Sars Seminars 2009/2010
Sars Seminar Room (222 A2) at 13:00

Date Speaker Title and Abstract
30.Sept Satoko Awazu
Jiang Group
Gene-breaking transposon mutagenesis in Ciona intestinalis.
Here we report the approach of a new gene tagging, identification and mutagenicity (“gene-breaking”) method for the ascidian Ciona intestinalis utilizing Minos transposon system. This modular approach consists of two distinct and separable molecular cassettes, gene-finding cassette and transcriptional termination mutagenicity cassette which are assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic region. First we tested the availability of each element within the cassettes in Ciona by electroporation of the vector variants fused with reporter GFP to see their transient expressions in the embryo. Second, to generate the mutagenesis founder animals, Minos transposon vector containing two molecular cassettes and synthesized Minos transposase mRNA were introduce into eggs by microinjection. Animals were cultured until sexual maturation and crossed with wild-type to obtain F1 generation. So far we have established 10 pseudo-positive “mutator” lines which contain “gene-breaking” cassettes insertion into the genome.
04.Nov Friederike Hoffmann
Adamska Group
Reproduction and development of the sponge Sycon ciliatum.
The calcareous sponge Sycon ciliatum is currently established as a new model system for developmental biology in our group. However, basic biology description of this species, which is an essential prerequisite for planning and interpretation of molecular work and laboratory culture, is scarce. This study therefore provides histological description of different cell types in the target species, and of its life cycle throughout the year.

Histological protocols were established, which enabled identification of different cell types and detailed description of reproduction and early development in the target species. To unravel the reproductive cycle during the year, several local populations were investigated throughout 2008 and 2009. In spite of some variations among years and sites, the following picture emerges: Sycon ciliatum is an annual, hermaphroditic species which reproduces from April to October. Development time (from fertilisation to spawning of larvae) is 2-4 weeks, while minimum generation time is 10 weeks. During May and June, the reproductive effort is high, with 100% of the population being reproductive. Water temperature and food availability might be important factors regulating both development and generation time.

These results now facilitate planning of field collection, optimisation of sponge cultivation protocols and interpretation of results of in situ hybridization experiments.
11.Nov Bo Dong
Jiang Group
Control of Cell Elongation and Lumen Formation during Notochord Tubulogenesis
Cell elongation is a simple yet fundamental process that allows cells to adopt new shape and perform certain function. During Ciona notochord tubulogenesis, dramatic individual cell elongation takes place that furthers lengthens the notochord and embryo after convergent extension. We found that a deep furrow forms at the center of basal surface of each notochord cell during this elongation. Actin and myosin co-localized at the basal furrow and formed a contractile ring. The movement of actin bundles from edges of ring to the deeper center and discontinuous myosin localization indicated actinomyosin network that could generate pulsed contraction. We demonstrated that the force generated at this site caused cell to lengthen. Accompanying the cell elongation, notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at the opposite ends. Extracellular lumens are subsequently deposited at these surfaces. Unexpectedly, we found that extracellular lumen formation was actin-dependent. We showed that actin dynamics and organization at cortical region of apical domains were essential for directional apical vesicles trafficking and docking on membrane. Furthermore, actin-dependent polarized-distributed microtubule network was important for lumen development. Interestingly, at the later stage microtubule network made a 90º rotation during adjacent lumen joining. This process was required for the correct orientation of actin-based protrusion and subsequent lumen coalescence. Based on these data, we propose a mechanism for in vivo extracellular lumen formation in ascidian notochord.
24.Nov Coen Campsteijn
Thompson Group
Regulation of cell cycles in Oikopleura Dioica.
Controlled division of a cell involves complex interplay between the components that drive the cell cycle, with a number of checkpoint criteria that must be sequentially met to ensure correct progression. Besides conventional mitotic cycling, cells can undergo meiosis (reduction in ploidy) but can also commence endoreduplication (a.k.a. endocycling, endomitosis), a process where cells execute consecutive S-phases without intervening mitoses, thus progressively increasing their ploidy. Endocycling is conserved from plants to mammals, with widespread functions in differentiated tissues, growth, development and stress response. At the same time, evidence is accumulating implicating increased cell ploidy through uncontrolled entry into endocycles as an early event during tumorigenesis. At this point, molecular insight into endocycling is rather sketchy, in part due to the lack of suitable modelsystems.

