Sars Seminars 2010/2011
Sars Seminar Room (222 A2) at 13:00

Date Speaker Title and Abstract
25.october Bo Dong
Jiang Group
Dissection of actin organization and dynamics in cell shape change and polarized vesicle trafficking using Ciona notochord as a model
Actin organization and dynamics are pivotal to numerous fundamental cellular processes such as cytokinesis, morphology, motility, intracellular vesicle and organelle trafficking.

At the stage IV of tubulogenesis, ascidian notochord cells elongate 2.5 fold by cell shape change and become a short cylinder with approximately equal diameter and length. Through fluorescent protein fusion marker and time lapse movies, dynamic actin activity is displayed in this equatorial region of notochord cells. Actin filaments emerge at anterior and posterior cell boundary and migrate toward the midline in traveling waves; and then condense into continuous and thick bundles when they reach equatorial region. As they approach the middle line, the circumferential lengths of the bundles decrease, following the shape of the basal furrow. Excitingly, contractions generated by this dynamic actomyosin ring can be visualized with the process of membrane bleb. To elucidate the regulatory mechanisms of actin dynamics in contractile ring, we have developed nicely fusion protein markers and high resolution live notochord cell time lapse imaging techniques to visualize all of the events above. I will report recent results on the activities of actin binding proteins. Cofilin, which mediates the disassembly of actin filaments, is localized in a limited region and form a narrow ring at the middle of equator. Tropomyosin, which regulates actin-myosin interaction, is localized at the sub-equatorial regions flanking cofilin. Expression of a cofilin mimicked mutant disrupts F-actin organization, suppresses furrow formation, and blocks cell elongation. Furthermore, data show that a-actinin is essential for organization of actomyosin network in circumferential midway of notochord cell. Its mutant indicates that focal adhesion and extracellular matrix provide regulatory pathway to signal contracting events in notochord cell. . Based on these data, we propose that Ciona notochord use an equatorial actomyosin contractile ring to drive bi-directional cell elongation. The contractile ring is assembled at regions halfway between the anterior and posterior ends of the cell and the equator, and migrates to the equator. As they migrate they first encounter, at the sub-equatorial regions, tropomyosin and myosin, which regulate their contractility, and then the cofilin at the equator, which disassembles the filaments. The contraction is regional and periodical. We hypothesize that the interaction between local contractions at a large time scale and viscoselastic cytoplasm can generate sustained equatorial constriction and cell elongation.

