La position des membres le long du corps est reproductible chez une même espèce mais est très variable entre différentes espèces. Comment les membres acquièrent leur position et quel mécanisme est à l'origine de ces variations est à ce jour non élucidé. De part leur rôle dans la mise en place des axes embryonnaires, les gènes Hox sont depuis longtemps suspectés de jouer un rôle dans ce processus. Cependant les différentes preuves disponibles à ce jour restent indirectes et corrélatives. Chez l'embryon de poulet, je montre que la position des membres est établit précocement au cours du développement, lors de la gastrulation. Je démontre que la formation de la lame latérale (i.e. le tissue d'origine des membres) est un processus graduel et que l'activation séquentielle des gènes Hox spécifie ce tissue en domaines du membre et du flanc. Dans un second temps, une combinaison d'actions activatrice et répressive des gènes Hox sur le programme d'initiation du membre, actions liées à leur organisation colinéaire, est critique pour l'organisation de la lame latérale en domaines du membre et du flanc. Enfin, en étudiant des embryons de différentes espèces d'oiseaux présentant des variations dans la longueur de leur cou et donc dans la position de leur ailes (le poulet, l'autruche et le diamant mandarin), je montre que des changements relatifs dans la séquence d'activation colinéaire des gènes Hox au cours de la gastrulation sous-tendent les variations naturelles de la position de l'aile. L'ensemble de ces résultats montre que les gènes Hox jouent un rôle direct et précoce dans le positionnement des membres et propose un model général de mise en place d'un organisme par ces gènes. / Limb position along the main body axis is highly consistent within one species but very variable among tetrapods. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the anteroposterior axis remains largely unknown. Hox genes have long been suspected to play a role in this process, however supporting evidences are mostly correlative and a direct role has yet to be demonstrated. Here, using bird embryos, I show that limb position is established very early during development, during the process of gastrulation. I find that the formation of the Lateral Plate Mesoderm (i.e. the embryonic compartment from which limbs will form) is a progressive process and that co-linear activation of Hox genes sequentially patterns it along the antero-posterior axis. Subsequent combinatorial activation and repression activities of Hox genes on limb initiation are particularly critical to pattern the LPM into limb- and non-limb-forming domains. Finally, by analyzing chicken, zebra finch and ostrich embryos which exhibit variation in their forelimb position, I show that relative changes in the timing of co-linear Hox gene activation during gastrulation underlie variation in limb position. Altogether these result shed light on the cellular and molecular mechanism that regulate limb position by showing a direct and early role for Hox genes in this process during gastrulation and provide a mechanism for variation in body plan organization observed in tetrapods.

