identification of conserved neuronal gene regulatory mechanisms.
Novel ascidian photoreceptors and the origin of vertebrate eyes
Vertebrates have evolved two types of eyes; the lateral eyes (paired eyes) and the median eyes (pineal or parietal eyes). The ascidian larva has an eye-spot (ocellus) in its brain. Understanding evolutionary relationships between the ascidian photoreceptors and the vertebrate eyes is a key to uncover the origin and evolution of the vertebrate eyes. In the ascidian Ciona intestinalis, we have characterized and examined expression patterns of homologues of genes involved in function or development of the vertebrate eyes. The results suggest that ascidians have photoreceptor systems more similar to those of vertebrates than to those of other invertebrates. The larval ocellus expresses a vertebrate-type opsin gene (Ci-opsin1) and the surrounding brain cells express visual cycle genes similar to those found in the retinal pigment epithelium of vertebrates. We recently discovered novel photoreceptor cells in the primordial pharynx and adhesive organ of the larva, in which mRNA and protein of Ci-opsin2, another vertebrate-type opsin, are localized. A number of genes related to eye function and development are also expressed in part of the primordial pharynx and atrial primordia, suggesting that adult photoreceptors develop in these regions, possibly oral and atrial siphons. Based on comparisons of the developmental origins, gene expression patterns, and functions of eyes between vertebrates and ascidians, we propose a hypothesis that the larval ocellus and the novel photoreceptors (of the oral siphon or adhesive organ?) are homologous to the vertebrate median eye and lateral eyes, respectively. The last common ancestor of urochordates and vertebrates may have possessed distinct precursors of the lateral eyes and the median eye of vertebrates. We also discuss a possible evolutionary link between the eye and hypophysis of vertebrates.
Behavioural genetics in ascidian larva ; from photoreceptor to motorneuron
Motoyuki Tsuda, Takehiko Kusakabe, Isao Kawakami, Takeo Horie, Daisuke Sakurai, Kyoko Inada and Hideo Iwamoto.
The ascidian larva has a characteristic diurnal pattern of behaviour consisting of an initial period when it swims upward followed by a period when it swims downwards. The anterior sensory vesicle of the ascidian larvae contains a single large vesicle in which lies two distinct types of pigment cells, otolith and ocellus. Laser ablation experiments clearly showed that the anterior pigment cell is used for gravity detection and is responsible for upward swimming behaviour while the posterior pigment cell contains photopigments responsible for downward swimming.The action spectrum of the photic behaviour suggested that rhodopsin in the ocellus is responsible for the photic behaviour of the larvae. Understanding the role of genes in photic behaviour is greatly enhanced by understanding how they affect the function of the neurons that underlie behaviour. Antibody against Ci-opsin 1 stained the outer segements of photroreceptor in ocellus, but the larvae developed from eggs injected with a Ci-opsin1 MO lost expression of Ci-opsin 1 as well as photoresponse in swimming behaviour. In addtion to Ci-opsin1, we studied behavioural genetics which are expected to be involved for photic behavior of ascidian larvae. Neural networks from eye to motor neuron were visualized by WGA transgene expressed under the control of photoreceptor specific promotor elements.
Realtime measurment of neural network activity controlling swimming in the larvae of the ascidian (C. intestinalis)
Brown ER, Bone Q, Okamura Y. Neurobiology Laboratory, Stazione Zoologica 'Anton Dohrn', Naples, Italy; MBA Laboratory, Plymouth, UK, Okazaki National Institutes, Japan.