Lumen formation is the hallmark of the tubulogenesis in diverse tube formation model systems. In Ciona intestinalis notochord tubulogenesis each notochord cell produces extracellular luminal pockets at both the anterior and the posterior end. We show that a distinct population of actin filaments localizes at the cortex of the apical/luminal domains. Whereas the disruption of microtubule network results in a partial block of lumen formation, depolymerization of actin filaments by latrunculin B abolishes lumen formation completely. Expression of a dominant negative ERM, a transmembrane protein that links the apical membrane with cortical actin, blocks extracellular lumen formation. Treatment with actin depolymerization inhibitor jasplakinolide also blocks extracellular lumen formation. Surprisingly however, large intracellular lumens are produced in jasplakinolide-treated notochord. We hypothesize that both the localization of apical cortical actin filaments and their dynamics are essential for the trafficking of secretory vesicles at the cortex before docking. To explore the regulatory mechanism of the actin dynamics, we disrupt actin depolymerizing factor cofilin and N-WASP. Both cofilin and N-WASP localize at the apical/luminal cortex, with N-WASP being more proximal to the apical membrane, and cofilin also present in the cytoplasm. The expression of a phosphomimetic cofilin mutant completely blocks the lumen formation without producing any intracellular lumen. In contrast, similar to the result of jasplakinolide treatment, expression of a dominant negative N-WASP results in a switch from extracellular lumen formation to intracellular vacuole formation. Based on these results, we present a molecular model for apical actin filaments and their dynamics in lumen formation and notochord tubulogenesis. We propose that the cortical actin filaments provide tracks proximal to the luminal membrane for the trafficking of secretory vesicles. These filamemts are anchored at the apical cortex via ERM. The cortical filaments are highly dynamic, and act as tracks for proximal luminal vesicle trafficking via a treadmilling mechanism. In the absence of actin dynamics, the migration of secretory vesicles is stalled and vesicles coalesce intracellularly through a homophilic vesicle fusion process.
03.november Simon Henriet
Chourrout Group
Recent retrotransposons in the compact genome of Oikopleura
The activity of mobile DNA has a strong influence on the evolution of genome architecture. In particular, the activity of retrotransposons is believed to have a dramatic effect on the size of the genome. The genome of the planctonic tunicate Oikopleura dioica is extremely compact and lacks most of retrotransposons clades usually found in chordates. In contrast, O. dioica host a well diversified family of LTR retrotransposons, the Tor elements, whose members have kept intact gag and pol ORFs. About half of these elements have a third ORF which potentially encodes a viral envelope (Env) protein, suggesting that Tor elements have been recently acquired by infection-mediated transfer in the Oikopleura genome. Results from a survey of Tor genomic insertion sites and the mapping of new Tor insertions show a high presence/absence polymorphism, supporting the recent acquisition of Tor copies in the Oikopleura genome. We detected Tor RNA essentially during the embryonic stages of development. Northern blot and 5'-RACE analysis show that Tor elements express a long mRNA which is a candidate template for the reverse transcription, and a shorter mRNA carrying the env ORF. Differing from other retroelements, Tor carry an internal promoter driving the expression of env mRNA, possibly with a tissue-dependent activity. After transient expression in mammalian cells, we found that Tor Env proteins were heavily glycosylated, suggesting they could function as surface receptor acting during the horizontal transfer of Tor elements. Taken together, our results support a model where Tor elements are active in the Oikopleura genome. The presence/absence polymorphism likely reflects a situation where the presence of individual Tor copies is under high turnover, as a result of combined genomic constraint, control of Tor expression and horizontal transfer of Tor elements carrying an env gene.
11.november Nagayasu Nakanishi
Rentzsch Group
To be rescheduled
30.november Pavla Navratilova
Thompson Group