Le but de toute étude en biologie évolutive du développement est l’étude des mécanismes développementaux à l’origine des diversifications morphologiques. Dans ce contexte, j’ai décidé de me focaliser sur l’émergence des Vertébrés au cours de l’évolution, par la mise en œuvre d’études comparatives entre différents modèles de Deutérostomiens. Le travail réalisé durant ma thèse est subdivisé en trois projets: (i) j’ai abordé le lien entre l’évolution du cerveau chez les Chordés et les modifications de la signalisation à l’acide rétinoïque (AR) au cours du développement. En particulier, j’ai examiné les rôles de l’AR au cours du développement du cerveau chez la lamproie Lampetra fluviatilis, et j’ai comparé les résultats obtenus chez cette espèce aux mécanismes développementaux agissant chez l’amphioxus, un Chordé invertébré, et chez les modèles gnathostomes classiques. Les données obtenues lors de ces analyses comparatives ont permis une meilleure compréhension de l’évolution de la régionalisation cérébrale chez les Vertébrés. (ii) j’ai étudié l’évolution des séquences régulatrices présentes au sein des clusters de gènes hox, connus pour agir dans la régionalisation du système nerveux des Chordés. L’identification d’éléments non-codants conservés ainsi que d’éléments de réponse à l’AR (RARE) potentiels dans des clusters hox de Chordés, combinée à la caractérisation de RAREs in vivo en cellules murines a permis une vision intégrée de l’évolution du contrôle des gènes hox par l’AR, chez les Chordés. (iii) j’ai analysé l’évolution des microARNs chez les Chordés en comparant les répertoires microARN chez plusieurs espèces de Deutérostomiens. Cette étude a permis d’émettre de nouvelles hypothèses quant à l’émergence des microARNs chez les animaux. Toutes ces analyses ont abordé différents aspects de l’évolution des Chordés avec pour objectif la proposition d’une vision intégrée des mécanismes moléculaires à l’origine de l’émergence des Vertébrés. / The aim of the evolutionary developmental biology is to study the developmental mechanisms at the base of morphological diversification. In this context, I decided to focus my attention on the emergence of vertebrates during evolution by carrying out comparative analyses in several deuterostome models. The work carried out during of my thesis can be subdivided into three major projects: (i) I addressed the link between brain evolution and modifications in retinoic acid (RA) signaling during chordate development. In particular, I investigated the roles of RA signaling in brain development in a jawless vertebrate, the lamprey Lampetra fluviatilis, and compared the results with developmental mechanisms in the invertebrate chordate amphioxus and classical developmental model systems in jawed vertebrates. Data obtained from these comparative studies provided insights into the evolution of brain patterning in vertebrate evolution. (ii) I investigated the evolution of the regulatory landscape of hox gene clusters that are known to be fundamental for the patterning of the chordate central nervous system. The identification of conserved non-coding elements and putative RA response elements (RAREs) in hox clusters of different chordate species combined with the in vivo characterization of functional RAREs in mouse F9 cells provided an integrated view of the evolution of RA-dependent hox cluster regulation in chordates. (iii) I studied the roles of microRNAs (miRNAs) in chordate evolution by comparing the miRNA complements of different deuterostome species. This analysis provided novel insights about the general mechanisms of miRNA emergence in animals and highlighted species-specific miRNA complement amplifications in different deuterostome lineages. In sum, these studies have tackled different aspects of chordate evolution from an evo-devo perspective, aiming at an integrated view of the molecular mechanisms underlying vertebrate diversification.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22641号 / 医博第4624号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The ParaHox cluster contains three Hox‐related homeobox genes. The evolution of this sister of the Hox‐gene clusters has been studied extensively in metazoans with a focus on its early evolution. Its fate within the vertebrate lineage, and in particular following the teleost‐specific genome duplication, however, has not received much attention. Three of the four human ParaHox loci are linked with PDGFR family tyrosine kinases. We demonstrate that these loci arose as duplications in an ancestral vertebrate and trace the subsequent history of gene losses. Surprisingly, teleost fishes have not expanded their ParaHox repertoire following the teleost‐specific genome duplication, while duplicates of the associated tyrosine kinases have survived, supporting the hypothesis of a large‐scale duplication followed by extensive gene loss.

Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory regions. Since these elements are subject to stabilizing selection they evolve much slower than adjacent non-functional DNA. These so-called phylogenetic footprints can be detected by comparison of the sequences surrounding orthologous genes in different species. In this paper we present a new method and an effcient software tool for the identifcation of corresponding footprints in long sequences from multiple species. This allows the evolutionary study of the origin and loss of phylogenetic footprints if suffcient number and appropriately placed species are included. We apply this method to the published sequences of HoxA clusters of shark, human, and the duplicated zebrafish and Takifugu clusters as well as the published HoxB cluster sequences. We find that there is a massive loss of sequence conservation in the intergenic region of the HoxA clusters, consistent with the finding in [Chiu et al., PNAS 99, 5492-5497 (2002)]. We further propose a simple model to estimate the loss of sequence conservation that can be attributed to gene loss and other structural reasons. We find that the loss of conservation after cluster duplication is more extensive than expected by this model. This suggests that binding site turnover and/or adaptive modification may also contribute to the loss of sequence conservation. We conclude that this method is suitable for the large scale study of the evolution of (putative) cis-regulatory elements.

The statistical analysis of phylogenetic footprints in the two known horn shark Hox clusters and the four mammalian clusters shows that the shark HoxN cluster is HoxD-like. This finding implies that the most recent common ancestor of jawed vertebrates had at least four Hox clusters, including those which are orthologous to the four mammalian Hox clusters.

Osteoarthritis the quality and span of life in horses. Previous studies focused on nasal cartilage as a possible source for autologous chondrocyte implantation (ACI) in cartilage defects in humans. “HOX gene-negative” nasal chondrocytes adapted articular HOX patterns after implantation into caprine joint defects and produced cartilage matrix proteins. We compared the HOX gene profile of equine chondrocytes of nasal septum, anterior and posterior fetlock to identify nasal cartilage as a potential source for ACI in horses. Cartilage was harvested from seven horses after death and derived chondrocytes were cultured in a monolayer to fourth subcultivation. HOX A3, D1, D8 and chondrocyte markers COL2 and SOX9 were analyzed with qPCR in chondrocytes of three different locations obtained during passage 0 and passage 2. HOX gene expression showed no significant differences between the locations but varied significantly between the horses. HOX genes and SOX9 remained stable during culturing. Cultured nasal chondrocytes may be a target for future research in cell-based regenerative therapies in equine osteoarthritis. The involvement of HOX genes in the high regenerative and adaptive potential of nasal chondrocytes observed in previous studies could not be confirmed.

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