Etude fonctionnelle de l'induction neurale chez le céphalochordé Branchiostoma lanceolatum

Neural induction is the process through which embryonic ectodermal cells become neural. Many studies on the mechanisms controlling this process have been made, but because of its complexity, many questions remain unanswered. In this thesis, I have focused my interest on the study of neural induction in an evolutionary context studying this process in the cephalochordate amphioxus, one of the closest relatives of vertebrates. I have highlighted that amphioxus, as vertebrates, possesses an organizer. I have demonstrated a conservation of the role of BMP and FGF signals in the induction of the epidermis and the regionalization of neural tissue respectively. However, in contrast to vertebrates, FGF signal does not appear to be a major player in neural induction. Instead, an important role of Activin/Nodal signaling pathway has been demonstrated. These results support, first, the conservation of several aspects of this mechanism in all chordates, and second, they suggest the involvement of the Activin/Nodal signaling in this process, something previously unknown in vertebrates. The phylogenetic position of amphioxus and the overall conservation of this process between cephalochordates and vertebrates allow us to suggest that the ancestor of chordates formed its neural tissue through mechanisms highlighted in this study. These results also allow us to propose new studies in vertebrates for establishing a putative role of the Activin/Nodal signaling during this process, a role previously completely unknown.L’induction neurale est le processus au travers duquel les cellules ectodermiques de l’embryon deviennent neurales. De nombreuses études sur les mécanismes contrôlant ce processus on été réalisées mais du fait de sa complexité, de nombreuses questions restent sans réponse. Au cours de ce travail de thèse, je me suis intéressé à l’étude de l’induction neurale sous une perspective évolutive en étudiant ce processus chez le céphalocordé amphioxus, l’un des plus proches parents des vertébrés. J’ai pu mettre en évidence que, comme les vertébrés, l’amphioxus possède un organisateur. J’ai également confirmé une conservation du rôle des voies de signalisation BMP et FGF respectivement dans l’induction de l’épiderme et la régionalisation du tissu neural. Cependant, au contraire des vertébrés, le signal FGF ne semble pas être un acteur prépondérant de l’induction neurale. Au contraire, un rôle important de la voie de signalisation Activine/Nodal a été mis en évidence.Les résultats obtenus soutiennent d’une part la conservation de certains aspects de ce mécanisme chez tous les chordés, et suggèrent d’autre part l’implication de certains acteurs comme la voie Activine/Nodal jusque là inconnue chez les vertébrés. La position phylogénétique de l’amphioxus et la conservation globale de ce processus entre les céphalochordés et les vertébrés nous permettent de suggérer que l’ancêtre des chordés formait du tissue neurale au travers des mécanismes mis en évidence dans cet étude. Ces résultats nous permettent également de proposer de nouvelles études chez les vertébrés visant à établir un rôle putatif de la voie Activine/Nodal au cours de ce processus, rôle jusque la complètement inconnu

Developmental cell-cell communication pathways in the cephalochordate amphioxus: actors and functions

The laboratory of H.E. was supported by the CNRS and the ANR16-CE12-0008-01 and S.B. by the Institut Universitaire de France. The laboratory of I.M.L.S. is currently supported by Wellcome Trust ISSF grant 204821/Z/16/ZDuring embryonic development, cells of metazoan embryos need to communicate in order to construct the correct bodyplan. To do so, they use several signals that usually act through interactions between ligands and receptors. Interestingly, only a few pathways are known to be fundamental during animal development, and they are usually found in all the major metazoan clades, raising the following question: how have evolution of the actors and of the functions of these pathways participated in the appearance of the current diversity of animal morphologies? The chordate lineage comprises vertebrates, their sister group the urochordates, and the cephalochordates (i.e. amphioxus). Urochordates are quite derived relative to the chordate ancestor, whereas cephalochordates and vertebrates share many morphological traits. Thus, comparing embryonic development between vertebrates and cephalochordates should give us some insight into the ancestral characters present in chordates and into the morphological evolution in this clade. However, while much is known about the function of different signalling pathways in vertebrates, data are still scarce in the literature for cephalochordates. In this review, we summarize the current state of the field concerning the expression of actors and the function of the major cell-cell communication pathways, including Hedgehog (Hh), Notch, Nuclear Receptor (NR), Receptor Tyrosine Kinase (RTK), Transforming Growth Factor-β (TGF-β) and Wingless/Int (Wnt), in amphioxus.Publisher PDFPeer reviewe

Sequencing and Analysis of the Mediterranean Amphioxus (Branchiostoma lanceolatum) Transcriptome