After hatching, the larvae of sessile tunicates swim before undergoing metamorphosis. We have begun an analysis of the 'input-output' properties of the neural net controlling swimming behaviour ín Ciona intestinalis by making electrical recordings from the muscle fibres of the tail, from the outputs of the nerve cord and from 'inputs' from the isolated tail. The objective of this study is to understand how the nervous system of the ascidian larva controls swimming behaviour and settlement. Our recordings show that 'random or spontaneous' swimming is mediated by a pathway (p1) that is sensitive to blockers of ionotropic glutamate receptors and has a firing rate of around 20 Hz. A second pathway (p2), is activated by step-down light responses and becomes apparent 20-24 hours post fertilization (pf). p2 is insensitive to ionotropic glutamate receptor blockers and has an output period that is extended by GABA receptor antagonists. Activation of p2 apparently cross inhibits p1 while no evidence was found for cross inhibition by p1 over p2. We conclude that in early (< 20 hs pf) larvae, a glutamate sensitive p1 pathway dominates behaviour while in > 20 hrs pf larvae a light sensitive pathway that cross inhibits p1 when activated allowing ´random swimming´ between light stimuli. Thus at least two pathways control swimming behaviour in Ciona larve.
Session 6.1: Oogenesis, fertilization and early development
Meroistic oogenesis in Oikopleura dioica
P.Ganot & E.M.Thompson, Sars
A unique feature of insect oogenesis is that nurse cells
and oocytes originate from the same precursor. We find that a similar phenomenon
appears to occur during oogenesis in the dioecious urochordate Oikopleura dioica. A key originality in Oikopleura
is the intimate association of meiotic chromatin and nurse chromatin during
oocyte maturation. After a proliferation period, progenitor nuclei differentiate
into the meiotic and the polyploid nurse chromatin compartments. These two
chromatin regions are arranged by pairs in a syncitium. Eventually, a subset
of the meiotic chromatin will cellularise to form the mature oocyte blocked
Sperm-triggered calcium oscillations in ascidians
*Levasseur, M. and **McDougall, A. **Biologie
Cellulaire, Station Marine, Villefranche sur
Fertilisation of ascidian and mammalian eggs results in the generation of a series of transient rises in cytosolic calcium - termed calcium oscillations. These oscillations play an indispensable role in ensuring meiotic exit and subsequent entry into embryogenesis in both ascidians and mammals. It is not yet clear how these oscillations are regulated such that they commence shortly after fertilisation and only cease when meiotic exit is achieved; in both ascidians and mammalian zygotes the calcium oscillations are prolonged when CDK1 activity is kept elevated with Delta 90 cyclin B1 or full-length cyclin B1. In the ascidian, where eggs are arrested at metaphase of meiosis I, the oscillations are generated via an IP3-dependent mechanism. We have measured responsiveness to IP3-mediated calcium release following resumption of the meiotic cell cycle using caged IP3, and have also examined whether responsiveness changes when the activity of the major cell cycle kinase, CDK1, is either inhibited or maintained. We found that responsiveness is unaltered throughout meiotic maturation but fell very rapidly (t1/2=2.9 min +/- 1.2) following extrusion of the 2nd polar body, i.e. after the point in the ascidian meiotic cell cycle when calcium oscillations have already ceased. We also found that inhibition of CDK1 in the unfertilised egg has no effect on IP3 responsiveness, but when CDK1 activity was maintained beyond the time of 2nd polar body extrusion, when it has normally declined, responsiveness was also maintained. These results show that at least two mechanisms can control the response to IP3. One mechanism involves CDK1 activity while the second mechanism is regulated by the meiotic cell cycle and is independent of CDK1.