Profiling of chromatin modification in Oikopleura dioica.
Covalent modification of histones plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of meaningful combinations. This histone code is being resolved in higher vertebrate genomes and there is whole-genome data on yeast and few invertebrate model organisms. We took challenge and advantage of the chordate Oikopleura dioica compact genome to study the combinatorial complexity of its histone modification pattern to answer how the epigenome is organized and to help to improve the genome annotation. Taken the unique endocycling mode of cell cycle in the adult Oikopleura, we might also contribute to understanding the role of histone code in the cell cycle regulation. This project has started with optimization of the ChIP method for Oikopleura material and is now in the stage of whole-genome ChIP-chip profiles collection and analysis. I will present some preliminary results showing that the transcription-related histone code is conserved from yeast to mammals and even fast Oikopleura evolution did not alter its function.
01.december Anne Zakrzewski
Comparing the formation of chaetae in Lophotrochozoa on the molecular and structural level
Chitinous chaetae are one of the most diagnostic characters of annelids. Generally they are produced by different cell types, which contribute to the formation process. Of these, the chaetoblast controls the structure of growing chaetae by basal apposition of ?-chitin on the surface of dynamic microvilli. Similar structures are known from Echiura, Myzostomida, but also from Brachiopoda and certain members of Mollusca and Bryozoa. Homology of these structures with annelid chaetae is controversially discussed. In order to better resolve their evolution we complement structural comparisons with molecular characterization of cell types involved in chaetogenesis in the annelid Platynereis dumerilii. By the combination of transmission electron microscopy, immunohistochemistry, and in situ-hybridization in larvae of P. dumerilii, we have determined 23 genes that are specifically expressed by either chaetoblasts or follicle cells, as well as transcripts that are shared between these cell types. Most interestingly, the gene expression studies even yield new insights into molecular aspects of chaetogenesis. Due to their tissue specificity, finally all 23 genes provide a highly specific set of molecular markers for chaetogenesis in annelids and introduce a new level for comparative investigation of chaeta-like structures. Major comparative investigations in other lophotrochozoans have been started.
08.december Sven Leininger
Expression of germ cell markers in the calcareous sponge Sycon ciliatum
The segregation of germ cells from somatic cells is a crucial step during development in all sexually reproductive animals. Whether a cell becomes a component of the germ line or undergoes differentiation into another cell type is determined by a complex molecular mechanism. Several conserved proteins are responsible for protecting the genome of germ line cells to ensure the heritage of a non-modified and undamaged genotype. Prominent representatives of pan-metazoan germ line markers are vasa, nanos, pl10 and piwi. Activity of these genes is generally restricted to cells of the germ lineage in higher bilaterians, but they were found to be expressed also in multipotent stem cells in basal metazoans (e.g. cnidarians, ctenophores, sponges, acoels and planarians) that are capable of asexual reproduction and regeneration. The abovementioned genes were recently shown to be expressed in archeocytes and choanocytes of demosponges, cell types bearing stem cell character and capable of differentiation into germ cells. In this seminar, I will present the isolation and characterisation of vasa, piwi, nanos and pl10 genes from the calcareous sponge Sycon ciliatum. Their cellular expression is currently investigated throughout the entire life cycle of this sponge, including embryogenesis and metamorphosis. Comparison of expression patterns of the demosponge and calcaronean germ line markers demonstrates dramatic differences between these two poriferan clades. Our results add to the growing knowledge on molecular principles of “stemness” and germ lineage determination in basal metazoans.
15.december Supat Thongjuea Lenhard Integration of ChIP-seq and 3C-seq Reveal Long-range Regulation of Myb Transcription During Erythriod Development