The chordate Oikopleura dioica is known to shift from mitotic cycling to near-systemic endocycling during development. Additionally, its female gonad simultaneously supports meiotic progression of oocytes and endocycling of nurse nuclei within a single continuous cytoplasm. Endocycling could ensue from alternative expression of known cell cycle regulators, but an additional interesting possibility is that O. dioica has evolved its gene complement to adapt to these processes and their transitions (e.g. specific gene variants).

In an initial approach to pinpoint genes that might be implicated in regulation of endocycling, we identified a panel known cell cycle regulatory gene variants in O. dioica (~100) and assessed their expression profile in three different experimental settings (developmental progression, developmental arrest and sex-specificity). Our results point towards central roles for components of two conserved regulatory pathways, i.e. the mitotic entry network and the G1-phase cell cycle entry network in establishing and maintaining endocycling. At the same time, our work shows that O. dioica displays an exceptional evolution in complexity of key regulators of these regulatory pathways. We are currently addressing mechanistic regulation and molecular connectivity between the factors identified.

Although our qPCR results have validated our experimental approach, qPCR is inherently biased as its output is restricted to a selected fraction of the O. dioica transcriptome. We have therefore established an O. dioica genome-wide tiling microarray. To date we have used this array to repeat the above experimental approaches and are analyzing the resulting expression data in collaboration with the group of John Manak (University of Iowa/CCG). This unbiased approach allows us to identify further cell cycle regulators of interest and focus our efforts, to perform functional clustering of genes and to explore underlying transcriptional networks (together with Gemma Danks and Boris Lenhard from CBU/BCCS). At the moment we are developing further uses of this powerful tool to gain additional insight into endocycling (and other cell cycles), general gene regulation and animal development.
02.Dec Andrea Trentani
Glover Group
Reconstructing the Oikopleura dioica CNS
The tunicates (or urochordates) (e.g. the ascidians, the thaliaceans and the appendicularians) are our closest invertebrate relatives. Appendicularians are planktonic organisms that retain a swimming tadpole shape throughout their life. Oikopleura dioica, one such appendicularian, is characterized by its simplified life habit and anatomical organization. It has a very small genome - the smallest ever found in a chordate - and a short life cycle (of ca. 5 days). Embryos and adults are transparent and consist of a small number of cells. Like vertebrates, tunicates possess a notochord and a central nervous system (CNS). In recent years, urochordates have become popular model organisms in efforts to deduce information about the common chordate ancestor. Oikopleura dioica, in particular, possesses a relatively simple CNS (120-130 neurons) which comprises a cerebral ganglion in the head, a caudal ganglion in the proximal part of the tail and a nerve cord along the tail length. The relative simplicity of this system provides a unique opportunity to investigate neurotransmitter-specific neuron types and their synaptic connections in detail. This in turn may provide valuable insight into the understanding of brain regionalization and functions in other chordates.

During this seminar, I will provide my attempts at 3D reconstruction of Oikopleura dioica's CNS with particular focus on its caudal ganglion and nerve cord. A detailed description of the number and location of neurons within these two structures in adult animals (3/4 days old) will also provided. Furthermore, a new method to visualize synaptic circuits and investigate the neuronal network architecture (known as “brainbow”) will be presented.
09.Dec Alexandra Moosmann
Thompson Group

The diverse histone complement of Oikopleura: A model to study epigenetics signatures established by histone variants and histone modifications.
The basic structural matrix of chromosomes, chromatin, involves complex interactions between DNA and proteins. Many nuclear functions are carried out on the heart of chromatin structure, the nucleosome, consisting of 147 bp DNA wrapped around an octameric unit of small disc-shaped proteins, the histones H2A, H2B, H3 and H4 which mediate the folding of DNA into chromatin. Linker histone H1 binds DNA close to the nucleosomal dyad axis and further stabilizes the chromatin fiber. Through histone posttranslational modifications (PTMs) and ATP-dependant chromatin remodeling the accessibility of nucleosomal DNA to nuclear machinery is positively and negatively modulated.