Polarities of ascidian eggs and embryos compared with those of Drosophila, C. elegans, sea urchins, Xenopus and mouse
Christian Sardet, François Prodon, Gerard Pruliere, Solenn Patalano, Janet Chenevert. BioMarCell, UMR 7009,CNRS/UPMC, Station Zoologique, Villefranche sur Mer, 06230, France firstname.lastname@example.org
Ascidian eggs like those of sea urchin, Xenopus and mouse, clearly display an Animal-Vegetal (A-V) polarity acquired during oogenesis and maturation. The Dorso-Ventral (D-V) and Antero-Posterior (A-P) polarities of the embryos of ascidians, Xenopus, AND C elegans are set up after fertilization, as a result of cell cycle driven cytoskeletal reorganizations triggered by fertilization. A-P polarity in C elegans and in ascidians, and D-V polarity in Xenopus become fixed when the meiotic cell cycle ends as sperm asters and their growing astral microtubules interact with the cortex. These interactions cause characteristic translocations of cytoplasmic and cortical domains which localize determinants, causing them to be partitionned into specific blastomeres following asymmetric cleavages. Major players in these processes are the PAR polarity proteins which are polarized before (Drosophila, Xenopus) or soon after fertilization and/or at the time of first unequal cleavages (C elegans, ascidians). Germ plasms, key indicators of vegetal or posterior polarity in Drosophila, Xenopus and C elegans are also localized in ascidian eggs and embryos. In Drosophila, Xenopus and ascidians molecular determinants of polarity and differentiation have been identified as maternal mRNAs localized in the egg cortex. Ascidian maternal determinants (including mRNAs) are distributed in the cortex of oocytes along an A-V gradient as in Xenopus. In both organisms specific 3’UTR sequences and ER networks seem to play a major role in the localization of vegetal cortical mRNAs (macho-1, Veg1...). We will discuss the significance of these cellular and molecular similarities and differences and also examine the importance of early cleavage patterns in the establishment of embryonic polarities.
Involvement of IkBa/NF-kB pathway in regulation of ascidian extra-embryonic apoptosis
B.Maury1, JP.Chambon1, M.Weill2, P.Fort3, J.Soulé4, A.Berthomieu2, L.Roger1, M.Cantou5, P.Mangeat1, S.Baghdiguian1,2
1UMR-CNRS-5539 Montpellier, 2UMR-CNRS-5554 Montpellier, 3UPR-CNRS-1086 Montpellier, 4Bergen SARS Centre, 5Station Biologique Sète
In Ciona, extra-embryonic cells elimination during hatching seems to be under the control of a caspase-independent apoptosis. In an attempt to understand the mechanisms underlying this intriguing cell death, we used TUNEL and immuno-fluorescence labelling on embryos at 5, 12 and 18 hpf. Among the different apoptosis controlling pathways, we particularly focused on the IkBa/NF-kB pathway, because of its well-recognized role in the control of caspase-independent apoptosis. In silico protochordate genome analysis leads conclusively to the presence of two distinct Rel/NF-kB genes (one encodes a potential p105 precursor and the other encodes two proteins (Ci-p65, Ci-p30) generated from differential splicing). In addition, two distinct IkB genes encode homologues of mammalian IkBa/e. Ciona p105, p30 and IkBa/e present a high level of similarity with vertebrate homologues all along their sequences, while p65 is conserved with the human p65 only in its N-terminal domain. p105 and p65 are predicted to be nuclear proteins, while p30 is cytoplasmic. Using RT-PCR, direct sequencing and Western blotting, we showed that Ci-p65 and Ci-p30 are actually expressed at mRNA and protein levels in Ciona fertilized eggs. By immuno-fluorescence labelling, Ci-IkBa or Ci-IkBa/Ci-p65 were found to accumulate in apoptotic nuclei whereas the nuclear presence of free Ci-p65 is detected only in non-apoptotic cells. The same localisation pattern was also observed in Ascidiella aspersa and Phallusia mammillata. Interestingly, Ci-p65 mRNA disappear after 4 hpf and Ci-p65 proteins during the period time of massive apoptosis in extra-embryonic cells (between 12 and 18 hpf). Taken together, these results argue for the functional existence of a highly conserved IkBa/NF-kB survival pathway that controls apoptosis from protochordates to mammals.