Christmas Break

12.january Chiara Sinigaglia
Identification of genes involved in aboral patterning and apical organ formation in Nematostella vectensis
Nematostella vectensis larvae present at the aboral pole an organ, called apical organ. Apical organs are sense organs characterized by a tuft of cilia, found in marine ciliated larvae of both protostomes and deuterostomes. The possible homology of apical organs within bilaterians and between cnidarians and bilaterians has been long debated, since only few characteristics are in common. The problem can be considered also in a broader perspective, looking more extensively at the aboral region of the larva: homology might be restricted to aboral patterning mechanisms or to specific cell type of the apical organ. We used microarray analysis to identify a set of genes expressed in the apical organ area, comprising both differentiation genes and transcription factors. These genes provide a basis for a comparative gene expression analysis. Currently, I am focusing mainly on the aboral patterning question, employing a candidate gene approach. To begin the reconstruction of the gene interactions shaping the aboral area, we started from some known anterior or anterior neural marker genes: six3/6, foxQ2, irx, soxB1. Their expression patterns were analysed in wild type and perturbed conditions, focusing on early stages. The perturbation experiments were carried out using antisense morpholinos, directed against the UTR or the start codon of the selected genes. The phenotypes and the perturbed expression patterns are put in relation with the wild type condition, in order to determine the relationships between genes.
19.january - 11:00 Yoshimasa Sagane
Cellulose- and chitin-based extracellular structures in Oikopleura dioica: Identification of GPI anchored cellulase-like protein, and characterization of chitin-based extracellular structures.
Cellulose and chitin are polysaccharides that function to support and protect the organism. The cell walls of plants are composed of cellulose. The cell walls of fungi and the exoskeleton of arthropods are composed of chitin. Urochordates are the only metazoans that possess biosynthesis ability of both cellulose and chitin. Ascidians have one cellulose synthase (CesA) gene, whereas duplicated larvacean genes (CesA1 and CesA2) have evolved distinct temporal and functional specializations. Spatial-temporal expression analysis revealed that CesA1 produces long cellulose fibrils along the larval tail, whereas CesA2 is responsible for the cellulose scaffold of the post-metamorphic filter-feeding house. Extracellular cellulose microfibrils produced by the pre-metamorphic CesA1 duplicate have a role in notochord and tail morphogenesis. Furthermore, we characterized the distinctive cellulose microfibril scaffold of the filter-feeding house. Specialized cortical F-actin arrays template microfibril orientation and glycosylphosphatidylinositol (GPI)-anchored proteins in lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules deliver and maintain CesA complexes to specific cell membrane sites rather than orienting their movement through the membrane. By in silico analysis, we found six genes that encode cellulase-like proteins having glycosyl hydrolase family 9 domains. Of these, three proteins are predicted to possess a C-terminal GPI-anchoring signal. Now we focus on these genes as factors involved in construction of cellulose-based architecture.

