Notre objectif est de comprendre les mécanismes qui déterminent les axes embryonnaires. Ces mécanismes sont à l'origine de la polarisation progressive de l'information maternelle pendant l'ovogénèse, la maturation et les premiers clivages. Les clefs de cette compréhension résident dans les processus de localisation et de l'activation traductionnelle des ARN essentiels pour le développement. Le projet repose sur l'expertise de 3 équipes (Sardet/Houliston/McDougall) et leur réseau de collaborations (Nishida / Standart / King / Deshler / Woodland / Osborne / Manuel). Les résultats espérés sont une compréhension de la conservation des mécanismes des évènements de polarisation chez 2 organismes chordés modèle (ascidie, Xénope) et un nouveau modèle, le cnidaire Clytia, méduse au cycle maitrisé (programme EST engagé). Le projet s'articule autour des thèmes suivant : 1) Localisation des ARNm. Les mécanismes impliqués dans la localisation des ARN messagers dans les plasmes germinaux et dans le cortex des ovocytes de Xénope et d'ascidies seront comparés a ceux mis en œuvre chez Clytia. Nous utiliserons des méthodes d'imagerie haute résolution d' ARN fluorescents in vivo et d' hybridation/immunolocalisation in situ (ARNm Vg1, Xcat2, PEM1, macho1,..). Ces ARNs et leurs proteins de liaison identifiées grace aux approaches de protéomique / génomique avec nos partenaires seront simultanément analysées à l'aide d'anticorps spécifiques et de marquages fluorescents. La manipulation des séquences d'ARN et l'étude du comportement des proteins de liaison à l'ARN permettront d'analyses les relations entre localisation et controle traductionel des ARNm. 2) Maturation meiotique et polarisation des ovocytes. Nous étudierons les liens entre les facteurs de régulation du cycle et la polarisation dans l'ovocyte d'ascidie basé sur notre observation récente que la polarité s'établit lors de la migration du fuseau meiotique vers le cortex. Chez le Xénope, nous examinerons le role des évènements de phosphorylation controlés par les kinases meiotiques dans l'ancrage des ARNm au cortex végétatif et la dérepression concomitante de leur traduction. 3) Réorganisations cytoplasmiques et corticales liées aux facteurs du cycle cellulaire. Cette question sera principalement examinée sur les œufs de la méduse Clytia en utilisant des inhibiteurs spécifiques et la microinjection localisée de régulateurs du cycle (notament Cdc2/cyclinB et ses kinases activatrices). Nous suivrons la localisation et la dynamique des régulateurs de la mitose fluorescents (constructions GFP) grace aux méthodes d'imagerie confocale rapide et déconvolutions. Nous analyserons également le role des vagues de contraction de surface (SCWs) dans l'établissement de la polarité chez les ascidies et chez Clytia. 4) Regulation de la polarité cellulaire au cours du développement précoce. Nous poursuivrons nos recherches sur le role des proteine de polarité s PAR dans les embryons d'ascidies et nous aborderons leur identification et role chez Clytia. Nous préciserons la nature des interactions entre les PAR, les structures corticales et microtubulaires pour comprendre les mécanismes présidant aux divisions inégales (stades 8-64) qui donnent naissance aux lignées germinales et musculaires du tetard d'ascidie. Outre une contribution aux couts de fonctionnement nécessaire nous demandons les financements pour 2 Post Docs qui travailleront entre les équipes et avec les collaborateurs. Au niveau des methodologies, le projet requiert un microscope confocal rapide et sensible qui complémentera notre plateforme d'imagerie et permettra d'analyser in vivo les proteines et ARNm fluorescents impliqués dans les processus de polarisation des oeufs, ovocytes et embryons.

