Abstracts
Sars Internal Seminars 2006
Sars Seminar Room (222 A2) at 13:00



Date Speaker Abstract
29.nov Fekadu Yadetie Genome-wide overview of trans-splicing and operons in Oikopleura dioica
Spliced-leader (SL) RNA trans-splicing to 5' ends of mRNAs occurs in some animal phyla such as nematodes, hydra and tunicates. In nematodes and recently in tunicates, it has been reported that a fraction of trans-spliced genes are also transcribed as polycistronic pre-mRNAs. Trans-splicing and polycistronic transcription have also been shown to occur in the appendicularian tunicate O. dioica (Ganot P., Kallesoe T., Reinhardt R., Chourrout D., Thompson EM., Mol. Cell. Biol. 2004). About 25% of O. dioica mRNAs are trans-spliced to a single 40 nucleotides SL RNA. Here, the extent of occurrence of operons in the O. dioica genome, and some features such as the types of genes and functional categories enriched in operon gene set will be discussed. A method for large scale determination of coupled 5' and 3' ends of SL trans-spliced mRNAs will also be described. By analysis of the trans-spliced transcriptome (ESTs) and scaffolds of a preliminary genome assembly, we find that the majority (at least 90%) of trans-spliced mRNAs are transcribed from operons. A rough estimate is that 20-25% of O. dioica genes are organized in operon like clusters. As in Caenorhabditis elegans, putative operons in O. dioica are enriched for genes involved in fundamental cellular processes, mainly protein metabolism (synthesis, modification, folding, transport, sorting and degradation), RNA processing and energy metabolism. In most cases, genes co-transcribed from the same operons have no obvious functional relationships. However, ribosomal protein genes are notable exceptions as the majority of them appear as 2-6 gene clusters in the same operons, far more often than for C. elegans.
22.nov and
01.dec
Vibeke Kyrkjebø
Thesis Defence
Large Auditorium (2144), Data building, HIB 10:15
Characterization of Nuclear Respiratory Factor 1 using transgenic zebrafish.

Nuclear respiratory factor 1 (NRF1) is a transcription factor essential for the embryonic development of vertebrates. The zebrafish mutant nrf is characterized by apoptotic photoreceptor death during embryogenesis followed by late larval lethality. In zebrafish nrf1 is initially expressed throughout the CNS at a high level at days 1 and 2 and later fades to levels undetectable by in situ hybridization. This study shows that the initial phase of expression is crucial for photoreceptor maintenance, but not for their initial development, as homozygous mutant cells can form normal photoreceptors in a wild type background. By utilizing reporter expression of a marker insertion near the wild type allele of nrf1 combined with transgenic rescue, I show that a single heat shock induced pulse of nrf1 expression at any time between 24 and 54 hours post fertilization is sufficient to rescue the mutant phenotype and delay photoreceptor degeneration until larval stages. Moreover, no ectopic defects are detected after ubiquitous expression of the gene, suggesting that Nrf1 serves no detectable instructive role during embryogenesis. These results suggest that Nrf1 plays a permissive role in zebrafish photoreceptor maintenance and is crucial for the formation and survival of the outer nuclear layer, but is not strictly necessary for the initial development of individual photoreceptors. Gene expression comparison analysis identifies several up and downregulated genes in the nrf mutant, suggesting that the intraflagellar transport machinery of the photoreceptor connecting cilium might be defect.
22.nov Vibeke Kyrkjebø Characterization of Nuclear Respiratory Factor 1 using transgenic zebrafish.
This is a pre disputas seminar - please see above for abstract
15.nov Edi Renfer I-Sce I Mediated Transgenesis in Nematostella - a Progress Report.
We are working on a project to create transgenic Nematostella. Our goal is to be able to generate a variety of transgenic lines for gene expression studies in Nematostella. In this progress report, a new method of transgenesis is presented, using a homing endonuclease (I-SceI). We show that this meganuclease indeed increases the integration rates in injected Nematostella embryos and therefore might be developed into a good standard method for transgenesis in our animal.
08.nov Jana Mikhaleva Fox-genes expression patterns.
