Chourrout Group - Molecular Genetics of Protochordates

We are interested in how macroevolution proceeds to create the diversity of large animal taxons. This question cannot be readily addressed in most taxons through a genetic approach, but it may receive partial answers through the comparison of annotated genome sequences. Candidate determinants of developmental innovations can be elected for functional studies within individual model organisms under comparisons.

Our focus is placed on the chordate phylum, in which coexist extremely simple and complex organisms. We have contributed to establish Oikopleura dioica as a model system. Oikopleura is a pelagic tunicate with very short life cycle (4-5 days at 20°C), and it is cultivable in the lab for many successive generations. It is highly fecund and transparent at both the embryo and adult stages. For these reasons, Oikopleura offers good opportunities for genetic and embryological studies. We found that Oikopleura has a very compact genome, smaller than the genome of any other bilaterian animals only 65-70 megabases long, with around 15,000 genes, a typical gene number for invertebrates. We have been interested in several features related to the extreme genome compaction, such as the very short introns and intergenic sequences, which culminate with the polycistronic transcription of numerous genes. The compaction appears as a lineage-specific secondary event, as suggested by the absence of most entire clades of ancient retrotransposons in the Oikopleura genome. We keep strong interest in elucidating the forces driving genome compaction.

Dense packing of exons (red rectangles) in the Oikopleura genome. The region with a yellow frame represents one of numerous operons.
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However, the very small genome size in itself has also represented a chance to obtain the full genome sequence at high coverage, with direct contributions from two genome centers (Genoscope and Max-Planck Institute for Molecular Genetics). The annotation is still in a preliminary phase, but the genome sequence has already revealed information of considerable interest for the evolution of tunicates and other chordates.

For example, Oikopleura gene sequences have been crucial in a recent study conceived by Herve Philippe (Montreal), which ultimately transformed our view of the chordate phylogeny. Using appropriate methods and resources, we have learned that tunicates but not cephalochordates are the closest living relatives of vertebrates. A general implication of this exciting finding is that the ancestor of chordates may be more complex than often thought, and that tunicates may have been simplified from this ancestor. An important line of our data on developmental genes of Oikopleura indeed supports this view fairly well.

New phylogeny of chordates, where tunicates is the sister group of vertebrates
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First, the homeobox gene complement of Oikopleura shows a loss of at least 26 gene groups compared to other bilaterians such as Drosophila or human, despite a relatively "standard" total number of individual homeobox genes. The overall homeobox gene diversity has been severely amputated, but approximately 15 of the remaining gene groups have been amplified at an unusual level. Preliminary data suggest their contribution in patterning the oikoplastic epithelium which is in charge of the house production after metamorphosis. We increase our efforts on understanding the genetic mechanisms underlying the emergence of this function, through the study of various transcription factors and signalling pathways.

Second, among the homeobox genes, we have completed a full study of the Hox gene complement which showed rapid evolution in tunicates including a total disintegration of the Hox cluster in Oikopleura. We found that the expression patterns of Oikopleura Hox genes are markedly tissue specific in four tissues of the tail, but keep a certain level of spatial colinearity. This is not entirely suprising, as individual Hox transgenes in the mouse essentially show expression similar to those of endogenous Hox genes from the Hox clusters. The Hox cluster breakdown may have more to do with the absence of temporal colinearity as well as cell lineage-dependent expression of developmental genes in general. We are devoting strong efforts to develop methods of gene manipulation in Oikopleura, so to be able to understand the function of its Hox genes and other developmental determinants.

Expression patterns of Oikopleura Hox genes at 4 hours post-fertilisation
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Research on other animals

We have been strongly involved into recent research on the homeobox gene complement of cnidarians, an outgroup of bilaterians, because they must give insight into the old history of the Hox and ParaHox gene complement. This work is resulting from a collaboration with Ulrich Technau's group at the Sars Centre, and the major input from Frederic Delsuc (CNRS Montpellier).We have taken benefit of the genome data generated for the sea anemone Nematostella and the freshwater polyp Hydra, and performed a set of experiments including physical mapping, expression studies and elaborate phylogenetic analysis. We indeed produce a novel scenario for the history of both clusters, with a ProtoHox cluster comprising only two anterior genes, and considerable divergence of cnidarians and bilaterians after their split.

