Chourrout Group - Molecular Genetics of Protochordates

Chourrout Group December 2014

Note: This text aims at remaining very general and does not contain literature citations

We are interested in how macroevolution proceeds to create the diversity of large animal taxons. 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(Figure 1). Oikopleura is a pelagic tunicate with very short life cycle (6 days at 15°C), and it is bred in the lab for many successive generations. It is highly fecund and transparent at both the embryo and adult stages. Gene expression can be knocked down and knocked out. For these reasons, Oikopleura offers good opportunities for genetic and embryological studies.

Fig. 1 Oikopleura dioica is the only larvacean with separated sexes (A). It can be collected virtually anywhere in the
ocean (B) but it can be bred in captivity for hundreds of generations (C). Its development and life cycle last for several
hours (D) and several days, respectively. Recently, methods of gene expression knock down were established
(Omotezako et al., 2013 and our group: Mikhaleva et al. 2015). (E left: control larvae expressing a homeodomain
transcription factor; E right: larvae after injection of dsRNA; F: after injection of dsRNA from the Brachyury
gene, a majority of larvae have a truncated tail as expected for a loss of Brachyury function).

Oikopleura has an extremely compact genome, whose relatively thorough analysis has revealed divergent genome architecture and an important turn over of developmental genes (Figure 2). We have been interested in several features related to 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. We keep strong interest in the mechanisms driving an unusually rapid evolution, and genome changes can be historically traced using several other larvacean genomes (Figure 3). We found original indications in the Oikopleura genome that support two previously poorly documented mechanisms of intron gain.

Fig. 2 Due to unusually rapid evolution, the genome architecture in Oikopleura dioica is highly divergent from
those of other chordates. The genome is very compact (70 Mb) and distributed over 2 autosomes and two
well differentiated sex chromosomes (left).The result of a considerable number of chromosomal rearrangements
is the total loss of Hox gene clustering (left) and an almost total loss of old syntenic associations (right).

Fig. 3 Oikopleura dioica is probably the most cosmopolitan larvacean and well adapted to
laboratory conditions. We also sequenced and begun the analysis of genomes from
a variety of other larvaceans, including other populations of O. dioica.

Our second and increasing interest is the evolution of tunicate late development. The Oikopleura genome sequence has been an essential piece of information for establishing the new phylogeny of chordates (Figure 4). Tunicates form the sister group of vertebrates, in contrast to older views, and most likely resulted from an anatomic simplification of ancestral chordates. Such simplification occurred multiples times during animal evolution and we are willing to understand how it proceeded at the molecular level, by focusing on conserved key developmental genes and their pathways. The Oikopleura Hox genes are the main focus for this part. Nine of them were found in the genome at nine distinct locations and their expression patterns suggest that the function of several of them has little to do with a role in AP axis patterning (Figure 5).

Fig. 4 The genome sequence of Oikopleura dioica has contributed to revisit phylogenetic relationships
among chordates, and to propose that tunicates are the sister group of vertebrates (left, full lines).
As cephalochordates and vertebrates grossly resemble each other and are anatomically more complex
than tunicates, a current view is that tunicates are the outcome of a simplification process. Phylogenetic
trees indicate comparatively rapid evolution in tunicates and particularly in larvaceans (right).

Fig. 5 A number of conserved developmental genes are studied to study the evolution of larvaceans
since they split from common ancestors with other groups. The expression of several Hox genes in
O. dioica (left) is markedly tissue- (or even cell-) specific, suggesting that their role has evolved
towards cell specification. The transparency of O. dioica embryos allowed to build with 4D
microscopy a fate map (right) which is similar to those of ascidians, with an even earlier
restriction of cell fate. Cell lineage is totally invariantamong three distinct embryos.

In parallel to development simplification, the evolution of tunicates has been accompanied by a number of innovations. The most spectacular one for larvaceans, that is definitely lineage specific, is the house, a very sophisticated extracellular structure used by the animals as a filter feeding apparatus. It is repeatedly synthesized (and replaced during the life cycle) by most epithelial cells of the trunk, that are organized in territories that are morphologically and functionally specialized. Our current activity is to identify transcription factors and pathways that are involved in the formation of this extraordinary cell layer. We already identified several candidate transcription factors and showed for two of them their direct involvement (Figure 6).

Fig. 6 Oikopleura dioica adult (yellowish color) in its house (A), the complex extracellular structure
made by larvaceans for filter feeding purpose (house compartments drawn in B). Most of the
trunk epithelium (= oikoplastic epithelium) synthesizes the house components that are cellulose
and a rich variety of glycoproteins named oikosins. This epithelium is organized in territories
of polyploid cells that can be recognized individually (C), because they grow through endocycles
in the absence of mitotic divisions. Each territory produces its own set of oikosins. Our group
is studying the development of the oikoplastic epithelium, which involves an early, strong
and highly regionalized expression of multiple homeodomain transcription factors (D).


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