Despite a loss of homeobox diversity, Oikopleura keeps a high total number of homeobox genes, because quite few remaining gene groups have been specifically amplified in Oikopleura. We have systematically performed expression studies of these amplified groups and could recognize it in multiple cases expression in the trunk epithelium of the larva. Such patterns are rare for genes belonging to non-amplified groups of homeobox genes. For verification of our hypothesis, that amplified genes have expression pattern in epithelium more often, we selected another big group of genes - Fox-genes. They are characterized by an evolutionary conserved DNA-binding domain of winged helix structure. Currently, 20 out of 22 different Fox genes are cloned in Oikopleura dioica. Only for 12 genes zygotic signal was detected and described. Analysis of expression has shown that in amplified groups of Fox-genes pattern of expression equal distribute in epithelium surface and in inner organs.

Morpholino injections into Oikopleura eggs.
Based on results of the in situ screen of homeobox genes we would like to make functional study of the genes, which have expression pattern in epithelium. For establishment of morpholino injection technique we selected Brachyury gene. Morpholino oligonucleotides against Pax37B gene were injected in Oikopleura eggs also. In both cases I observed arrest of development at 2 hpf stage, which correspond to middle gastrula.
25.oct Mary Laplante To be rescheduled
18.oct Alexandra Schulmeister Histone H3 variants in Oikopleura dioica.
Eukaryotes package their DNA into chromatin fibers to achieve compaction, enabling their entire genome to fit into the nucleus, while allowing DNA transcription, replication and repair when necessary. The core nucleosomal subunit of chromatin consists of 147 bp DNA wrapped around an octameric unit of small basic proteins, the histones H2A, H2B, H3 and H4. Several mechanisms exist to make nucleosomal DNA more accessible to the cellular machinery. All of these are affected directly by modifications in the amino acid sequence of histones; either by posttranslational modifications (PTMs) or by the introduction of histone variants. PTMs can dramatically alter the character and reactivity of amino acids and therefore influence chromatin structure through electrostatic mechanisms and the ability of non-histone proteins to interact with chromatin. Histone variants allow variation in the composition of individual nucleosomes, but also allow the cell to expand its PTM profile. With the exception of histone H4, each of the histone subtypes is represented by several different variants. In particular, H3 has a key role in organizing the nucleosome from a structural perspective by organizing the two fold symmetry of the eukaryotic nucleosome, making contact with H2A and specific contact with nucleosomal DNA. Oikopleura dioica has some interesting life history features with respect to histone gene organization, histone gene regulation, and spatial and temporal use of histone variants. With our study on the different H3 variants of Oikopleura dioica we aim to shed further light on the function of different H3 variants, their modifications and the evolutionary constrains that may have led to repeated origins of H3 subtypes. We have identified six different H3 variants expressed in O. dioica, the S-phase dependent H3.2, the replacement variant H3.3, a putative centromeric H3 OdCENP and three H3 genes of unknown function that exhibit a male specific expression profile. The number of male specific variants is surprising, since only one testis specific H3 has been found in rat and human so far and little is known about the role of H3 variants in the chromatin remodeling of male sperm. Performing immunostainings and capped mRNA transfections we have begun to localize some of the OdH3s to further elucidate their function. Additionally, mass-spec analysis of Oikopleura histones are currently being pursued to identify PTMs and their quantities in different developmental stages. This approach will also allow us to identify possible specific PMTs for the male specific H3s of Oikopleura in order to understand the function of their specific residue changes.
11.oct Michael Saina Investigating the role of DPP signalling in the starlet sea anemone.
The establishment of metazoan body plans is highly dependent on multiple cell signaling pathways. These play crucial roles not only during embryonic development but are also important for other processes from gametogenesis to cell cycle control. One of the best investigated pathways is the TGF-ß pathway. It has been shown that one particular member of the TGF-ß superfamily, the molecule dpp/BMP2/4 is crucial for the establishment of the dorso-ventral axis in bilaterian animals. The homologue of dpp/BMP2/4 in Nematostella vectensis has been shown to be expressed asymmetric in an axis perpendicular to the oral-aboral body axis. It is not yet clear what the function of this molecule in the starlet sea anemone is. To shed light on this question, I will present some first results of morpholino-based knock down experiments.
27.sept Kari Ersland To be rescheduled
20.sept Anna Zofia Komisarczuk
Becker Group
FGF signaling in zebrafish.