Nous proposons de mettre en place un crible pan-génomique sans précédent, dont le but est d'identifier de nouveaux gènes impliqués dans plusieurs voies de signalisation majeures (insuline/IGF, Hedgehog, JNK, JAK/STAT), contrôlant le développement, la morphogenèse, ou la croissance tissulaire chez la drosophile. A cette fin, nous exploiterons une collection unique de lignées transgéniques exprimant des ARN double-brin sous contrôle du système inductible Gal4/UAS, permettant une extinction génique par effet RNAi ( silencing ) de façon tissu-spécifique. Ce crible RNAi pan-génomique est rendu possible grâce à une collaboration internationale exclusive impliquant nos 3 laboratoires et celui du Dr R. Ueda (National Institute of Genetics, Japon). En partenariat avec la société Mitsubishi, le Dr Ueda génère des lignées transgéniques permettant l'expression de constructions RNAi dirigées contre chacun des 14.000+ gènes du génome de la drosophile. Nos laboratoires ont ou développent actuellement des lignées GAL4 portant des marqueurs spécifiques des voies de signalisation d'intérêt. Ces lignées seront croisées aux lignées RNAi afin d'induire des invalidations spécifiques de chacun des gènes, qui seront testées pour des défauts phénotypiques ou des altérations de l'activité des voies de signalisation. La fonction de chaque nouveau gène candidat sélectionné sera étudiée selon des approches classiques mises en place de façon routinière dans les différents laboratoires du projet. Ce projet ambitieux requiert la mise en place d'une structure commune permettant la manipulation, le tri et le croisement de centaines de lignées en flux constant. Cet effort majeur ne peut être réalisé que sous la forme d'une collaboration entre équipes voisines impliquées dans des interactions régulières. Un effort commun de crible génétique a déjà porté ses fruits dans le passé et les membres de nos équipes ont l'habitude de travailler ensemble. L'expertise complémentaire des équipes constituantes sera ensuite exploitée pour l'analyse détaillée des gènes impliqués dans : le contrôle de la mise en place du patron de développement (Thérond, Noselli), la prolifération et la croissance tissulaire (Léopold, Thérond, Noselli), la morphogenèse tissulaire (Noselli, Thérond, Léopold). Ce crible RNAi pan-génomique inédit permettra de réaliser de nombreuses avancées dans les domaines de la morphogenèse et de la croissance, qui sont des sujets majeurs de la Biologie soutenus par cet appel d'offre.

The JNK (Jun N-terminal Kinase) signaling pathway is a fundamental regulator of epithelial morphogenesis and has also been involved in immunity, cell death, wound healing, regeneration and some human cancers. In Drosophila embryos, JNK controls dorsal closure, an epithelial morphogenetic movement consisting of the collective and coordinated migration of two epithelial sheets and their fusion at the dorsal midline. Activation of the JNK pathway in cells at the front of migration (leading edge) is essential in this process. We performed a genomic analysis using microarrays to identify genes that are activated by JNK during dorsal closure. Thirty-one genes were validated experimentally, while only two were known previously. Eight of these genes showed a specific JNK-dependent expression in the leading edge. Surprisingly, this expression is not uniform, but seems to follow the segmented pattern of the embryonic epidermis. At the onset of dorsal closure, the segments are fully formed due to the action of the primary segmentation genes engrailed, wingless and hedgehog. Once established, the segmental compartments are fixed and do not show plasticity. We recently characterized a unique case of breaking of the segment boundary during dorsal closure. Specific cells (called Mixer cells) cross the border and enter the adjacent posterior compartment, releasing tissue tension as dorsal closure proceeds. This cell mixing behavior is mediated by a JNK-induced developmental reprogramming involving de novo expression of engrailed. The research project is divided in three tasks. We first want to characterize the function of the 31 new JNK target genes during dorsal closure using classical genetics and UAS-GAL4-induced RNAi in fly embryos. Molecular tools (specific antibodies, expression vectors …) and genetic tools (mutants, recombinants …) will be developed for these essential genes and dorsal closure defects will be linked to cellular modifications (cytoskeleton, adhesion, polarity …) of the leading edge cells to understand their function. Second, we would like to analyze the influence of the segmentation genes on the JNK transcriptional response at the leading edge. We will develop a quantitative analysis of the expression at the leading edge based on fluorescent in situ hybridization. This will allow us to determine the segmental profile for each gene, and then to test which of the segmentation genes contributes to the non-uniform expression along the segment. The third task of the project aims to understand how the JNK pathway controls cell reprogramming/mixing and to find the function of this unique event taking place during dorsal closure. We will carry out a targeted RNAi screen (chromatin, kinases, phosphatases) in the embryo to identify the molecular and cellular mechanisms that are involved. In addition, we would like to directly answer the question of how the Mixer cell is reprogrammed, i.e. how the JNK pathway regulates the expression of engrailed. Previous studies in Drosophila have shown that the chromatin, and in particular the repressor proteins of the Polycomb family, regulates the expression of engrailed, and that the JNK activity is able to relieve the negative action of these proteins during the regeneration process. Therefore we will test the effect of JNK on the Polycomb proteins in the Mixer cells for the expression of engrailed. Overall, this study intends to better understand the role of the JNK pathway in epithelial morphogenesis and developmental reprogramming, as well as the interaction between dorsal closure (i.e. morphogenesis) and the segmentation (i.e. patterning) of the embryo. As JNK is involved in oncogenesis in vertebrates, as well as in wound healing and regeneration, a better understanding of the function of the JNK pathway in a model system should provide new insights into these syndromes.