Chitin staining using chitin binding protein revealed chitin-based extracellular structures in the digestive tract (esophagus, stomach, vertical intestine, median intestine, and rectum) and in the ciliary funnel. Chitin-based structures in the digestive tract cover the luminal epithelium, whereas that in ciliary funnel covers the entrance of the duct connecting the oral cavity and the anterior ganglion. In the O. dioica genomic database, at least seven chitin synthase genes were found. In situ expression patterns revealed the responsible genes in each tissue. These structures may protect the digestive and nervous system from viral and bacterial invasions.
24.January Andrea Trentani
Neuromuscular connectivity in the appendicularian Oikopleura dioica
Appendicularians are pelagic tunicates that remain free-swimming throughout life. Tunicates are receiving increasing focus in the context of vertebrate brain evolution, as they derive from the base of the chordate lineage tree. Past research has focused on the development of motoneurons (MNs) and neuromuscular connections in the appendicularian Oikopleura dioica, with special emphasis on MN differentiation and the developmental appearance of efferent projections to the tail muscles (Søviknes et al., 2007). This work revealed that Oikopleura, in contrast to ascidian tunicates such as Ciona, has a vertebrate-like organization of neuromuscular connections, with segmentally distributed, bilaterally symmetrical cholinergic neuromuscular projections, but did not address the organization of neuromuscular synapses.

In this seminar, new evidence will be presented regarding the organization and distribution of Oikopleura dioica peripheral fibers originating from the caudal nerve cord. Using a panel of anti-tubulin antibodies, alpha-bungarotoxin and the synaptic activity marker FM1-43, new findings show that tubulin-positive fibers can be followed to corymbiform terminals on muscles. These terminals in turn colocalize with alpha-bungatoxin labeling on the muscle surface and can be vitally labeled with FM1-43, indicating synaptic vesicle turnover. Finally, the structure and distribution of two additional and separate classes of peripheral projections will be presented. Together, these new findings add to our comparative insight into the diversity of efferent projections within the protochordate radiation.
27.january Lisbeth Olsen
Germ line development in Oikopleura dioica
02.february Elsa Denker
Tubulogenesis in Ciona notochord requires the formation of atypical “bi-apical” cells through two discrete par3/par6/aPKC domains.
Biological tubular structures are found in a wide variety of organs and organisms, allowing to carry fluids, absorbing and exchanging molecules between several compartments… (Lubarsky and Krasnow, 2003). The processes through which they form, or tubulogenesis processes, are amongst the most interesting and challenging morphogenetic processes for developmental biologists, because they imply many changes in cell state and behavior (Baer et al., 2009, Bryant & Mostov 2008). We have recently shown that Ciona notochord is a good model to study tubulogenesis, because it occurs in a structure composed of only 40 big cells, that no longer divide, its tubulogenesis only involving remodelling of cell shape and of tissue configuration, neighbour exchanges and no cell membrane rupture (Dong et al., 2009). Moreover we have shown that this tubulogenesis process is quite original compared to the other ones described before.

Cell polarization is a critical event in morphogenesis, because it drives the overall 3D organisation of the cell components with respect to positional cues (Tanos & Rodriguez-Boulan 2008, Bryant and Mostov 2008). Over the last decades, studies from embryogenesis, cultured cells and cancer research, have highlighted the role of several very conserved key complexes that define and maintain cell polarity. The Par3/Par6/aPKC is one of them. I have investigated the subcellular localization of the members of the complex in developping Ciona notochord cells, and have shown a strong correlation between their distribution and the specification of an apical domain where the lumen will appear, and, at the onset of lumen formation, with the formation of tight junctions around the lumen (monitored by the localization of the ZO1 marker). Conversely, usual basolateral markers such the homologues of Lethal giant larvae, Discs large and Scribble, get excluded from the apical domain and remain baso-lateral. Adherens junction markers get down-regulated from the forming apical domain and get resticted to the lateral domain.