Many signaling processes during organ formation are controlled in time and space by the expression of particular growth factors which activate Ras / MAP kinase pathways. To work in the right way signaling pathways mediated by receptor tyrosine kinases (RTKs) have to be tightly regulated by positive and negative factors. Sprouty proteins are an important group of RTK inhibitors. Sprouty 1 is expressed in several signaling centers during embryogenesis, and starts at the tail bud stage in a tail bud and polster. It is expressed in the embryonic midbrain - hindbrain boundary, a known domain of FGF8, where expression persists until adulthood, and hindbrain. Sprouty 1 is also expressed in optic stalk, and ventral retina, branchial arches, trigeminal ganglion, epiphysis, pronephric duct and fins, where it largely overlaps with FGF8 expression.

The zebrafish embryonic, larval and adult pigment pattern consists of several horizontal stripes, made by specific distribution of three types of pigment cells: melanophores, xantophores and iridophores. The pigment cells come from the neural crest, derived from the ectoderm shortly after gastrulation. Recent publications show that cell - cell interactions play a key role in the formation of this pattern, but the particular details of these interactions have not yet been clarified. There are many known zebrafish pigment mutants with defects in stripes formation. We present the phenotypic result of retroviral insertion in the neighborhood of fgf8 and fbxw4 genes. Insertions and deletions in this area in zebrafish, mouse and human were previously described, and these mutations are known as hagoromo, dactylaplasia and split hand/foot malformation 3, respectively. None of the insertions disturb the coding sequence of either of these genes, but the presence of multiple highly conserved non coding elements in the area suggest that these are position effect mutations affecting the expression of fgf8. The spatial relationship of fgf8 and fbxw4 is conserved in ciona genome, pointing to a fundamental aspect of chordate fgf8 regulation.
13.sept Sutada Munpgakee
Chourrout Group
Genome Duplication in Atlantic Salmon revealed by the study of Hox clusters
To examine the effects of recent genome duplication in salmonid fish (25-100 Mya), Hox cluster organisation is a powerful marker. Using a high quality BAC library from the SGP (Salmon Genome Project), BAC clones containing most Hox clusters have been isolated and fully sequenced. HoxA clusters were sequenced through genome walking. We have thus far found a total of 110 Hox genes in 13 clusters which we named HoxAaα, Aaß, Abα, Baα, Baß, Bbα, Bbß, Caα, Caß, Cbα, Cbß, Daα and Daß. Interestingly, most Hox genes issued from the previous genome duplication (ray finned-fish radiation) have been conserved in salmon with strong purifying selection. These include posterior and middle genes in both HoxCb clusters and also HoxD1a, which were so far not revealed in any known teleost fish. However, a few other Hox genes have been lost in the salmon lineage or became pseudogenes. The clusters that evolve faster than others tend to lose more genes, such as the HoxBbß cluster. The mode of genetic inheritance in salmon (tetrasomy and disomy) can explain the variable rate of evolution, with for example a little divergence between HoxBaα and Baß clusters due to continuous meiotic interaction. Sequencing salmon Hox genes supports earlier phylogenetic analysis of salmonids and related teleosts. Following by the rapid genome rearrangement period after genome duplication, subfunctionalization is likely favored for the retention of duplicate genes.
06.sept 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 poorly understood process where cells execute consecutive S-phases without intervening mitoses, thus progressively increasing their ploidy. Oikopleura Dioica is known to shift from mitotic cycling to systemic endocycling during development. Additionally, its female gonad simultaneously supports meiotic progression of oocytes and endocycling of nurse nuclei within a single continuous cytoplasm. As such, Oikopleura Dioica presents an excellent modelsystem to study different types of cell cycles, their transitions and their co-occurrence. 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). As an initial survey to identify genes that might be implicated in endocycling, we have begun to identify known cell cycle regulatory gene variants in O. dioica and assess their developmental expression profile. So far, our results point towards central roles for Cyclin D variants in establishing systemic endocycling. Additionally, expression of various genes appears restricted to the female gonad (as compared to the male gonad). The role of several candidate genes will be pursued by overexpression and misexpression, as well as knock-down experiments complemented by (cross-)rescue experiments. To this end, we are trying to establish stable transgenesis using both endogenous and exogenous transposons.
30.august Holger Bielen
Technau Group
Two ancient brachyury paralogs in Hydra:
Identification of Interaction partners and structure function-analysis in Xenopus animal cap assays.