Histone variants alter the composition of individual nucleosomes and expand the PTM repertoire. With the exception of histone H4, several different variants of each histone subtype have been reported for most organisms. The Oikopleura histone complement comprises 47 genes encoding 32 different variants and shows the same degree of diversity in histone variants as the mammalian with an even larger number of male specific variants. Many studies examining histone modifications have concluded that the combination of modifications on nucleosomes may be the key to determining chromatin structure and function in a variety of organisms. There is evidence, however, that some histone modifications and their dynamics may have different interpretations and roles across divergent evolutionary groups of organisms. Performing quantitative RT-PCR and mass-spectrometry we assessed the expression profiles for all variants throughout Oikopleura development, verified expression for specific variants on protein level and identified many conserved PTMs on Oikopleura histones. Spermatogenesis in many organisms, requires incorporation of histone variants that precede subsequent histone removal and replacement by protamines. Recent findings suggest that some histone variants are retained in mature sperm marking the paternal genome.

We have further begun to study the centromeric variant CenH3. CenH3 is essential for recruitment of kinetochore components and accounts for the highly differentiated structure of centromeres that regulates chromosome segregation during cell division. Epigenetic regulation of centromere identity is not well understood since localization of CenH3 can occur independent of DNA sequence and the cell cycle timing of CenH3-nucleosome assembly differs significantly among organisms. Od CenH3 fused to eGFP localizes to the outer regions of centromeres in the monocentric metaphase-chromosomes of Oikopleura and overexpression of CenH3 leads to missegregating chromatids.. Interestingly, we find that the centromeres of prometaphase-, metaphase- and anaphase chromosomes in Oikopleura become significantly enriched in serine 28 phosphorylated H3 (H3S28P), a modification that has not been found to be present at centromeres in other animals. Conducting further CenH3-eGFP expression experiments together with cell cycle inhibitors we aim to study the function of centromeric H3S28P in mitosis and a possible link between centromere phosphorylation and CenH3 deposition.

16.Dec Nagayasu Nakanishi
Rentzsch Group
Incredible complexity of sea anemone nervous system development and how it came to be.
In this talk, I will 1) describe nervous system development in the sea anemone Nematostella vectensis, and 2) discuss its evolutionary histories.
06.Jan Simon Henriet
Chourrout Group
The genetic activity of Tor retrotransposons in the compact genome of Oikopleura.
The planctonic tunicate O. dioica has an extremely compact genome. Whereas O. dioica genome lacks most of known retrotransposons clades, we found one flourishing family of Gypsy-like elements, namely the Tor elements, with apparently intact open reading frames (ORF) for the reverse transcriptase (RT) gene. In about half of these elements, a third ORF is found which may potentially encode an envelope (Env) protein. The well diversified family of Tor elements sharply contrasts with the small number of other potentially autonomous retroelements present in the genome. Since vertebrate genomes are devoid of Gypsy elements carrying an env gene, by showing recent acquisition of Tor elements we would strongly support the hypothesis of a secondary reduction of the genome. To get better insights into the activity of Tor elements, we have studied the insertion sites and the expression of two elements Tor-3G and Tor-4H.