The question of how differential gene expression is established in the embryo is at the heart of developmental biology. Gene regulatory networks, which describe the interconnections between regulatory genes and signaling components at different stages of development, are powerful tool to understand how distinct territories of gene expression are established within the embryo. A few system-level approaches of early developmental processes have been successful in vertebrates, but these are made difficult by the large number of cells present in a vertebrate embryo, their high diversity, the complex morphogenetic movements that occur during vertebrate development as well as by the partial redundancy of their genomes. Here we propose to attempt a large scale analysis of the processes responsible for regionalization of the embryo along the dorso-ventral axis in a model that is much simpler than vertebrates: the sea urchin embryo. Sea urchins are non chordate deuterostomes and this position among the invertebrates makes them an interesting group to study the mechanism of axis specification and the evolution of developmental mechanisms. Unlike vertebrates, sea urchin embryos have a relatively small number of cells, are fully transparent and the morphogenetic movements that accompany gastrulation are easy to follow. A rich panoply of techniques is available for functional analysis including microinjection of morpholino oligonucleotides for gene loss of function, overexpression of mRNA and pharmacological treatments for gain of function, transgenesis by microinjection of plasmidic DNA, etc. Finally, the sea urchin embryo has a prolific history of experimental embryology and a wealth of biological knowledge is available on its development. The genome of Strongylocentrotus purpuratus has been sequenced, revealing that echinoderms have a rich genetic repertoire and a low level of genetic redundancy. For all these reasons, the sea urchin embryo has turned out to be a model of choice for the analysis of gene regulatory networks during early development. The project builds on previous work from our laboratory in identifying key elements of the gene regulatory network driving dorsal-ventral patterning such as Nodal and BMP2/4 and in identifying key transcription factors and signaling factors acting upstream and downstream of these signals. However, until recently, our studies focused on small scale analyses and on the roles of individual genes in cell fate specification. This project instead proposes to use a large scale approach to understand several key features of an important developmental process common to all bilateria : the establishment of dorso-ventral polarity. The first aim of this project is to assemble a gene expression atlas of all the regulatory factors involved in dorso-ventral axis formation then a map describing the regulatory interactions between the most important players. This will be achieved by using a combination of expression studies (in situ screen, micro array screens), perturbation analysis (loss and gain of function), and promoter analysis of the most important genes (identification of cis-regulatory modules by phylogenetic footprinting). The second aim of this project is to understand how Nodal and BMP2/4 work to organize the whole dorso-ventral axis of the embryo. While Nodal has long range organizing activity in rescue experiments, preliminary experiments indicate that Nodal does not work as a morphogen but suggest instead that it requires a relay to specify dorsal fates. The third aim of this project is to understand the molecular mechanisms and to identify the maternal factors that activate the dorso-ventral gene regulatory network. While redox gradients and spatially restricted activation of p38 MAP kinase signaling have been implicated in the early steps of dorso-ventral axis formation upstream of Nodal, the link between these events and the transcriptional machinery responsible for initiating nodal expression remain to be established. We propose to characterize the activating transcription factors that initiate nodal expression and to characterize the function and regulation of a transcriptional repressor that we identified and which appears to be a major determinant of the spatial expression of nodal and an excellent candidate to integrate signals from Redox./MAP kinase signaling. The long term goal is to draw a detailed picture of which genes are regulated by which transcription factors, how extracellular signals regulate the activity of these transcription factors and how these genes work together to form a transcriptional network that drives morphogenesis of the embryo. This work is important since not only it will improve our undestanding of a complex developmental process, but it will also illuminate the evolution of mechanisms regulating nodal expression, and more generally, the mechanisms responsible for dorsal-ventral patterning in deuterostomes.