BACKGROUND: The basally divergent phylogenetic position of amphioxus (Cephalochordata), as well as its conserved morphology, development and genetics, make it the best proxy for the chordate ancestor. Particularly, studies using the amphioxus model help our understanding of vertebrate evolution and development. Thus, interest for the amphioxus model led to the characterization of both the transcriptome and complete genome sequence of the American species, Branchiostoma floridae. However, recent technical improvements allowing induction of spawning in the laboratory during the breeding season on a daily basis with the Mediterranean species Branchiostoma lanceolatum have encouraged European Evo-Devo researchers to adopt this species as a model even though no genomic or transcriptomic data have been available. To fill this need we used the pyrosequencing method to characterize the B. lanceolatum transcriptome and then compared our results with the published transcriptome of B. floridae. RESULTS: Starting with total RNA from nine different developmental stages of B. lanceolatum, a normalized cDNA library was constructed and sequenced on Roche GS FLX (Titanium mode). Around 1.4 million of reads were produced and assembled into 70,530 contigs (average length of 490 bp). Overall 37% of the assembled sequences were annotated by BlastX and their Gene Ontology terms were determined. These results were then compared to genomic and transcriptomic data of B. floridae to assess similarities and specificities of each species. CONCLUSION: We obtained a high-quality amphioxus (B. lanceolatum) reference transcriptome using a high throughput sequencing approach. We found that 83% of the predicted genes in the B. floridae complete genome sequence are also found in the B. lanceolatum transcriptome, while only 41% were found in the B. floridae transcriptome obtained with traditional Sanger based sequencing. Therefore, given the high degree of sequence conservation between different amphioxus species, this set of ESTs may now be used as the reference transcriptome for the Branchiostoma genus

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations

Functional study of neutral induction in the cephalochordate Branchiostoma lanceolatum

L’induction neurale est le processus au travers duquel les cellules ectodermiques de l’embryon deviennent neurales. De nombreuses études sur les mécanismes contrôlant ce processus on été réalisées mais du fait de sa complexité, de nombreuses questions restent sans réponse. Au cours de ce travail de thèse, je me suis intéressé à l’étude de l’induction neurale sous une perspective évolutive en étudiant ce processus chez le céphalocordé amphioxus, l’un des plus proches parents des vertébrés. J’ai pu mettre en évidence que, comme les vertébrés, l’amphioxus possède un organisateur. J’ai également confirmé une conservation du rôle des voies de signalisation BMP et FGF respectivement dans l’induction de l’épiderme et la régionalisation du tissu neural. Cependant, au contraire des vertébrés, le signal FGF ne semble pas être un acteur prépondérant de l’induction neurale. Au contraire, un rôle important de la voie de signalisation Activine/Nodal a été mis en évidence.Les résultats obtenus soutiennent d’une part la conservation de certains aspects de ce mécanisme chez tous les chordés, et suggèrent d’autre part l’implication de certains acteurs comme la voie Activine/Nodal jusque là inconnue chez les vertébrés. La position phylogénétique de l’amphioxus et la conservation globale de ce processus entre les céphalochordés et les vertébrés nous permettent de suggérer que l’ancêtre des chordés formait du tissue neurale au travers des mécanismes mis en évidence dans cet étude. Ces résultats nous permettent également de proposer de nouvelles études chez les vertébrés visant à établir un rôle putatif de la voie Activine/Nodal au cours de ce processus, rôle jusque la complètement inconnu.Neural induction is the process through which embryonic ectodermal cells become neural. Many studies on the mechanisms controlling this process have been made, but because of its complexity, many questions remain unanswered. In this thesis, I have focused my interest on the study of neural induction in an evolutionary context studying this process in the cephalochordate amphioxus, one of the closest relatives of vertebrates. I have highlighted that amphioxus, as vertebrates, possesses an organizer. I have demonstrated a conservation of the role of BMP and FGF signals in the induction of the epidermis and the regionalization of neural tissue respectively. However, in contrast to vertebrates, FGF signal does not appear to be a major player in neural induction. Instead, an important role of Activin/Nodal signaling pathway has been demonstrated. These results support, first, the conservation of several aspects of this mechanism in all chordates, and second, they suggest the involvement of the Activin/Nodal signaling in this process, something previously unknown in vertebrates. The phylogenetic position of amphioxus and the overall conservation of this process between cephalochordates and vertebrates allow us to suggest that the ancestor of chordates formed its neural tissue through mechanisms highlighted in this study. These results also allow us to propose new studies in vertebrates for establishing a putative role of the Activin/Nodal signaling during this process, a role previously completely unknown

Sequence and assembly statistics.

<p>Sequence and assembly statistics.</p

Size distribution of annotated (blue) and non-annotated (red) contigs.

<p>Non-annotated contigs are highly present within the shorter contigs (less than 500 bp).</p

Biological Process.

<p>Gene Ontology (GO) assignment of the <i>Branchiostoma lanceolatum</i> transcriptome for the Biological Process category (total of 54,218 terms).</p

Molecular Function.

<p>Gene Ontology (GO) assignment of the <i>B. lanceolatum</i> transcriptome for the Molecular Function category (total of 85,213 terms).</p