Our analysis shows a high degree of insertion polymorphism of Tor element in the Oikopleura genome. Taken together with the high conservation of Tor copies, this observation suggests an ongoing activity. The preferential insertion of Tor-3G into exons and its developmentally restricted expression suggest adaptations to optimize its expression. Like retroviruses, Tor-3G and Tor-4H express a long RNA carrying a sequence repeated in 5’ and 3’ ends, such RNA being a candidate template for the RT. However, Tor-3G and Tor-4H differ from known retroviruses by transcribing their env RNA from an internal promoter. Preliminary results from cell-free expression show that the Tor-3G Env could carry post-translational modifications usually found in cell surface receptors. Taken together, our results support the hypothesis that Tor elements were acquired recently by the Oikopleura genome, possibly by ways of infection. Further research will aim at understanding the function of Env in the horizontal transfer of Tor elements.
13.Jan Reidar Andreson
Comparative genomics of calcification pathways
The current rate of ocean acidification is a serious concern for many calcifying organisms as they may not be able to adapt with rapidly evolving environmental conditions. Thus it is more difficult or requires more energy for calcifying organisms to form biogenic calcium carbonate. The information on the common origins of calcification pathways and molecular mechanisms in different taxa is still sparse and fragmentary. The availability of the whole genome sequence of coccolithophore E. huxleyi and the forthcoming availability of genomic and EST resources for other calcifying organisms will allow to generate longer transcript models, identify new homologs for the increasing number of genes, more thorough phylogenomic analyzes of lateral and horizontal gene transfer, and understand the relation between newly discovered gene families and cell life cycle phases. The specific transcriptional regulation analysis of the calcification-related signaling with the computational genomics methods by identifying conserved regulatory elements and detect transcription factors is another possibility to contribute in given topic.
20.Jan Yoshimasa Sagane
Thompson Group
Cellulose and chitin based extracellular structures in larvacean Oikopleura dioica.
Extracellular matrices play important but poorly investigated roles in morphogenesis. Extracellular cellulose is central to regulation of pattern formation in plants but among metazoans, only tunicates are capable of cellulose biosynthesis. Cellulose synthase (CesA) gene products are present in filter-feeding structures of all tunicates and also regulate metamorphosis in the ascidian Ciona. Ciona CesA is proposed to have been acquired by lateral gene transfer from a prokaryote. We identified two CesA genes in the sister class, larvacean, Oikopleura dioica. Each has a mosaic structure of a glycoslyltrasferase-2 domain upstream of a glycosyl hydrolase family-6 cellulase-like domain, a signature thus far unique to tunicates. Spatial-temporal expression analysis revealed that Od-CesA1 produces long cellulose fibrils along the larval tail whereas Od-CesA2 is responsible for the cellulose scaffold of the post-metamorphic filter- feeding house. Knockdown of Od-CesA1 inhibited cellulose production in the extracellular matrix of the larval tail. Notochord cells either failed to align or were misaligned, the tail did not elongate properly, and tailbud embryos also exhibited a failure to hatch. Knockdown of Od-CesA2 did not elicit any of these phenotypes and instead caused a mild delay in prehouse formation. Phylogenetic analyses including Od-CesAs indicate a single lateral gene transfer event from a prokaryote at the base of the lineage conferred biosynthetic capacity in all tunicates. Ascidians possess one CesA gene whereas duplicated larvacean genes have evolved distinct temporal and functional specializations. Extracellular cellulose microfibrils produced by the pre-metamorphic Od-CesA1 duplicate have a role in notochord and tail morphogenesis.

On the other hand, we found an unknown extracellular chitin structure in the nerve system in O. dioica. The structure covers the entrance of ciliate funnel connecting the buccal cavity and brain. Additionally we found the chitin-based peritrophic membrane that covers the gastrointestinal epithelial tissues. The chitin-based extracellular structures in O. dioica seem to have a barrier function to protect from the entry of unnecessary substances and from bacterial infection.
27.Jan Lisbeth Olsen
Chourrout Group
An unorthodox mitochondrial genome in Oikopleura dioica?
10.Feb Sven Leininger
Adamska Group

Development, Gastrulation & Regeneration in the Calcareous Sponge Sycon ciliatum
Sponges represent one of the evolutionarily oldest metazoan phyla. Although they are often considered not to possess true tissues or organs, their embryonic development and metamorphosis from larvae into adult animals are characterized by complex cell division patterns and morphogenetic movements strikingly similar to these in Eumetazoans. In “higher” animals, these processes are governed by concerted action of transcription factors, cell adhesion molecules and signalling pathway components. Most of genes encoding these conserved proteins are present in the genome of the demosponge Amphimedon queenslandica. Moreover, many of them are expressed during its embryonic development in patterns suggesting they play similar developmental roles in sponges and Eumetazoans. To date, knowledge on developmental molecular mechanisms is still limited for the highly diverse poriferan phylum.

Especially, whether a gatrulation-like process occurs during sponge development is highly debated. While many authors argue there is no gastrulation in sponges, others speculate it happens either during embryonic development or later during metamorphosis. In order to shed light on this issue we cloned a brachyury homologue from the calcareous sponge Sycon ciliatum (SciBra). The T-box transcription factor Brachyury is known to be a conserved player in the majority of animals being specifically expressed during gastrulation. Remarkably, brachyury is absent in A. queenslandica, the only sponge where molecular developmental studies have been available so far. We analysed the spatial and temporal expression of Brachyury in the course of embryonic development and metamorphosis of S. ciliatum by in-situ hybridisation. SciBra expression was not detectable in any embryonic stage, but was strongly expressed during metamorphosis. Therefore, our results suggest that gastrulation is present in calcareous sponges when epithelial-mesenchymal transition occurs during the conversion of the larva to the juvenile organism. Future functional analyses using transgenic methods and knock-down experiments are needed to reveal the specific role of brachyury during sponge development.

