Coordinated spatial and temporal expression of Hox genes during embryogenesis in the acoel Convolutriloba longifissura

Background: Hox genes are critical for patterning the bilaterian anterior-posterior axis. The evolution of their clustered genomic arrangement and ancestral function has been debated since their discovery. As acoels appear to represent the sister group to the remaining Bilateria (Nephrozoa), investigating Hox gene expression will provide an insight into the ancestral features of the Hox genes in metazoan evolution. Results: We describe the expression of anterior, central and posterior class Hox genes and the ParaHox ortholog Cdx in the acoel Convolutriloba longifissura. Expression of all three Hox genes begins contemporaneously after gastrulation and then resolves into staggered domains along the anterior-posterior axis, suggesting that the spatial coordination of Hox gene expression was present in the bilaterian ancestor. After early surface ectodermal expression, the anterior and central class genes are expressed in small domains of putative neural precursor cells co-expressing ClSoxB1, suggesting an evolutionary early function of Hox genes in patterning parts of the nervous system. In contrast, the expression of the posterior Hox gene is found in all three germ layers in a much broader posterior region of the embryo. Conclusion: Our results suggest that the ancestral set of Hox genes was involved in the anteriorposterior patterning of the nervous system of the last common bilaterian ancestor and were later co-opted for patterning in diverse tissues in the bilaterian radiation. The lack of temporal colinearity of Hox expression in acoels may be due to a loss of genomic clustering in this clade or, alternatively, temporal colinearity may have arisen in conjunction with the expansion of the Hox cluster in the Nephrozoa

Unravelling body plan and axial evolution in the Bilateria with molecular phylogenetic markers

SETTING THE PROBLEM The emergence of dramatic morphological differences (disparity) and the ensuing bewildering increase in the number of species (diversity) documented in the fossil record at key stages of animal and plant evolution have defied, and still defy, the explanatory powers of Darwin’s theory of evolution by natural selection. Among the best examples that have captured the imagination of the layman and the interest of scores of scientists for 150 years are the origins of land plants from aquatic green plants, of flowering plants from seed plants, of chordates from non-chordates and of tetrapod vertebrates from non-tetrapods; and the conquest of the land by amphibians; the emergence of endotherms from ectotherm animals; the recurrent invention of flight (e.g. in arthropods, birds and mammals) from non-flying ancestors; and the origin of aquatic mammals from four-legged terrestrial ancestors. Key morphological transitions pose a basic difficulty: reconstruction of ancestral traits of derived clades is problematic because of a lack of transitional forms in the fossil record and obscure homologies between ‘ancestral’ and derived groups. Lack of transitional forms, in other words gaps in the fossil record, brought into question one of the basic tenets of Darwin’s theory, namely gradualism, as Darwin himself acknowledged. Since Darwin, however, and especially in the past 50 years, numerous examples that may reflect transitional stages between major groups of organisms have accumulated

The nervous system of Xenacoelomorpha: a genomic perspective

Xenacoelomorpha is, most probably, a monophyletic group that includes three clades: Acoela, Nemertodermatida and Xenoturbellida. The group still has contentious phylogenetic affinities; though most authors place it as the sister group of the remaining bilaterians, some would include it as a fourth phylum within the Deuterostomia. Over the past few years, our group, along with others, has undertaken a systematic study of the microscopic anatomy of these worms; our main aim is to understand the structure and development of the nervous system. This research plan has been aided by the use of molecular/developmental tools, the most important of which has been the sequencing of the complete genomes and transcriptomes of different members of the three clades. The data obtained has been used to analyse the evolutionary history of gene families and to study their expression patterns during development, in both space and time. A major focus of our research is the origin of 'cephalized' (centralized) nervous systems. How complex brains are assembled from simpler neuronal arrays has been a matter of intense debate for at least 100 years. We are now tackling this issue using Xenacoelomorpha models. These represent an ideal system for this work because the members of the three clades have nervous systems with different degrees of cephalization; from the relatively simple sub-epithelial net of Xenoturbella to the compact brain of acoels. How this process of 'progressive' cephalization is reflected in the genomes or transcriptomes of these three groups of animals is the subject of this paper