The T-box transcription factor Brachyury plays a crucial role in early mesoderm formation and convergent extension in all vertebrates. Its circumblastoporal expression is remarkably well conserved throughout the animal kingdom and likely to be involved in the regulation of gastrulation movements. In Hydra, the Brachyury homologues HyBra1 and HyBra2 are expressed very early during head formation in all developmental conditions and might have a role in axis elongation and head differentiation. This project focuses on the characterization of protein interacting partners of Brachyury in Hydra, which might be involved in the regulation of these important developmental processes. An extensive yeast 2-Hybrid screen and subsequent GST Pull Down assays have been performed to isolate and verify four putative HyBra1 interacting factors called GoBra1-4 (Gang of Brachyury). One of these factors, the cnidarian otx is like brachyury highly conserved from Hydra to Humans and represented by two paralogous genes in the Hydra genome. We have found evidence that the interactions of HyBra1/2 with CnOtx1/2 are conserved even in vertebrates. The Xenopus animal cap assay, was used to compare the activity of the Hydra Brachyury paralogues with the endogenous orthologue. Co injection of HyBra2 and cnotx into animal caps showed a clear synergistic effect, which suggests their role as co factors in an in vivo environment.

Functional analysis in Hydra is still a challenging task, since neither RNAi nor Morpholino approaches have yielded reproducible data. The most promising approach is the efficient generation of transgenic Hydra lines by embryo microinjection, which results in mosaic patches of expression of the gene of interest.
21.june Hiroshi Kikuta
Becker Group
Genomic regulatory blocks form the basis of synteny in vertebrate chromosome evolution.
Multiple syntenic blocks conserved across all vertebrates are larger than expected by chance compared to the overall synteny conservation. Genes in those syntenic blocks are often spanned by multiple highly conserved non-coding elements, several of which have been shown to act as long-range enhancers. By insertion into the zebrafish genome of a viral vector that can sense long-range enhancers, we find that cis-regulatory information can be available at a distance of 100kb or more from the target gene in a number of blocks of conserved synteny, often with unrelated genes between the enhancer and its target gene. Reporter insertions distal to developmental regulatory genes pax6a/b, rx3, id6, fgf8 and micro RNAs mir9-1 and mir9-5 mimic the expression pattern of these genes, rather than that of any other gene in the area, regardless of insertion position within the block. This suggests that cis-regulatory elements are distributed over large intervals up- or downstream of their target genes, and that cis- regulatory information for a single developmental gene dominates each syntenic block. Using the duplicated zebrafish pax6 loci as an example, we show that upon duplication of syntenic blocks, unrelated neighboring genes are lost from one copy by neutral evolution, while the underlying non coding sequence is conserved, suggesting that long- range regulatory sequences together with their target genes form genomic regulatory blocks (GRBs), which constrain the evolution of genome architecture and are at the basis of vertebrate synteny. We further show that heritable diseases have been mapped to these genomic regulatory blocks. Chromosomal breakpoints within the pax6 regulatory block outside of the transcriptional unit have been shown to cause aniridia. In the case of the FGF8 gene, we present evidence that human split hand/foot malformation 3 has possibly been misattributed to noncoding lesions in the unrelated downstream gene encoding dactylin: we show that this gene is part of the fgf8 genomic regulatory block. The data provide further support to the fragile breakage model of chromosome evolution.
14.june Xianghui Fu
Thompson Group
Cloning, expression, and possible function of microRNAs from Oikopleura dioica.
In the past few years, microRNAs (miRNAs) have emerged as a major class of regulatory genes that control many biological processes in development, differentiation, growth and metabolism. In animal kingdom, miRNAs have been identified in protostomes and veterbrates so far. Thus, miRNA information from other animal species, especially from tunicates that are regarded as the closest living relatives of vertebrates recently, will throw insights on miRNA functions, as well as their regulation. In Oikopleura doica, important proteins for miRNA biogenesis are found, indicating miRNA pathway is conserved in this species. By employing small RNA cloning strategy, 61 miRNAs are identified in Oikopleura dioica. Developmental expression analysis defines distinct miRNA classes with different developmental expression pattern. Interestingly, as to genomic location, about 50 % Od miRNAs are predicted in the antisense strand of protein-coding gene, whose expression, however, shows no relationship with its correspondingly miRNA expression. More surprisingly, Od miRNAs show very limited conservation with known miRNAs, whereas 24 and 20 miRNAs are identified from Ciona intestinalis and Ciona savignyi, respectively, by homology search. Furthermore, to elucidate the temporal and spatial expression, in situ hybridization (ISH) for a few miRNAs is performed. Finally, some putative miRNA targets are predicted by blast in EST and genome database. One possible target of miR-611, GCNF (germ cell nuclear factor), is further investigated. miR-611 is specifically expressed in D6 female and it seems to be a material miRNA. In embryogenesis, its expression is decreased as development proceeds. As the only member of nuclear receptor superfamily VI, GCNF plays a critical role in reproduction, neurogenesis, stem cell differentiation, and so on. Two GCNF genes are identified in O.dioica. 3’-UTR of both GCNF genes contain putative target sites for miR-611. The expression of GCNF is studied carefully by real-time PCR and RNA ISH, whereby the possible function of miR-611 on neurogenesis and reproduction is discussed.

