Evolution of retinoic acid receptors in chordates: insights from three lamprey species, Lampetra fluviatilis, Petromyzon marinus, and Lethenteron japonicum

International audienceBackground : Retinoic acid (RA) signaling controls many developmental processes in chordates, from early axis specification to late organogenesis. The functions of RA are chiefly mediated by a subfamily of nuclear hormone receptors, the retinoic acid receptors (RARs), that act as ligand-activated transcription factors. While RARs have been extensively studied in jawed vertebrates (that is, gnathostomes) and invertebrate chordates, very little is known about the repertoire and developmental roles of RARs in cyclostomes, which are extant jawless vertebrates. Here, we present the first extensive study of cyclostome RARs focusing on three different lamprey species: the European freshwater lamprey, Lampetra fluviatilis, the sea lamprey, Petromyzon marinus, and the Japanese lamprey, Lethenteron japonicum.Results : We identified four rar paralogs (rar1, rar2, rar3, and rar4) in each of the three lamprey species, and phylogenetic analyses indicate a complex evolutionary history of lamprey rar genes including the origin of rar1 and rar4 by lineage-specific duplication after the lamprey-hagfish split. We further assessed their expression patterns during embryonic development by in situ hybridization. The results show that lamprey rar genes are generally characterized by dynamic and highly specific expression domains in different embryonic tissues. In particular, lamprey rar genes exhibit combinatorial expression domains in the anterior central nervous system (CNS) and the pharyngeal region.Conclusions : Our results indicate that the genome of lampreys encodes at least four rar genes and suggest that the lamprey rar complement arose from vertebrate-specific whole genome duplications followed by a lamprey-specific duplication event. Moreover, we describe a combinatorial code of lamprey rar expression in both anterior CNS and pharynx resulting from dynamic and highly specific expression patterns during embryonic development. This ‘RAR code’ might function in regionalization and patterning of these two tissues by differentially modulating the expression of downstream effector genes during development

Evolution of chordate development : a story of retinoic acid, hox genes and microRNAs

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

Evolution of chordate development (a story of retinoic acid, hox genes and microRNAs)

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.LYON-ENS Sciences (693872304) / SudocSudocFranceF

microRNA complements in deuterostomes: origin and evolution of microRNAs

Although numerous studies have emphasized the role of microRNAs (miRNAs) in the control of many different cellular processes, they might also exert a profound effect on the macroevolution of animal body plans. It has been hypothesized that, because miRNAs increase genic precision and are continuously being added to metazoan genomes through geologic time, miRNAs might be instrumental for canalization of development and morphological evolution. Nonetheless, an outstanding question remains: how are new miRNAs constantly evolving? To address this question, we assessed the miRNA complements of four deuterostome species, chosen because of their sequenced genomes and well-resolved phylogeny. Our comparative analysis shows that each of these four species is characterized by a unique repertoire of miRNAs, with few instances of miRNA loss. Moreover, we find that almost half of the miRNAs identified in this study are located in intronic regions of protein coding genes, suggesting that new miRNAs might arise from intronic regions in a process we term intronic exaptation. We also show that miRNAs often occur within cotranscribed clusters, and describe the biological function of one of these conserved clusters, the miR-1/miR-133 cluster. Taken together, our work shows that miRNAs can easily emerge within already transcribed regions of DNA, whether it be introns or preexisting clusters of miRNAs and/or miRNAs and protein coding genes, and because of their regulatory roles, these novel players change the structure of gene regulatory networks, with potential macroevolutionary results

The roof plate boundary is a bi-directional organiser of dorsal neural tube and choroid plexus development

The roof plate is a signalling centre positioned at the dorsal midline of the central nervous system and generates dorsalising morphogenic signals along the length of the neuraxis. Within cranial ventricles, the roof plate gives rise to choroid plexus, which regulates the internal environment of the developing and adult brain and spinal cord via the secretion of cerebrospinal fluid. Using the fourth ventricle as our model, we show that the organiser properties of the roof plate are determined by its boundaries with the adjacent neuroepithelium. Through a combination of in ovo transplantation, co-culture and electroporation techniques in chick embryos between embryonic days 3 and 6, we demonstrate that organiser properties are maintained by interactions between the non-neural roof plate and the neural rhombic lip. At the molecular level, this interaction is mediated by Delta-Notch signalling and upregulation of the chick homologue of Hes1: chairy2. Gain- and loss-of-function approaches reveal that cdelta1 is both necessary and sufficient for organiser function. Our results also demonstrate that while chairy2 is specifically required for the maintenance of the organiser, its ectopic expression is not sufficient to recapitulate organiser properties. Expression of atonal1 in the rhombic lip adjacent at the roof plate boundary is acutely dependent on both boundary cell interactions and Delta-Notch signalling. Correspondingly, the roof plate boundary organiser also signals to the roof plate itself to specify the expression of early choroid plexus markers. Thus, the roof plate boundary organiser signals bi-directionally to acutely coordinate the development of adjacent neural and non-neural tissues