Functional experiments are now required to definitely support the function of these molecules, but these results already bring us the following informations:

1) for the first time, the mechanisms for apico-basal cell polarization have been investigated in urochordates and again the same players appear to be invloved; moreover interstingly, the configuration of the junctions and the position of the polarity protein with respect to them is very similar to what is found in vertebrates, and the overall process is very similar to what has been described for lumen formation in cultured MDCK cells in isochoric configuration (Ferrari et al., 2008).

2) however the major new feature, that has been not described in any other context, is that each notochord cell has two apical domains, that seem to be driven by the formation of 2 discrete par3/par6/apkc patches in opposite points of the cells.

3) according to the current view on the phylogeny of chordates and the internal phylogeny of urochordates (Delsuc et al., 2008, Tsagkogeorga et al., 2009), we can suggest that having a tubular notochord and thus apico-basal polarity is not the ancestral configuration in chordates, but could be ancestral to urochordates, suggesting that in the urochordate lineage there was a co-option of this polarization system to generate the notochord tube, allowing a new strategy for tail expansion and maybe swimming behavior, even if this remains poorly understood.
09.february Jan Inge Øvrebø
Regulation of multiple cell cycle variants: amplified cyclins and CDKs in the urochordate, Oikopleura dioica
At the heart of the cell cycle lie the cyclin dependent kinases (CDKs), which control key events such as DNA replication and mitotic cell division. As their names imply the CDKs require activating subunits, the cyclins, in order to maintain kinase activity. In general the canonical cell cycle consists of four distinct phases; G1, S, G2 and M phase, but there are also other variants of the cell cycle such as meiosis, which give rise to gametes, and endoreduplication, in which cells increase their ploidy without completing cell division. Examples of multiple cell cycle variants are found in the marine urochordate Oikopleura dioica. The animal develops through rapid mitotic divisions during early development, but most cells shift to endocycles prior to metamorphosis. Studies of Oikopleura endocycling epithelial cells reveal various cell sizes and bilateral symmetry, which have been shown to be carefully controlled through G-phase length. Another fascinating example of cell cycle regulation is found in the ovary, which consists of a giant multinuclear cell containing endocycling nurse nuclei and meiotic nuclei ,seeding oocytes, all connected by a shared cytoplasm supported by an actin scaffold, whose organisation is termed a coenocyst. To better understand the underlying mechanisms of the cell cycle we have scrutinised the Oikopleura genome for cyclins and CDKs and predicted their function and annotation. This work has revealed significant amplifications of the CDK1 complement, unknown among other metazoans, as well as the cyclin b and D families. These alterations are discussed in terms of what is known about the molecular regulation of endocycles in insects, mammals and plants.
16.february Matteo Ugolini
Immunity genes in the Oikopleura lineage
Invertebrates, which lack a complex adaptive cell-based immune system, possess other mechanisms of biological host defense to respond to invading pathogens such as bacteria, fungi or viruses which are typically present in their habitats. Given the absence of lymphocytes or an antibody-based humoral immune responses, the innate immunity represents the first, and only, line of host defense and is thought to have emerged at an early stage in the evolution with the later evolved adaptive immunity being a distinctive feature of jawed vertebrates. Evidences for the ancestral origin of innate immunity came from Nematostella, Hydra, sea urchins, amphioxus and Ciona which showed an elaborate repertoire of potential immune receptors, regulators and effectors with homology to known key molecules found in the defense systems from arthropods to mammals. Such comparisons stressed also the role of ecological circumstances on the evolution of the immune system. Lineage-specific diversification of immune recognition and perhaps responses is shaped by the different habitats, environments and lifestyles and transcends phylogenetic relatedness in way that solution for internal defense in one group could be completely different from the solution obtained in other groups. Among the marine invertebrates in the recent years increasing attention has been focused on the marine zooplanktonic appendicularian Oikopleura dioica as emerging model organism for a wide range of crossdisciplinary research. Despite its important evolutionary position as member of the subphylum Urochordata so far the host-pathogen interactions, organization and components of its immune system are still unknown. Given the high concentration of viruses and bacteria in the sea water we expect O. dioica to possess a basic if not more complex system to sense and overcome infection by pathogenic viruses The availability of the genome sequence at a high coverage allowed an extensive search for homologues and representatives of several key proteins families involved in the defense system. In Oikopleura, the proteins families typically involved in the defense system in protostomes and deuterostomes are very small, if not completely absent. In particular (i) only a few receptor/effector classes have been found, (ii) no large arrays of Pathogen Recognition Receptors (PRRs) could be identified (i.e. TLRs) and notably (iii) there is a complete lack of components of a complex cytokine signaling network. Nevertheless a few elements with homology to components of the antiviral and antibacterial machineries in other species can still be traced in the Oikopleura genome and in particular: 1. Two Peptidoglycan Recognition Proteins (PGRP) 2. A unique putative toll-like receptor which displays structural features of vertebrate TLRs 3. An exceptionally expanded family of putative Phospholipase A2 effectors Marine invertebrates are especially suited to understand the evolutionary forces that shape the genetic diversity of the innate immune armamentarium as they are constantly subjected to selective pressure from variable physical condition of the aquatic habitat (eg. UV irradiation, salinity, trophic conditions) and from interaction with pathogens. By investing the immune system of O. dioica and the specific solutions elaborated in this lineage we can shed light on the evolution of immunity in early Chordates
Lucas Lèclere
Netrin and SFRP protein families: Evolutionary origins and functions during early development in the cnidarian Nematostella vectensis
Netrin and SFRP (Secreted frizzled related proteins) are two groups of multidomain signaling proteins that are both key regulators of animal development. In Bilateria, Netrin pathway regulate axon guidance during central nervous system formation, and SFRPs act as inhibitor of the Wnt signaling and are involved in antero-posterior patterning. To get insight into the origin and evolution of these two groups of proteins, we performed a functional study in the non bilaterian Nematostella vectensis (Cnidaria, Anthozoa). Our preliminary results indicate that Netrin is not involved in axon guidance but in the overall patterning of the embryo, while SFRP seems involved in patterning of the aboral pole. We also performed a phylogenetic study of these two proteins and show that the particular domain architectures of each of these proteins have been present in the eumetazoan ancestor and evolved a second time independently in the metazoan lineage. These phylogenetic reconstructions are the first evidences that, in addition to gene duplication, independent evolution of domain architecture within one evolutionary lineage is a mode of diversification of multidomain protein families.
02.march Gunasekaran Subramaniam
Role of TOR signaling in growth and longevity of Oikopleura dioica
Caloric intake and ageing are tightly coupled processes. Excessive calories and increasingly sedentary lifestyle result in obesity and predispose individuals to major risks of age related diseases. Studies have shown that TOR (Target of Rapamycin) signalling, a nutrional signalling pathway, is coupled to growth and ageing by regulating cell division and protein synthesis. In ageing cells, high TOR activity culminates in increased protein translation and subsequently results in a high percentage of unfolded or misfolded protein, endoplasmic reticulum stress and massive production of free radicals which are responsible for damage to DNA, proteins and membranes, thus causing senescence or ageing.