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Gene colocalization and phosphorylated Pol II redistribution during development may contribute to achieving high-level gene expression in Oikopleura epithelium.
The eukaryotic nucleus is structurally and functionally compartmentalized and gene localization within the nucleus has an important effect on gene regulation. Oikopleura epithelium, a specialized single-layered organ with sustaining high-rate of house protein synthesis, provides an excellent model system to investigate the role of nuclear architecture in gene regulation. To validate the attractive hypothesis that functionally/physiologically related genes are co-regulated by nuclear colocalization, nuclear localization of five representative oikosin genes, e.g. oikosin1, oikosin2, oikosin3, oikosin5, and oikosin7, is defined. Interestingly, oikosin3 and oikosin5 genes with identical expression pattern, which are not linearly related in the genome, are colocalizated when they are actively expressed. In contrast, oikosin genes with different or partially overlapped spatial expression pattern display distinct nuclear localization. In addition, the whole oikopleura genome seems to be amplified during endoreplication, since no obvious gene-copy difference can be observed for distinct oikosin genes investigated here. Then the distribution of Pol II is determined. It shows that phosphorylated Pol II, but not unphosphorylated Pol II, is relocated during 12~14 hr, when metamorphosis happens. Finally, not all amplified oikosin gene copies are associated with phosphorylated Pol II and some amplified gene copies also display association with phophorylated Pol II in non-expressed region of epithelium. These results illuminate the developmentally dynamic of nuclear compartmentalization in epithelium. On one hand, limited phosphorylated Pol II is redistributed in the nucleus to form transcriptional factories. On the other hand, co-regulated genes, such as oikosin 3 and oikosin 5, are associated with the same transcriptional factory mediated by certain cis-element and possess the potentiation for transcription. Both developmentally dynamic processes may contribute to achieving high-level gene expression in epithelium.
07.june Jens Fritzenwanker
Technau Group
Investigation of the role of churchill in Nematostella vectensis.
My recent project is dealing with the over all question of how mesoderm arose during evolution. The development of mesoderm in vertebrates is controlled by the FGF pathway. Many genes of the FGF signalling cascade and many downstream targets, also those which are responsible for mesoderm induction in vertebrates, are already present in basal cnidarians like Nematostella. The gene churchill seems to be, next to the FGF signal, a second improtant factor in the regulation of mesoderm formation in vertebrates. Churchill seems to work like a switch between mesoderm formation and neural tissue formation by interacting with the FGF signalling cascade. Suprisingly the so thought vertebrate specific churchill gene is also present in Nematostella. If churchill and the FGF signalling cascade are already linked in Nematostella and which function they might have in terms of mesoderm formation is the main interest of my project.
31.May Anne-Mette Søviknes
Chourrout Group
Mapping the neurons of the Oikopleura central nervous system: A platform for future genetic manipulation studies.