New scenario of Hox/ParaHox cluster genesis inferred from the comparison of bilaterian and cnidarian gene complements.
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We are doing significant research on the development of the Atlantic salmon, a very important commercial fish species. We have been identifying all Hox clusters born through a recent lineage specific duplication and are currently looking at the divergence of the newborn clusters after duplication, both at the structural and functional levels. We finally have important work on salmon genes involved in the development of the pituitary gland, including the Pitx family and the Prop gene, including phylogenetic analysis, expression studies and manipulation of the gene function. This latter aspect involves zebrafish as a model system.

More details in the following publications:

• Chourrout D, Delsuc F, Chourrout P, Edvardsen RB, Rentzsch F, Renfer E, Jensen MF, Zhu B, de Jong P, Steeele RE, Technau U (2006) Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements. Nature, in press.
• Delsuc F, Brinkmann H, Chourrout D, Philippe H (2006) Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature,439, 965-968.
• Edvardsen RB, Seo HC, Jensen MF, Mialon A, Mikhaleva J, Bjordal M, Cartry J, Reinhardt R, Weissenbach J, Wincker P, Chourrout D (2005) Remodelling of the homeobox gene complement in the tunicate Oikopleura dioica. Curr. Biol., 15, R12-13.
• Søviknes AM, Chourrout D, Glover JC (2005) Development of putative GABAergic neurons in the appendicularian urochordate Oikopleura dioica. J Comp Neurol., 490,12-28.
• Weill M, Philips A, Chourrout D, Fort P (2005) The caspase family in urochordates : distinct evolutionary fates in ascidians and larvaceans. Biol Cell., 97, 857-866.
• Seo HC, Edvardsen RB, Maeland AD, Bjordal M, Jensen MF, Hansen A, Flaat M, Weissenbach J, Lehrach H, Wincker P, Reinhardt R, Chourrout D (2004) Hox cluster disintegration with persistent anteroposterior order of expression in Oikopleura dioica. Nature, 431, 67-71.
• Edvardsen RB, Lerat E, Maeland AD, Flaat M, Tewari R, Jensen MF, Lehrach H, Reinhardt R, Seo HC, Chourrout D (2004). Hypervariable and Highly Divergent Intron/Exon Organizations in The Chordate Oikopleura dioica. J Mol Evol. 59, 448-457.
• Ganot P, Kallesoe T, Reinhardt R, Chourrout D, Thompson EM (2004). Spliced-leader RNA trans splicing in a chordate, Oikopleura dioica, with a compact genome. Mol Cell Biol., 24, 7795-7805.
• Volff JN, Lehrach H, Reinhardt R, Chourrout D (2004). Retroelement dynamics and a novel type of chordate retrovirus-like elements in the miniature genome of the tunicate Oikopleura dioica. Mol Biol Evol., 21, 2022-2033.
• Nilsen IW, Myrnes B, Edvardsen RB, Chourrout D (2003). Urochordates carry multiple genes for goose-type lysozyme and no genes for chicken- or invertebrate-type lysozymes. Cell Mol Life Sci, 60:2210-2218.
• Seo HC, Kube M, Edvardsen RB, Jensen MF, Beck A, Spriet E, Gorsky G, Thompson EM, Lehrach H, Reinhardt R, Chourrout D (2001) Miniature genome in the marine chordate Oikopleura dioica. Science, 294:2506.

Collaborators

The group is collaborating on other projects through specific collaborations and external funding by the Research Council of Norway:

• Patrick Wincker, Jean Weissenbach (Genoscope, Evry, France)
• Richard Reinhardt, Hans Lehrach (Max-Planck Institute MPIMG, Berlin)
• Jean-Nicolas Volff (University of Wuerzburg, Germany)
• Herve Philippe (University of Montreal, Canada)
• Frederic Delsuc (CNRS, Montpellier, France)
• Giacomo Cavalli (CNRS, Montpellier, France)
• Philippe Fort (University of Montpellier, France)
• Eric Thompson (Sars Centre)
• Ulrich Technau (Sars Centre)
• Joel Glover (University of Oslo & Sars Centre, Norway)
• Sigurd Stefansson (University of Bergen, Norway)

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