Another fascinating feature of sponges is their enormous capability to regenerate lost tissue after injury or even the regeneration of their whole body from dissociated cells. Similar phenomenons are known from other basal metazoans, e.g. Hydra, where morphogenetic rearrangements and their underlying molecular processes during regeneration are described in detail. In Sycon species no cellular or molecular data regarding regeneration have been available so far. Labelling experiments using fluorescent marker dyes showed that transverse sections of Sycon can regenerate both the osculum and the basal part, preserving previous axial polarity. This suggests an intrinsic polarity of the sponge which is kept during regenerative processes. Currently, we are searching for genes encoding factors that are responsible for this polarity in S. ciliatum in order to analyse their role during regeneration and to compare their activity during embryonic development.

17.Feb Chiara Sinigaglia
Rentzsch Group
Genetic Control of Apical Sense Organ Formation in the Sea Anemone Nematostella vectensis
Apical organs are sense organs characterized by a tuft of cilia, and located at the aboral pole of marine ciliated larvae of both protostomes and deuterostomes. Since they are absent in the major model organism, such as C. elegans, Drosophila and vertebrates, apical organs are poorly understood. They are thought to be required for the detection of suitable conditions for metamorphosis and/or for directed swimming.

Apical organs are present also in some Anthozoan larvae. There are only few studies on apical organs of cnidarian planulae larvae: Chia and Koss (1979) showed that in the sea anemone Anthopleura elegantissima the apical organ is characterized by a number of monociliated cells forming an apical tuft, and resembling sensory cells, with an adiacent nerve plexus. The homology between the cnidarian and bilaterian organs has been long debated.

This project aims to understand the genetic mechanisms involved in the formation of apical organ in Nematostella vectensis, in terms of genetic regulatory network (GNR). However, it is important to consider also the genes involved in the patterning of the aboral region, that could give an insight in the homology question. To do so, we followed two approaches:
- analysis of expression patterns of known anterior- and neural marker genes (candidate gene approach);
- search for new apical organ-enriched genes, with a microarray analysis. The microarray analysis was based on a recent paper showing that fibroblast growth factor (FGF) signalling is involved in the formation of the apical organ in N. vectensis. In particular, NvFGFa1 promotes, whereas NvFGFa2 prevents its formation. The knockdown of the two FGFs has an opposite effect and provided an opportunity to identify apical organ specific or enriched genes: these genes should be enriched in NvFGFa2 morphants, but reduced in NvFGFa1 morphants.

Initial results from both approaches will be discussed.
24.Feb Gunasekaran Subramaniam
Thompson Group
Growth Regulation Of Oikopleura dioica via Endocycling
Oikopleura dioica, serves as an excellent model to understand the molecular and cellular mechanisms that govern development. Oikopleura dioica has an unusually short life cycle involving rapid growth through extensive recourse to endoreduplication/endocycling. During the earlier stages of it’s development, the cells in the organism enter into the endoreduplication cycle. Once Oikopleura dioica reaches maturity it terminates the endoreduplication cycling in the cells.

Oikopleura, during the course of it’s development grows largely by increasing the size of it’s cells. These endocycling cells are highly polyploid (34C to 1300C), having undergone repeated rounds of endoreduplication. Thus cells in Oikopleura, during development step into endocycling to meet the demands of a growing cell and become highly metabolically active which results in a larger cell size. Because cell size for a given cell type is generally proportional to the amount of nuclear DNA, endoreduplication constitutes an effective strategy of cell growth. Not much is known about the mechanisms which set in, to turn on endocycling in the development stages of Oikopleura dioica, as a means to regulate growth.

My study attempts to explore TOR signaling as one of the possible mechanisms which might be responsible for regulating endocycling and hence growth in Oikopleura dioica. The TOR signalling pathway is known to control cell growth by activating an array of anabolic processes including protein synthesis, transcription, and ribosome biogenesis, and by inhibiting catabolic processes such as bulk protein turnover (autophagy) and mRNA degradation, all in response to nutrients (Schmelzle and Hall, 2000). Since endocycling is arrested during starvation in drosophila (Britton et al 1998), we hypothesize that the nutritionally modulated TOR signaling controls the endocycling of cells by twiddling with the cell cycle regulatory machinery, to promote growth in Oikopleura dioica during the early stages of development.