The evolution and regulation of the chordate ParaHox cluster

The ParaHox cluster is the evolutionary sister of the Hox cluster. Like the Hox cluster, the ParaHox cluster is subject to complex regulatory phenomena such as collinearity. Despite the breakup of the ParaHox cluster within many animals, intact and collinear clusters have now been discovered within the chordate phyla in amphioxus and the vertebrates, and more recently within the hemichordates and echinoderms. The archetypal ParaHox cluster of amphioxus places it in a unique position in which to examine the regulatory mechanisms controlling ParaHox gene expression within the last common ancestor of chordates, and perhaps even the wider Deuterostomia. In this thesis, the genomic and regulatory landscape of the amphioxus ParaHox cluster is characterised in detail. New genomic and transcriptomic resources are used to better characterise the B.floridae ParaHox cluster and surrounding genomic region, and conserved non-coding regions and regulatory motifs are identified across the ParaHox cluster of three species of amphioxus. In conjunction with this, the impact of retrotransposition upon the ParaHox cluster is examined and analyses of transposable elements and the AmphiSCP1 retrogene reveal that the ParaHox cluster may be more insulated from outside influence than previously thought. Finally, the detailed analyses of a regulatory element upstream of AmphiGsx reveals conserved mechanisms regulating Gsx CNS expression within the chordates, and TCF/Lef is likely a direct regulator of AmphiGsx within the CNS. The work in this thesis makes use of new genomic and transcriptomic resources available for amphioxus to better characterise the genomic and regulatory landscape of the amphioxus ParaHox cluster, serving as a basis for the improved identification and characterisation of functional regulatory elements and conserved regulatory mechanisms. This work also highlights the potential of Ciona intestinalis as a ‘living test tube’ to allow the detailed characterisation of amphioxus ParaHox regulatory elements

Unravelling the Origins and Evolution of the Animal Kingdom using Genomics

There are ~35 classified phyla/sub-phyla within the Animal Kingdom; some of which haveunresolved relationships. The advent of genomics has made it possible to study new aspects of animalevolution, including comparative genomics (e.g., gene loss/gain, non-coding regions, synteny, etc),gene family evolution, and their evolutionary relationships using genome-wide data. No study to date has compared all the wealth of genomic data available to understand theevolution of the Animal Kingdom. Using a core bioinformatics pipeline and dataset to infer HomologyGroups (HGs), the losses and novelties of these HGs were proven integral to the diversification of theanimal kingdom. The same core pipeline was used to extract homeobox gene HGs, a key family usedto understand origin and diversification in animals. Gene trees were inferred from the core datasetHGs to determine the evolution of a gene family iconic in the study of animal body plans. Conservedanimal genes were also mined using the same pipeline and dataset. Animal phylogenomics is one ofthe most controversial areas in modern evolutionary science. Whilst many new methods have beendeveloped, no study to date has tried to assess the impact of gene age in the reconstruction ofevolutionary trees. The phyla with the largest count of HG losses also had the highest counts of HG novelties. Notall of these were strictly de novo, but the numbers suggest a re-manufacturing of the genetic materialfrom the genes reduced to those that were more recently diverged. A comprehensive classification of all the diversity of animal homeobox genes is lacking. Thegene trees showed complex patterns, with similar homeobox expansions between more distant species,and interlapping homeobox families. The highly conserved HGs recovered, for the animal phylogenies, well-established relationshipsbetween some phyla using maximum likelihood and Bayesian inference methods. Ctenophora wasconsistently recovered as sister to all other animals, and interesting relationships between ecdysozoansand lophotrochozoans. However, it was proven that it takes more than a highly conserved set of genesto infer a stable and correct phylogeny. Each of the additional methods used to extend the core bioinformatics pipeline revealed apattern of correlation, particularly among the fast evolving species, such as platyhelminthes, nematodes and tardigrades. These HG losers, and gainers also had lineage specific homeobox clades,and caused artefactual problems in the phylogenies