Conversely, reduction of TOR activity by Dietary Restriction (DR) confers extended lifespan (longevity) and protection from many age related diseases. Studies in yeast, C. elegans, drosophila and mouse have also shown that DR inhibits TOR signalling. Thus TOR signalling plays an important role in growth and longevity of cells in organisms. In the current study we exploit Oikopleura dioica, as a model organism to study the effect of TOR signalling on longevity since it has a short life span (6days at 160 C) and is very closely related to vertebrates phylogenetically.

Growth in Oikopluera dioica is rapid due to extensive recourse to endoreduplication during it’s short life cycle leading to polyploidy in cells. The present study explores the influence of TOR signalling on growth and longevity in Oikopleura dioica. In this study, blocking TOR signalling in Oikopleura using rapamycin, or growing them under Dietary Restriction revealed that the growth arrest by endocycling arrest. On the contrary, blocking TOR signalling in the mitotic gonad nuclei rendered them unaffected, allowing them to continue proliferation without entering meiosis and increased longevity.

Interestingly, studies with p27 in the somatic cells and mitotic gonad nuclei suggest that p27 is regulated by TOR signalling. Abrogating TOR signalling resulted in increased nuclear localisation of p27 in the somatic cells leading to cell cycle arrest whereas p27 was excluded from the mitotic gonad nuclei which continued to proliferate even when TOR signalling was inhibited. Surprisingly, increased mRNA and protein levels of Cyclin Db, a G1 regulator, during DR mediated TOR inhibition suggests that Cyclin Db is instrumental regulator during this inhibition. Further analysis revealed that Cyclin Db localised mainly to the mitotic gonad cytoplasm perhaps indicating it’s role in proliferation of mitotic gonad nuclei when TOR signalling is abrogated. Surprisingly, our findings reveal that the MAPK pathway is activated in the absence of TOR signalling which might be responsible for the germ cell nuclei proliferation. Additionally, the presence of AP1, a MAPK effector, binding sites in the promoter of Cyclin Db and colocalisation of Cyclin Db with p27, lead us to hypothesize that in the absence of TOR signalling by DR, MAPK pathway is activated leading to increased Cyclin Db synthesis, which then sequesters p27 in the cytoplasm and prevents cell cycle arrest. Thus the mitotic gonad continues to proliferate without entering meiosis and maturation thus bypassing the paradigm of senescence resulting in an increased life span in Oikopleura dioica. Thus, abrogating TOR signalling caused the somatic cells to arrest endocycling and become quiescent with increased p27 in nucleus whereas the effect of TOR abrogation on the gonad mitotic nuclei continuing to proliferate without maturing and ageing. Further characterization of the molecules downstream of TOR, and MAPK signalling with emphasis on effectors of the G1/S transition, will help to increase our understanding of TOR mediated regulation of somatic endoreduplication, oogenesis and longevity in Oikopleura dioica.
09.march Nagayasu Nakanishi
Neural development in Nematostella vectensis (Cnidaria, Anthozoa) and the early evolution of ectodermal neurogenesis in Metazoa
In Bilateria, the nervous system is invariably derived from the outermost layer of the embryo, the ectoderm, suggesting an ancient evolutionary origin of ectodermal neurogenesis. Yet in the sister group Cnidaria, whether a related mode of neurogenesis occurs is unknown. Here, we examined the mode of neurogenesis in the anthozoan cnidarian Nematostella vectensis by using the elav1::mOrange transgenic line, in situ hybridization (against Antho-RF and NvElav1 mRNAs), antibody and fluorescent dye staining (FMRFamide, GLWamide, tubulin, phalloidin, TOPRO/DAPI), morpholino-mediated gene knockdown, and confocal microscopy. We show that in the ectodermal epithelium of gastrulae and of growing tentacles during polyp formation, neural precursor cells occur apically and differentiate into sensory and ganglion cells as their cell bodies migrate basally. Early born ectodermal neurons send basal processes towards the aboral pole, converging to form a plexus at the base of the apical organ. Development of a subset of these early-born ectodermal neurons requires postmitotic expression of NvElav1, the ortholog to bilaterian neural Elav genes. Beginning at the early planula stage, NvElav1-expressing endodermal sensory cells develop from the endodermal epithelium, whose neurites form a complex basiepithelial network containing pairs of neuronal bundles flanking each strand of parietal muscle fibers. During late planula development, ectodermal neurons in the body column send transverse neuronal processes. Our findings combined with existing comparative data imply that ectodermal neurogenesis evolved prior to divergence of Cnidaria and Bilateria.
23.march Ivonne Sehring
Contractile ring formation and blebing during elongation of Ciona intestinalis notochord cells
Two different modes of local contraction seem to be involved in the elongation of Ciona intestinalis notochord cells: the force generated by an equatorial contractile ring (CR) and blebing. Equatorial organization of F-actin into a contractile ring is required for cytokinesis in eukaryotic cells. Interactions between actin and myosin II in the ring generate force that constricts the cell into two daughters. Current models for the formation of the contractile ring favor either localized de novo assembly or directional cortical transport toward the equator. The beginning of cell elongation in Ciona notochord cells is accompanied by the formation of a ring of actin filaments in the equatorial region, similar to the contractile ring during cytokinesis. These actin filaments seem to origin from the lateral domains, as we observe actin filaments undergo a directional flow from the lateral domains toward the equator. Fluorescence recovery after photobleaching experiments showed that actin in the CR is highly dynamic. We show that the actin regulating proteins actinin, tropomyosin and cofilin assemble at the equatorial contractile ring, and analyze the effect of constitutive active or inactive mutants on the actin ring and cell constriction. Phosphorylation of the regulatory light chain (MLC) of myosin II is a primary means of activating myosin II and is known to be crucial for the execution of cell division. Using specific antibodies and GFP-constructs, we localize myosin II in the equatorial region of notochord cells. The impact of the phosphorylation state is analyzed by phosphomimic and unphosphorylatable mutants. The formation of blebs is often observed during cytokinesis. During blebing, the membrane detaches from the cortex and inflates. Both actinin and cofilin are observed in blebs in notochord cells, followed by actin reassembly under the membrane during bleb retraction.
30.march Sabrina Schiemann
Rotifers - highly derived spiralians?
Rotifers are microscopic metazoans living in all kind of fresh- and seawater habitats. Although they are quite small they have a complex body plan. Their distinct features are a musculature pharynx called the mastax and a ciliated corona at the anterior end of the body. Most of the developmental research about cell fate determination done on rotifers is more than 60 years old. The results are contradictory and many questions still need to be answered. What is known so far is the fact that rotifers are direct developer with a determinative cleavage pattern. I will investigate to the question if rotifers have a derived spiral cleavage and show first results of the cell lineage analyze of the bdelloid rotifer Adineta vaga.
13.april Marcin Adamski
Preliminary assembly and analysis of Sycon ciliatum genome reveals string differences in developmental toolkit components between the Demospongia and the Calcaronea