To investigate the involvement of TOR signaling in regulating endoreduplication, Oikopleura was treated with Rapamycin, a fungicide known to block the TOR pathway, followed by Idu labeling. Results showed no Idu incorporation in the somatic endocycling cells of Oikopleura. This suggests that the endocycling in these cells was inhibited by the rapamycin mediated block in TOR signaling, implicating the involvement of the Rapamycin sensitive TOR pathway (raptor mediated TORC1) in regulating the endocyling in somatic cells. However Idu was incorporated in the mitotic undifferentiated intestinal cells , the mitotic nuclei of the early gonad and nurse endocyclic nuclei of late gonad suggestive of an alternate Rapamycin insensitive TOR pathway (rictor mediated TORC2) in those cells. To further elucidate the role of TOR signaling in regulating the endocycling in Oikopleura, stasis experiments were performed wherein the animals are cultured at high densities from spawn (growth arrest), followed by Idu labelling. There was a gradual reduction in Idu incorporation in the animals consistent with the shutting down of the TOR signaling in response to diminishing nutrition. However, Idu incorporation was restored upon redilution and addition of food thus reiterating the importance of TOR signaling in controlling the endocycling of cells in Oikopleura dioica. Having established the involvement of TOR signaling in regulating endocycling in Oikopleura, investigation of it’s effects on the cell cycle proteins using Q-PCR revealed that CKS1 and Cyclin Da are comparatively upregulated in the animals under stasis. Expression profiling of the TOR mRNA at different developmental stages of Oikopleura, revealed that a splice variant of TOR, OdTOR-S, is specifically upregulated during the endocycling stage, indicating it’s involvement in endocycling. Further characterization of the molecules downstream TOR, with emphasis on affectors of G1/S transition, will help increase our understanding of the TOR mediated regulation of endoreduplication to promote growth in Oikopleura dioica.
03.Mar Elsa Denker
Jiang Group
17.Mar Gemma Danks
Mining the Oikopleura genome and transcriptome.
24.Mar Lucas Leclère
Rentzsch Group
Evolution of axon guidance: Netrin signaling in the cnidarian Nematostella vectensis
Neurons use long processes (axons and dendrites) to establish connections to each other and to effector cells and the precise arrangement of these connections is essential for the formation of a functional nervous system. The pathfinding of axons is regulated by a process named axon guidance which in bilaterians is under the control of guidance molecules belonging to Netrin, Slit, Ephrin and Semaphorin pathways. Secreted molecules, as for example Netrin, have attractant or repellent effects on axons according to the type of transmembrane receptors present on neuron growth cones. This axon guidance process employing Netrin signalling has been shown to be conserved between arthropods, nematodes and vertebrates. However, very little is known in non-bilaterians animals. We identified genes of this signalling pathway in Nematostella vectensis, in particular the secreted molecules Netrin and RGM and the transmembrane receptors Unc5 and Neogenin/DCC. We studied the expression pattern of these genes and began functional experiments using morpholino antisense oligos. This study aims at providing insights into the evolution of this signalling pathway and more generally about evolution of axon guidance in metazoans.
07.Apr Julia Hosp
Thompson Group
Molecular scaffolding of the Oikopleura house
The tunicate Oikopleura dioica is a representative of the only class of animals able to synthesise cellulose. The trunk of the animal is surrounded by a monolayer of a highly polyploid epithelium that can be distinguished morphologically into several distinct fields of cells. As often as every four hours the epithelium secretes a highly complex and elegant filter house structure with different channels and chambers composed of cellulose microfibrils and protein components called Oikosins. Through a proteomics approach the array of house proteins was expanded to more than 50 with very distinct epithelial expression patterns. In the upcoming talk I will summarise the efforts and briefly describe the characteristics of these extracellular proteins with links to their potential functions.
14.Apr Sutada Mungpakdee
Chourrout Group
21.Apr Wei Deng
Jiang Group
28.Apr Marcin Adamski
Chourrout/Adamska Group
Evolutionary Marine Bioinformatics:
Part 1: Alternative splicing of Oikopleura dioica ancestral introns.
Part 2: Transcriptome resources in Sycon ciliatum, a calcareous sponge.