Programmed cell death and MAPK signalling in brain formation and evolution of urochordates
P. Sordino1, R.Tarallo1, E.Brown2, R. De Santis3, Departments of 1) Biochemistry and Molecular Biology, 2) Neurobiology, and 3) Cell Biology, Zoological Station ‘A.Dohrn’, Villa Comunale, 80121, Naples, Italy. email@example.com
With the presence of chordate-like traits in their larval bodies, ascidian taxa are sources of physiological and genetic insights into the evolutionary history of embryonic mechanisms in Vertebrates. Here we present a detailed illustration of Programmed Cell Death (PCD) in the ascidian species Ciona intestinalis. PCD is involved in a stereotyped series of cell death modules from hatching to metamorphosis. For example, in the selective elimination of Trunk Lateral Cells, neuronal clusters/compartments and caudal cells during tail resorption. To determine whether PCD interacts with other morphogenetic programmes, it was compared with cell division profiles. Immunochemical and pharmaceutical approaches provided evidences for a combinatorial modulation played by the Ras/MEK/ERK (MAPK) signal transduction pathway in apoptosis. In particular, distribution of MAPK factors in the larval CNS appears to dictate which neural divisions will survive metamorphosis and participate in the formation of the adult brain. This issue will be addressed by injection of a plasmid containing the synaptotagmin gene promoter fused to the GFP gene (gift of Y.Okamura), in order to follow neuronal cell fates during metamorphosis. Patterns and functions of PCD in Urochordates are discussed in terms of a complex network of death and survival events, with insights into morphogenesis and evolution of developmental mechanisms.
Rapid and environmentally-induced changes in gene expression during larval development and early metamorphosis in the ascidian Herdmania curvata
BM Degnan1, R Woods12, K Roper12, L Bebell1, MJ Garson1, MF Lavin2
1University of Queensland and
2Queensland Institute of Medical Research, Brisbane,
Within fours hours of hatching, Herdmania curvata larvae become competent to respond to environmental signals that induce settlement and metamorphosis. Initiation of metamorphosis requires activation of the MAPK pathway and signalling from Hemps, a novel EGF-like protein. Inhibition of MAPK and Hemps signaling stops metamorphosis at palp retraction and tail resorption respectively. We used a gene expression profiling approach with a microarray consisting of 4,800 cDNAs that has ~21% redundancy to assess gene activity during the acquisition of competence and early metamorphosis. Molecular profiling reveals that a significant portion of the genome, ~33%, is activated or repressed during larval development and early metamorphosis. Approximately 39% of the genes that are differentially expressed during early metamorphosis are affected in larvae treated with a neutralising anti-Hemps antibody that inhibits metamorphosis. These genes are implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and gene transcription. In situ hybridization analyses of a subset of genes that are induced in competent larvae reveal dramatic changes in localised expression occurring between larval hatching and competence. On coral reefs H. curvata larvae may come in contact with a wide range of allelochemicals produced by sessile competitors. Some chemicals act as inducers, others inhibitors. Using this cDNA microarray we assessed the effect of two poriferan allochemicals - an alkaloid and aminoalcohol - that inhibit H. curvata at the same stage of metamorphosis - 4 hours post-induction. We find that each allelochemical has a markedly different impact on gene expression, although they both rapidly repress the expression of genes implicated in innate immunity.
Genomics of an ascidian histocompatibility locus
Anthony W. De Tomaso & Irving L. Weissman Dept. of Pathol., Stanford Univ. Sch. of Medicine, Stanford CA, & Hopkins Marine Sta., Pacific Grove, CA. firstname.lastname@example.org
The colonial ascidian, Botryllus schlosseri, undergoes a naturally occurring transplantation reaction controlled by a single, highly polymorphic locus, analogous to the vertebrate MHC. When two colonies of B. schlosseri come into close contact, terminal projections of the colony vasculature, called ampullae, reach out from each individual and contact each other. Two outcomes can result from this interaction: either the ampullae will fuse together and form a single chimeric colony with a common vasculature, or the two colonies will reject each other in an active, blood-based inflammatory reaction during which the interacting ampullae are destroyed and the two colonies no longer interact. This histocompatibility reaction is controlled by a single, highly polymorphic mendelian locus called the Fu/HC. We are using map-based cloning strategy to isolate this locus. Currently, the Fu/HC is genetically resolved to < 1 Cm. The physical map now consists of a minimum tiling path of 850 Kb with three gaps remaining to be filled. 400 Kb of this region has been sequenced, and several genes identified. Current results in this project will be discussed.