Appendicularians are pelagic tunicates that remain free-swimming throughout life, in contrast sessile tunicates, witch are only free-swimming as larvae. Urochordates in general are receiving increasing focus in the context of vertebrate brain evolution, since they derive from the base of the chordate linage tree. In this project, “the mapping of the nervous system of Oikopleura dioica”, the development of the neurons in the major components of the nervous system is addressed. The development of the caudal nerve cord of the appendicularian Oikopleura dioica was assessed using differential interference contrast and confocal microscopy, phalloidin staining of actin, and in situ hybridization for the neuronal markers α-tubulin and choline acetyltransferase (ChAT). The caudal nerve cord first appears as a stream of α-tubulin mRNA-positive neurons that can be seen extending into the tail from the caudal ganglion as early as 4 hours after fertilization. Already at this stage a few actin-rich nerve fibers can be seen coursing longitudinally along the cord. As the tail grows in length, the caudal nerve cord extends, the longitudinal nerve fibers increase in number and become more fasciculated, and the neurons cluster at stereotyped longitudinal positions. The number of neurons reaches a relatively stable maximum of about 29 by 6 hours after fertilization. ChAT mRNA, a marker for motoneurons, is expressed in a subset of neurons in the caudal ganglion and caudal nerve cord in numbers consistent with an independent innervation of each segment of the tail musculature. The ChAT mRNA-positive neurons are detectable starting at 6 hours after fertilization in the caudal ganglion and a few hours later in the caudal nerve cord. Although the ChAT mRNA-positive neurons are not distributed segmentally along the caudal nerve cord, peripheral nerve fibers extend from the cord at roughly segmental intervals to the adjacent segmentally disposed muscle cells, coincident with the onset of ChAT mRNA expression. Recent results on FMRFamide immunostaining have given us indications that this is a neurotransmitter involved in the function of the nerve cord. Our results demonstrate that the nerve cord of O. dioica resembles the vertebrate spinal cord with respect to a longitudinally distributed pattern of motoneurons and a segmental organization of peripheral motor nerves to muscle. We have previously investigated regional patterns of neuronal differentiation by characterising the development of GABAergic and TH (tyrosine hydroxylase) mRNA positive neurons in the Oikopleura CNS. As a sum up of the results so far, a map of different neuron identities and their position during the CNS development will be presented. This map also help set the stage for gene expression studies aimed at understanding the evolution of developmental patterning in the different parts of the central nervous system. These results together with ongoing studies on birth dating of neurons in the major components of the CNS will make a platform for future genetic manipulation studies.
24.May Rita Angotzi
Chourrout Group
Cloning and evolution of Pitx genes with emphasis on fish pituitary development.
Gene duplication is considered as the main source of new genetic material leading to the evolution of new functions and consequent increase of biological complexity and diversity. Salmonids have undergone whole genome duplication some 30-100 million years ago and so far and represent an interesting model system to shed light on mechanisms of gene retention after recent duplication. Pitx genes encode homeodomain-containing transcription factor required for normal pituitary development in vertebrates; in the present study we have isolated Pitx genes and characterized their expression in both zebrafish and salmon. In zebrafish, we have isolated three Pitx genes, including Pitx1, the preservation of which was thus far uncertain. In salmon, six Pitx genes encoding proteins highly similar to their orthologous zebrafish counterparts Pitx1/2/3 were cloned; the highly conserved identity between the Pitx paralogs pairs indicates that they likely arose from lineage-specific genome duplication. In addition, two short splice variants of Pitx1-α and 1-ß respectively and one alternative 5’ UTR splice form of the Pitx2-α gene were identified. Comparative in situs hybridization studies on zebrafish and salmon pitx genes are being performed and current results indicate that (i) in contrast to zebrafish and mammals, both Pitx2 paralogs in Salmon, do not express in either the pituitary primordium or in the developed gland of embryos/adults; this suggests a degeneration of cis-regulatory elements for this gene in the salmon lineage (ii)in contrast to mammals, Pitx3 genes of fish are expressed in the pituitary gland (iii) in several cases, the developmental expression patterns of salmon Pitx duplicates complement each other to cover the expression of their zebrafish counterpart.

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Functional studies on Prop1 gene by morpholino knock-down approach.
Prop1 is a paired-like homeobox transcripton factor transiently expressed in the developing anterior pituitary of mouse embryos. This gene is essential for the expansion of the pituitary primordia and for the differentiation and function of hormone-producing cells. Deletion of Prop1 in mouse causes severe pituitary hypoplasia with failure of the entire Pit1 lineage. We have examined whether antisense morpholino oligonucleotides can suppress or "knockdown" the expression of Prop1 during development of the zebrafish pituitary. Effects on global pituitary morphology and Pit1 expression will be shown and discussed.
10.May Verena Hoppmann
Becker Group
The role of the GluR2 AMPA receptor subunit/Prickle as a modulator of retinal development in Zebrafish.