In part one I will present a look into splicing of Oikopleura introns. An earlier study of several gene families had shown an important divergence of intron positions between Oikopleura genes and other metazoans. Here, we performed analysis of alignments within orthologous proteins shared between Oikopleura dioica, Homo sapiens and Drosophila melanogaster. We asked if there is a selection pressure to preserve splice sites responsible for alternative splicing. We also tested a hypothesis that alternative splicing might rather happen in between protein domains and be responsible for domain skipping.

Part 2: The phylogenetic position of sponges, and especially the relationships within the clade itself remain contentious, with some data supporting monophyly of sponges and some suggesting that Demosponges, Clacareous sponges and Homoscleromorphs are not more closely related to each other than each is to Eumetazoa. While large scale sequencing projects have been performed for Demosponges and recently Homoscleromorphs, only a few small datasets exist for representatives of the Calcarea. I will describe current status of transcriptome resources being developed for Sycon ciliatum, an extremely abundant and broadly distributed calcareous sponge.
12.May Pavla Navratilova
Thompson Group
Study of Oikopleura chromatin architecture and male-specific transcriptional regulation
19.May Jean-Marie Bouquet
Appendicularia Facility
Effects of Ocean Acidification and Temperature Increase on Planktonic, Tunicate, Oikopleura Population Dynamics at Laboratory and Mesocosm Scales
Atmospheric CO2 is projected to double by 2100, resulting in increased temperatures, ocean acidification (OA) and changes in the balance of marine ecosystems. A lack of multifactorial studies means limited knowledge on the combined effects of these pressures on ecosystem structure and function. Laboratory data suggest that increasing temperature and decreasing pH, at magnitudes mimicking expected changes, may differentially impact the fitness of major zooplankton groups. Filter-feeding appendicularians ingest smaller sized food than copepods, by-passing the microbial loop, and directly transferring bacteria and nanoplankton to higher trophic levels. They are also vectors of global vertical carbon flux through trapping of prey in their frequently discarded houses. We obtained mono-factorial laboratory data suggesting that OA positively affects fitness parameters in Oikopleura. This may have important consequences for ecosystem function as the major zooplankton, copepods and appendicularians, have different trophic roles. To assess these effects in more complex natural ecosystems we have conducted a pilot mesocosm experiment use a 2x2x3 factorial design to assess effects of temperature, pCO2 and phytoplankton regimes on zooplankton population dynamics. Data will be presented showing the extent to which OA and temperature modulation of Oikopleura fitness parameters noted in the laboratory are also observed at the mesocosm scale.
16.June Sutada Mungpakdee
Chourrout Group
Characterization of sex chromosomes and sex determining genes in urochordate Oikopleura dioica Oikopleura dioica is an interesting model system to characterize the sex chromosome and study the process of sex separation, since its genome is very small (67 Mb) and it is the only dioecious tunicate. Tunicates are the closest living relatives of vertebrates, and therefore interesting for studies of sex determination near the invertebrate-vertebrate transition. The high coverage (14X) of shotgun reads allowed us to distinguish the X (gene-rich, repeat-poor) and the Y (gene-poor, repeat- rich) specific region. The ultra-scaffold of the X and Y chromosome were built by physical link from hybridization of 300 BAC clones on tiling array. The assembly was verified by segregation pattern of several markers and FISH show the existence of the sex chromosomes. By EST mapping, we have found eight giant genes on the Y chromosome. They are strongly expressed only in the testis during the late stages of spermatogenesis. The homology of these genes is low compared to genes of the Oikopleura genome and others. The relatively large Y chromosome compared to other organism implied that the appearance of Y chromosome in O. dioica is very recent.

It is known that the master gene of sex determination cascades is not conserved. However, the most downstream gene of the cascade is shared by distant bilaterians. This gene belongs to a group comprising Dmrt1 (doublesex and male abnormal 3-related transcription factor 1), Drosophila dsx (doublesex) and Caenorhabditis elegans mab-3 (male abnormal 3). To get insights into sex determination mechanisms in O. dioica, we have implemented a candidate gene approach. The search for Oikopleura genes homologous to various sex-determining genes of Drosophila shows the existence of two Dmrt genes (Dmrt38 and Dmrt117) at two very distant genome locations, as well as one tra2 gene (transformer2). Gene expression analysis by RT-PCR and ISH shows that tra2 and Dmrt38 appear as a valuable candidate sex determining gene in Oikopleura. We are currently manipulating its expression by over-expressing these two genes to elucidate their precise function.

Previous Sars Abstracts - 2006 - 2007/2008