Billie J. Swalla Biology Dept. and Friday Harbor Laboratories, Univ. of Washington, Seattle, WA 98195 email@example.com
Ascidian metamorphosis involves the remodelling of the ascidian tadpole larval body plan into a sessile adult. Metamorphosis can be divided into three phases: Larval competence, settlement, and adult body plan differentiation. We performed a series of three suppressive subtractive hybridisations between different stages of metamorphosis in the ascidian Boltenia villosa and were surprised to find the activation of innate immune system during the period of larval competence. Innate immunity confers a rapid response to pathogen-specific molecules and/or compromised self-tissues but also appears to be necessary for re-structuring of larval tissues during metamorphosis. A lectin mediated complement activation of an innate immune-related inflammatory response is necessary for two separate trans-epidermal blood cell migrations which occur during the swimming larval period and then immediately upon settlement. We characterized these trans-epidermal migrations by time-lapse photography and show that inhibiting the lectin mediated complement response also inhibits cell migration and remodelling into the adult body plan at metamophosis. We are currently investigating the complex interaction of the signals for larval competency and settlement and how these signals coordinate apoptosis in the tail with the activation of the innate immune system in the head. These results suggest a developmental role for innate immunity during metamorphosis and have interesting implications for the evolution of the innate immune system in marine invertebrates.
Morula cell activation upon the recognition of bacteria, in the compound ascidian Botryllus schlosseri
L. Ballarin, N. Franchi, F.
Cima, Dipartimento di Biol., Univ. di Padova,
Expression of placode- and crest-marking genes in larvacean embryos: Did vertebrates or eochordates invent cranial placodes and neural crest?
Susan Bassham and John
Postlethwait. Univ. of Oregon Inst. Of
What can larvaceans (Oikopleura dioica) tell us about the evolutionary history of the tripartite chordate brain?
C. Cañestro, S. Bassham, and J.H. Postlethwait, Eugene, Oregon
Maternal and zygotic functions of the gene mago nashi during Ciona intestinalis embryogenesis
J. H. Levialdi Ghiron2, L. Caputi1, I.Zucchetti2, R. De Santis2, P. Sordino1
Depts. of 1) Biochemistry and Molec. Biol., and 2) Cell Biol., Zoological Station ‘A.Dohrn’, Naples, Italy.
determinants in the ascidian Phallusia
Janet Chenevert (firstname.lastname@example.org ), François Prodon, Gerard Pruliere, Christian Sardet
BioMarCell, UMR 7009,CNRS/UPMC, Station Zoologique, Villefranche sur Mer, France
Role of some neurotransmitters in settlement and metamorphosis of Phallusia mammillata
Roberta Pennati, Giuliana Zega, Silvia Groppelli, Cristina Sotgia and Fiorenza De Bernardi. Dept. Of Biology, Univ. Of Milano, Italy Fiorenza.email@example.comThe swimming larva of solitary ascidians is a non feeding, mobile stage which selects the site of metamorphosis into the filter feeding, fixed juvenile. For this, the larva swims and settles in response to various stimuli, before undergoing metamorphosis. Little attention has been paid to define the neurotransmitter machinery underlying this process and the activity of the transitory larval organs with mechanosensory and photosensory function. By histochemical reactions and by immunofluorescence followed by confocal laser microscopy, we showed that serotonin is present in the photoreceptor complex surrounding the ocellus and in the adhesive papillae (Pennati et. al, 2001). AchE and ChAT are present in some primary neurones of adhesive papillae (Groppelli et al., 2001). We designed settlement and metamorphosis tests in which the swimming larvae, 2 hours post hatching, were put in presence of agonists and antagonists of specific rceptors of serotonin, Ach and dopamine. The results showed that dopamine prevents the onset of metamorphosis while serotonin seems to promote it. So it is possible to assume that there is a critical ratio between the levels of the two substances that triggers metamorphosis. It is interesting to note that, by immunolocalization, even dopamine is related to a larval sensory organ, the otolith. Thus it is intriguing to speculate that the coordinated actions of the two neurotransmitters, strictly related to the two sensory organs, regulate the length of larva stage in response to environmental stimuli (gravity, light). Probably the neurotransmitters exert their roles in modulating the exploratory period, settlement and metamorphosis, by acting through different receptors at different larval stages.