AMPA receptors (GluR1-4) are glutamate receptors that mediate the majority of fast synaptic transmission in the nervous system. They are composed of 4 subunits and form an ionchannel in the post-synaptic membrane. By integration of the GluR2 subunit into the receptor complex, the permeability of the receptor for Ca2+ is regulated. An editing site in this receptor subunit tunes this permeability. Receptor complexes comprising the edited GluR2 subunit are impermeable for Ca2+. The editing efficiency varies among species, different cell types and developmental stages. The unedited GluR2 subunit is mainly expressed during prenatal life. Extensive studies of the involvement of Ca2+ permeable AMPA receptors in neurodegenerative diseases have been performed and it turns out that downregulation of the GluR2 subunit or wrong editing are major reasons for neuronal cell death in several neurological insults. So far very little is known about the role of AMPA receptors and especially the editing of the GluR2 subunit during early development. Therefore we generated transgenic fish expressing fusion constructs of different types of the GluR2 subunit under the control of a heat shock inducible promoter. Overexpression of the unedited GluR2 subunit leads to severe damages. Those fish show a defect in motorneuron pathfinding, suffer from seizures and die before hatching. In a second part of this talk the role of the planar cell polarity gene prickle in the development of the zebrafish retina will be elucidated. Prickle is well known to be involved in convergence and extension movements during gastrulation and in planar cell polarity in drosophila omatida and bristle orientation on epidermal cells. Zebrafish has two copies of the prickle 1 gene. Both are expressed in the zebrafish retina during development but in different patterns, implying a subfunctionalization of those genes. Using an antisense morpholino approach, involvement of these prickle genes during patterning of the zebrafish retina were investigated.
03.May Grigory Genikhovich
Technau Group
The rise of cell types in Nematostella vectensis.
Cell proliferation, morphogenesis and differentiation are the key processes in multicellular organism development. In Nematostella, multiple cell types arise during planula development. In contrast to hydrozoans, with their interstitial stem cells system, there is no evidence of the presence of a multipotent stem cell, which would differentiate into gland, neuronal, stinging and other types of cells. In our attempt to clarify the question of presence or absence of the stem cell in Nematostella, we use genetic markers known to be expressed in the interstitial stem cell system of the freshwater Hydra: a Nematostella homologue of the Hydra Embryonic Ectoderm Development (the early embryonic marker of the interstitial cells) and a Zn finger protein-encoding gene, which we called Spotty. Full length coding sequences of these genes were obtained, and in situ hybridization with the Spotty probe showed expression in single cells in the ectoderm of the gastrula and planula larva and also in single endodermal cells of the planula. Expression data might reflect the fact that endodermal cells organize into a layer and start to differentiate later than the ectodermal cells.

Another important regulator of cell differentiation is a MADS box containing gene mef2. Isolation of the Nematostella mef2 homologue was reported earlier (Martindale et al., 2004). It is known, however, that mef2 can be alternatively spliced; and different splice variants can have different spatio-temporal expression patterns and be regulating different developmental processes. Our data suggest that this might also be the case with Nematostella mef2. We have proved that Nematostella mef2 is alternatively spliced and that different splice variants are differentially expressed in time during Nematostella embryonic and larval development. The role of particular N. vectensis mef2 variants remains to be elucidated.
05.April Holger Patzlaff
Technau Group
Functional analysis of brachyury, forkhead and snail during gastrulation of Nematostella vectensis.
We study the evolution of the mesoderm by investigating the role of “mesodermal” genes in the diploblast Nematostella vectensis. In particular we study their role during gastrulation, the time when the germ layers are separated. Three “mesodermal” genes, brachyury, forkhead and snail, were isolated previously and suggested to play an important role during the gastrulation in Nematostella (Fritzenwanker et al., 2004; Scholz and Technau, 2003), yet no functional data were available. To this end, we carried out knock-down experiments, in which gene-specific morpholinos were microinjected in zygotes of Nematostella. The specificity and efficiency of the morpholinos was beforehand tested in TnT in vitro assays. The knock down of snailA leads to arrest or delay in the formation of the preendodermal plate and the ingression of preendodermal cells. This implies a role for snailA in the regulation of epithelial-mesenchymal transition (EMT) during gastrulation. This effect could be rescued by coinjection of wt constructs with silent mutations in the Morpholino binding site. Conversely, the knock-down of brachyury and forkhead, suggested that these genes play a role in controlling the morphogenetic movements of the blastopore lip, i.e. of an epithelium, during gastrulation. These gene functions are reminiscent to those of bilaterian homologs, suggesting at least partially conserved molecular networks and gene functions between the Cnidaria and the Bilateria.