Easter Break

18.may Gemma Danks
Mining the transcriptome of the marine chordate Oikopleura dioica
25.may Gemma Richards
Transgenic approaches to studying neurogenesis in Nematostella
01.june Aina Børve
The Digestive Tract and Nervous System of the Nemertodermatid Meara stichopi
08.june Wei Deng
Functional study of the sulfate transporter prestin in notochord lumen formation
30.june Sofia Fortunato
Expansion of developmental transcription factor families in the calcareous sponge Sycon ciliatum
Sponges are likely the earliest branching multicellular animals, and represent a key phylum for evolutionary developmental studies. Recently, genome analysis of the demosponge Amphimedon queeslandica demonstrated presence and developmental expression of most Eumetazoan transcription factors (TF). However, it appears that the number of TFs is significantly lower in Amphimedon than in Cnidarians and Bilaterians, in line with the notion that developmental inventory of genes increased during evolution. To expand the understanding of sponge development and genome evolution, developmental TFs should be identified in other groups of sponges. The draft genome of the calcareous sponge Sycon ciliatum, reveals that the number of TFs in Sycon is larger than in Amphimedon. In this study, we have concentrated on genes belonging to Sox, Pax and Homeobox families. Phylogenetic analyses were performed on 12 HMG domains of Sox genes, 2 Pax, and 36 homeodomains of homeobox genes of S. ciliatum. Most of the SciSox genes are highly divergent and cannot be assigned to any Sox gene subfamily. The two SciPax genes grouped within the PaxB subfamily. Sycon homeodomain sequences grouped within known classes of homeobox gene families. Five of them are NK related genes, which are clustered in a monophyletic group. In situ hybridization analysis demonstrates that Sox and Pax genes are dynamically expressed during S. ciliatum embryogenesis, metamorphosis and in adult tissues. In addition, SciPaxBa seems to be expressed in few cells located on the side and tip of chambers. The ongoing analysis of Sycon genome, will provide more information on the presence and expression patterns of the homeobox genes. Our results indicate that compared to Amphimedon, the total number of some developmental TF families in S. ciliatum, were represented by twice or three times more genes, while other families (e.g, Paired) are reduced in Sycon. This highlights the differences between the two sponge lineages and underscores the necessity to analyze additional sponge genomes to reveal the evolutionary history of the developmental toolkit.

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