Morphogenesis and cellular differentiation of the neural gland complex in Ciona intestinalis
Carole Deyts1, Lionel Christiaen1, Danielle Caillol 2,3, Patrick Lemaire 2, Noruyi Satoh3, Franck Bourrat 1, Jean-Stéphane Joly 1 1 : UPR 2197 DEPSN IAF CNRS Av. de la Terrasse 91 198 Gif-sur-Yvette, France
2 : LGPD, IBDM, Univ. Marseille Luminy, 13 288 Marseille Cedex 09, France, 3 : Deceased
4 : Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan
Vasa positive germ plasm in ascidian eggs and embryos
Chakib Djediat, Philippe Dru, François Prodon, Christian Sardet(Sardet@obs-vlfr.fr) BioMarCell, UMR 7009 CNRS / UPMC, Station Zoologique, Villefranche sur Mer, 06230, France.
Isolation of an ascidian homologue of the vertebrate homeobox gene Rx
Salvatore D’Aniello*, Alessandra Memoli†, Marcella Corrado†, Monia
Teresa Russo†, Francesco Aniello‡, Laura Fucci‡,
Euan Brown* and Margherita Branno†
* Lab. of Neurobiol. and Comp. Physiology, Stazione Zoologica "Anton Dohrn" , Villa Comunale, 80121 Naples; †Lab. of Biochem. and Molec. Biol., Stazione Zoologica "Anton Dohrn" ,
‡Dept. of Genetics, General & Molec. Biol., Univ. di Napoli “Federico II", Naples
Transcript mapping and genome annotation of ascidian mtDNA using EST data
Carmela Gissi and Graziano Pesole Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Milano, Italy. e-mail: firstname.lastname@example.org email@example.com
Mapping Ciona savignyi
Matthew Hill (1), Di
Jiang (3), William Smith (3), Arend Sidow (1) In collaboration with: Shawn Hoon
(2), Balamurugan Kumarasamy (2), Elia Stupka (2), 1-Stanford University,
Stanford CA, USA;
2-Temasek Life Sciences Laboratory, Singapore; 3-University of California, Santa Barbara, USA
A genome-wide survey of the
genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic
comparisons of evolutionary conserved signaling components
Kohji Hottaa, Hiroki Takahashib
, Naoto Uenob, Takashi Gojoboria a: National Institute of
Characterisation of the upstream regulatory sequences of Ci-gsx, a gene expressed in the lateral sensory vesicle precursors.
Clare Hudson1,2,*, David Ferrier3, Peter Holland3, Hitoyoshi Yasuo2, Patrick Lemaire 1
1-IBDM, LGPD, Marseille, France. 2-UMR7009 Villefranche-sur-mer, France. 3-University of Oxford, UK.
Characterization of polymorphism in Ciona intestinalis
Dario Boffelli (firstname.lastname@example.org ), Claire Collins, Chris Detter, Paul Richardson, Eddy Rubin and David Keys. Joint Genome Institute, Walnut Creek, CA, 94606 USA.
Molecular cloning and sequence analysis of an ascidian egg b-N-acetylhexosaminidase with potential role in fertilization
Ryo Koyanagi and Thomas G. Honegger email@example.com firstname.lastname@example.org
Competence of the animal blastomeres to respond to neural induction : is the Wnt pathway implicated ?
Lamy Clément and LEMAIRE Patrick. LGPD/IBDM, Campus de Luminy, Case 907. F-13288 Marseille cedex 9,
Bud-dependent suppression of parent-specific programmed cell death during cyclic blastogenesis in Botryllus schlosseri.