29.March Yoshimasa Sagane
Thompson Group
The Appendicularian house: cytoskeleton-mediated architecture of cellulose microfibrils secreted from animal cells
Controlled orientation of cellulose microfibril deposition into the cell wall is critical to pattern formation in plant development; and long-standing, but much debated, models suggest that cellular microtubules play a key role by constraining the movement of cellulose synthase complexes in the plasma membrane. Though widespread in the plant kingdom, urochordates are the only animals capable of cellulose synthesis. We have characterized the distinctive cellulose microfibril architecture of the complex filter-feeding house of the appendicularian, Oikopleura dioica. Analysis of the O. dioica cellulose synthase gene was consistent with the hypothesis of a single gene transfer event from a prokaryote in the common ancestor of the urochordate lineage. We then examined cellular mechanisms for templating extracellular cellulose microfibrils in animal cells compared to current models in phylogenetically distant plants. Through targeted disruption of cytoskeletal elements, protein secretory pathways, and plasma membrane organization we elaborate a working model that shows some convergence but also significant differences with plant models. A specialized cortical F-actin filament array is implicated in templating cellulose microfibril orientation and glycosylphosphatidylinositol-anchored proteins in mobile lipid rafts may act as scaffolding proteins in microfibril elongation. Microtubules appear to be involved in delivery and maintenance of cellulose synthase complexes to specific sites on the cell membrane rather than in orienting movement of these complexes through the membrane. We are now attempting to isolate the plasma membrane protein(s) involved in guiding and scaffolding cellulose fibres during their elongation across epithelial cells and to determine the interactions of house structural proteins (oikosins) with these cellulose microfibrils in the extracellular space.
22.March Jean-Marie Bouquet
Appendecularia Facility
Chourrout Group
Partial automation of the culture technique of the Appendicularian Oikopleura dioica
Within the framework of development of the Appendicularian Oikopleura dioica (Larvacean, Fol, 1872) as a new research organism for genetic and evolution studies, and in parallel of the finalisation of techniques such as sperm cryopreservation and microinjection, the optimisation of our culture conditions have been the object of a permanent effort. The culture principle is simple, but the maintenance of synchronous populations of this delicate gelatinous planktonic organism require daily care, and is highly time consuming and manpower demanding (manual dilution and manual transfer). With our facility scale and staff, we produce yearly, keeping commonly 4 different populations, about 2500 matures per week, and can reach sporadically 5000-6000. The stability and quality of the production has progressed a lot, the last few years. But it was still imperative, in the optical of maintenance of multiple transgenic lines, in parallel of our regular production level, to drastically reduce the manual transferring steps of the culture technique. This is indispensable for the future of this model system here but also to be able to export it toward other institutes. We have now managed to partially automate the classic culture method. If the principle is still the same, 2 out of 3 manual transfer effected during Oikopleura dioica's 6 days life circle, can now be done automatically. We have shown after optimisation of the prototype, that this system is reliable, reproducible and that there is no significant difference using it, in term of animals density per beaker and fecundity, compare to our actual culture standard (100-110 animals per 6L beaker, 300-350 eggs per females).
15.March Pavla Navratilova
Becker Group
Identification of/ cis-/regulatory Regions in Zebrafish Genome
The enhancer discovery was started in intronic and flanking regions of the genes which are represented by YFP expressing enhancer trap zebrafish lines produced in our lab in last years. Examples of previously described genes found to be near insertion sites of the enhancer trap construct are pax6, sox11b or sox3. The research design is very straightforward with use of novel Tol2 transposon system. After the /in silico/ analysis the enhancer sequence is PCR amplified and cloned into the vector posessing the minimal promoter in front of the EGFP reporter gene. This construct is introduced into the zebrafish genome with use of Tol2 transposon system. The basic expression domains driven by enhancer can be observed in F0, but reproducible and ultimate proof will come from the resultant transgenic fish in the F1 generation. The advantage is that the system tests the enhancer in a genomic context and it's activity is possible to follow through embryo development.
08.March Fabian Rentzsch
Technau Group
BMP antagonists in Nematostella: implications for the evolution of axial patterning
The evolutionary origin of the anterior-posterior and the dorso-ventral body axes of Bilateria is under debate. At present it is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorso-ventral body axis of Bilateria. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin. Surprisingly, NvGDF5-like also had a strong dorsalizing activity in zebrafish suggesting that Gremlin, Chordin and GDF5-like proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Surprisingly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, wheras NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists do only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.

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