Computational analysis of transcriptional regulation in metazoans

This HDR thesis presents my work on transcriptional regulation in metazoans (animals). As a computational biologist, my research activities cover both the development of new bioinformatics tools, and contributions to a better understanding of biological questions. The first part focuses on transcription factors, with a study of the evolution of Hox and ParaHox gene families across meta- zoans, for which I developed HoxPred, a bioinformatics tool to automatically classify these genes into their groups of homology. Transcription factors regulate their target genes by binding to short cis-regulatory elements in DNA. The second part of this thesis introduces the prediction of these cis-regulatory elements in genomic sequences, and my contributions to the development of user- friendly computational tools (RSAT software suite and TRAP). The third part covers the detection of these cis-regulatory elements using high-throughput sequencing experiments such as ChIP-seq or ChIP-exo. The bioinformatics developments include reusable pipelines to process these datasets, and novel motif analysis tools adapted to these large datasets (RSAT peak-motifs and ExoProfiler). As all these approaches are generic, I naturally apply them to diverse biological questions, in close collaboration with experimental groups. In particular, this third part presents the studies uncover- ing new DNA sequences that are driving or preventing the binding of the glucocorticoid receptor. Finally, my research perspectives are introduced, especially regarding further developments within the RSAT suite enabling cross-species conservation analyses, and new collaborations with exper- imental teams, notably to tackle the epigenomic remodelling during osteoporosis.Cette thèse d’HDR présente mes travaux concernant la régulation transcriptionelle chez les métazoaires (animaux). En tant que biologiste computationelle, mes activités de recherche portent sur le développement de nouveaux outils bioinformatiques, et contribuent à une meilleure compréhension de questions biologiques. La première partie concerne les facteurs de transcriptions, avec une étude de l’évolution des familles de gènes Hox et ParaHox chez les métazoaires. Pour cela, j’ai développé HoxPred, un outil bioinformatique qui classe automatiquement ces gènes dans leur groupe d’homologie. Les facteurs de transcription régulent leurs gènes cibles en se fixant à l’ADN sur des petites régions cis-régulatrices. La seconde partie de cette thèse introduit la prédiction de ces éléments cis-régulateurs au sein de séquences génomiques, et présente mes contributions au développement d’outils accessibles aux non-spécialistes (la suite RSAT et TRAP). La troisième partie couvre la détection de ces éléments cis-régulateurs grâce aux expériences basées sur le séquençage à haut débit comme le ChIP-seq ou le ChIP-exo. Les développements bioinformatiques incluent des pipelines réutilisables pour analyser ces jeux de données, ainsi que de nouveaux outils d’analyse de motifs adaptés à ces grands jeux de données (RSAT peak-motifs et ExoProfiler). Comme ces approches sont génériques, je les applique naturellement à des questions biologiques diverses, en étroite collaboration avec des groupes expérimentaux. En particulier, cette troisième partie présente les études qui ont permis de mettre en évidence de nouvelles séquences d’ADN qui favorisent ou empêchent la fixation du récepteur aux glucocorticoides. Enfin, mes perspectives de recherche sont présentées, plus particulièrement concernant les nouveaux développements au sein de la suite RSAT pour permettre des analyses basées sur la conservation inter-espèces, mais aussi de nouvelles collaborations avec des équipes expérimentales, notamment pour éudier le remodelage épigénomique au cours de l’ostéoporose

Comparative transcriptomics enlarges the toolkit of known developmental genes in mollusks

Data used for the phylogenetic analysis of Hox and ParaHox genes, including the respective GenBank accession numbers. (DOC 31 kb

Xenacoelomorpha: The "simple" key to bilaterian ancestry?

Xenacoelomorpha (comprising Xenoturbellida, Acoela and Nemertodermatida) is a clade of marine worms whose position in the tree of life is still in debate. Several phylogenetic analyses have shown them to be placed at the base of all bilaterian animals (e. g. chordates, arthropods) or at a more derived position as sister group to the Ambulacraria (echinoderms and hemichordates) within the Bilateria. A key characteristic is the absence of traits found in other bilaterian animals. Orthogroups are groups of orthologous genes found in several organisms. Orthologues are assumed to retain the same function. These functions would be specific to the clade where an orthogroup is prevalent. I investigate a method to automatically establish and validate orthogroups specific to Bilateria, Protostomia and Deuterostomia. These genes could be relevant for the clades’ respective emergence and differences. These sets will also help to ascertain what genes/functions are absent from Xenacoelomorpha. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in RNA silencing and post-transcriptional regulation of gene expression. MiRNAs have not been extens- ively studied in the Xenaceolomorpha. I introduce a fully automatic miRNA detection pipeline to infer and confirm the existence of pre-miRNA sequences in the genome of Xenoturbella bocki as well as predict miRNA candidates from several xenacoel gen- omes. I report previously undetected miRNA families and opine that previous analyses on Acoelomorpha failed due to loss caused by the higher evolutionary rate when compared to the Xenoturbellida