1R. J. Lauzon, 2K. J. Ishizuka and 3I. L. Weissman email@example.com
1Dept. of Biol. Sciences, Union College, Schenectady, NY 12308; 2Hopkins Marine Station, Pacific Grove, CA 93950; 3Dept.s of Pathol. and Developmental Biol., Stanford Univ. School of Medicine, Stanford, CA 94305.
Hox regulatory elements in the ascidia Ciona intestinalis
Annamaria Locascio*, Alfonso Natale*, Alessandro Amoroso*, Laura Fucci† and Margherita Branno*
*Laboratory of Biochemistry and Molecular
Biology, Stazione Zoologica “Anton Dohrn”, Villa Comunale, 80121 Naples, Italy firstname.lastname@example.org
†Department of Genetics, General and Molecular Biology, University of Naples “Federico II”, Via Mezzocannone 16, 80134 Naples, Italy
Macho-like genes in the larvacean Oikopleura dioica.Kazuhiro W. Makabe1), Daniel Chourrout2), Hiroki Nishida3) 1. Dept. of Zoology, Grad. School of Sci., Kyoto Univ., Kyoto 606-8502, Japan. email@example.com 2.Sars International Centre for Marine Molecular Biology, N-5008 Bergen, Norway. 3.Dept. of Biology, Grad. School of Sci., Osaka Univ., Toyonaka 560-0043, Japan
Hair cells in ascidians: in search of the ancestor of the vertebrate lateral line.
Lucia Manni, Fabio Gasparini, Stefano Tiozzo, Federico Caicci, Giovanna Zaniolo, Paolo Burighel. Dipartimento di Biologia, Università di Padova, Italy firstname.lastname@example.org
Sperm-triggered calcium oscillations in ascidians
*Levasseur, M. and **McDougall, A. **Biologie Cellulaire, Station Marine, Villefranche sur Mer 06230, France. *CaMB, The Medical School, University of Newcastle, Newcastle NE2 4HH, UK email@example.com
Ascidian arrestin (Ci-Arr), the origin of the visual and nonvisual arrestins of vertebrates1Masashi Nakagawa, 1Takeo Horie, 1Hidefumi Orii, 2Eri Jojima, 2Norihiro Yoshida, 1Takehiro Kusakabe, 2Tatsuya Haga, 1Motoyuki Tsuda 1Department of Life Science, Himeji Institute of Technology; 2Institute for Biomolecular Science, Gakushuin University firstname.lastname@example.org
A cellulose synthase gene of ascidian origin
Keisuke Nakashima1*, Lixy Yamada2, Yutaka Satou2, Jun-ichi Azuma1 & Nori Satoh2
1Graduate School of Agriculture, 2Graduate School of Science, Kyoto University, sakyo, Kyoto 606-8502, Japan email@example.com
The ascidian mutant draemong disrupts atrial siphon and gill slit formation.
Yuki Nakatani and William C. Smith. MCD Biology, University of California, Santa Barbara, CA 93106, USA firstname.lastname@example.org
A role of BMP in epidermal sensory neuron differentiation
Ohtsuka, Y.1 and Okamura, Y.2
1Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Japan email@example.com 2Center for Integrative Bioscience, Okazaki National Research Institutes, Japan
Regulation and roles of Ca influx in Halocynthia larval development
1Okamura, Y. and 2Ohtsuka, Y. firstname.lastname@example.org
for Integrative Bioscience, Okazaki National Research Institutes, Japan,
2Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Japan
Origin and determination of the caudal epidermal sensory neurones in Ciona intestinalis.
Andrea Pasini, Sébastien Darras and Patrick Lemaire.
LGPD/IBDM, Parc Scientifique de Luminy, Case 907, F-13288 Marseille, France.
PAR polarity proteins localize to the ascidian Centrosome Attracting Body
Solenn Patalano, Christian Sardet, Gerard Pruliere, François Prodon, Janet Chenevert (Chenevert@obs-vlfr.fr )
BioMarCell, UMR 7009,CNRS/UPMC, Station Zoologique, Villefranche sur Mer, 06230, France.
Ascidians as a vertebrate-like model organism for physiological studies of Rho GTPase signaling
Alexandre Philips, Centre de Recherche de Biochimie Macromoléculaire, CNRS-UPR 1086, 1919 route de Mende, 34293 Montpellier Cedex 5, France
Polarisation of the egg cortex, cortical ER and mRNAs during early development in 3 ascidians (Phallusia, Ciona, Halocynthia)
Francois Prodon1 (email@example.com), Christian Sardet1, Philippe Dru1,
Kaichiro Sawada2, Hiroki Nishida21BioMarCell, UMR7009,
CNRS/UPMC, Station Zoologique, Villefranche-sur-mer, 06230, France.
2Tokyo Institute of Technology, Yokohama 226-8501, Japan
Regulatory elements controlling Ci-msxb tissue specific expression during Ciona intestinalis embryonic development
Monia Teresa Russo*, Aldo Donizetti*, Annamaria Locascio*, Salvatore
D’Aniello‡, Francesco Aniello†, Laura Fucci†
and Margherita Branno*
*Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica “A. Dohrn”, Villa Comunale, 80121 Naples, Italy.
‡ Laboratory of Neurobiology and Comparative Physiology,, Stazione Zoologica “A. Dohrn”, Villa Comunale, 80121 Naples, Italy.
† Department of Genetics, General and Molecular Biology, University of Naples “Federico II”, Via Mezzocannone 8, 80134 Naples, Italy
Polarities of ascidian eggs and embryos compared with those of Drosophila, C. elegans, sea urchins, Xenopus and mouse
Christian Sardet (firstname.lastname@example.org ), François Prodon, Gérard Pruliere, Solen Patalano, Janet Chenevert, BioMarCell, UMR 7009,CNRS/UPMC, Station Zoologique, Villefranche sur Mer, 06230, France
Mapping of GABA-positive neurons in Oikopleura dioica.
Anne Mette Søviknes, Joel Glover and Daniel Chourrout. Sars International Centre for Marine Molecular Biology. Bergen High Technology Centre, N-5008 Bergen, Norway. email@example.com
British Ciona intestinalis revealed high genomic variation in the species.
Miho M. Suzuki and Adrian Bird
Institute of Cell and Molecular Biology, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JR, UK.
A phylogenetic survey of programmed cell death in the subphylum Urochordata.
R. Tarallo1, R. De Santis2, and P. Sordino1 Departments of 1) Biochemistry and Molecular Biology, and 2) Cell Biology, Zoological Station ‘A.Dohrn’, Villa Comunale, 80121, Naples, Italy.
Expression of Bs-Pitx during the embryogenesis and blastogenesis of a colonial ascidian, Botryllus schlosseri
Tiozzo S.^, Christiaen L.*, Deyts C*, Joly J.S.*, Manni L.^, Burighel P^. ^Dept. of Biology, Univ. di Padova, Padova, ITALY *Group MNSC, UPR2197 - Inst. de Neurosci. “A.Fessard” CNRS, Gif-sur-Yvette (Paris, FRANCE
Spatial pattern of ERK activation during embryogenesis of Ciona intestinalis
Hitoyoshi Yasuo UMR7009/CNRS, Station Zoologique, Villefranche-sur-mer, France
Highly efficient expression of optimised Green Fluorescent Protein (GFP) transgenes in ascidian embryos created with a simple electroporation device
Robert W. Zeller, Dept. of Biol., San Diego State Univ., 5500 Campanile Drive, San Diego, CA 92182-4614, USA.
Expression of CiCD94, an NK cell receptor-like gene, during Ciona development
I. Zucchetti, R. Marino*, M.R. Pinto and R. De Santis Laboratory of Cell Biology and *Service of Technologies for the Study of Gene Expression, Stazione Zoologica “A. Dohrn”, Napoli, Italy