Invalidation du gène Gasp1 et étude de sa fonction chez la souris

Skeletal muscle formation is a complex process that involves the proliferation, alignment and fusion of myogenic precursor cells to form multinucleated myotubes that will subsequently mature into muscle fibers. A number of regulatory factors involved in these different steps have been identified. Among them, Myostatin (Gdf8), a member of TGFβ super-family is a negative regulator of myoblast proliferation and differentiation. The lack of a functional protein leads to remarkable muscle mass growth. Several Gdf8 inhibitors like the propeptide or the follistatin protein. Recently, Gasp1 was identified as a new myostatin partner, acting as a negative regulator of myostatin activity. To get more insights on Gasp1 functions, especially in myogenesis, we have used homologous recombination to effect the targeted disruption of the Gasp-1 gene in mouse embryonic stem cells. Gasp1-/- knock-out mouse provides us an excellent tool to conduct functional studies. Preliminary analyses show the viability of homozygotes without growth defects. In parallel, Gasp1 expression has been studied in spatio-temporal manner during embryogenesis by in situ hybridization on mouse embryo and quantified by quantitative PCR during myogenic proliferation and differenciation.La formation des muscles squelettiques est un processus complexe impliquant différentes étapes de différenciation, de prolifération, et de fusion ainsi que de nombreux facteurs moléculaires. Parmi ces derniers, la myostatine (Gdf8) est un régulateur négatif de la prolifération et de la différenciation des myoblastes, et l’absence d’une protéine fonctionnelle conduit à une remarquable augmentation de la masse musculaire. La myostatine, membre de la superfamille des TGFβ, est secrétée sous la forme d’un précurseur. De nombreuses études sur la voie de signalisation de la myostatine ont permis de caractériser différents inhibiteurs de Gdf8 comme par exemple son propeptide ou la follistatine. Récemment, Gasp1 a été identifié comme nouveau partenaire de la myostatine, agissant comme un régulateur négatif de l’action de Gdf8. Afin de mieux connaître le rôle de Gasp1, en particulier au cours de la myogenèse, nous l’avons invalidé chez la souris par recombinaison homologue dans des cellules souches embryonnaires. L’établissement des souris knockout Gasp1-/- nous permet maintenant d’aborder les aspects fonctionnels du gène Gasp1. Des études préliminaires révèlent que les homozygotes sont viables et ne présentent pas de défauts de croissance. D’autre part, l’expression de Gasp1 a été étudiée de manière spatio-temporelle au cours du développement embryonnaire grâce à des expériences d’hybridation in situ sur des embryons murins in toto et quantifiée en PCR quantitative au cours de la prolifération et différenciation myogénique

Invalidation du gène Gasp1 et étude de sa fonction chez la souris

Les mécanismes génétiques impliqués dans la myogenèse font l'objet de nombreuses études. Parmi les avancées récentes, l'identification du rôle de la myostatine a ouvert la voie de nouveaux traitements pour certaines myopathies. Des variants alléliques de Gdf8, gène codant la myostatine, ont été identifiés. La quantité de myostatine est contrôlée par divers mécanismes. Ainsi la protéine Gasp1 peut se lier à la myostatine ainsi qu'à son propeptide et contrôler l'obtention de la forme active. Les objectifs de ma thèse sont l'obtention d'une souris invalidée pour le gène Gasp1 et son exploitation afin de mieux comprendre les mécanismes contrôlant le taux de myostatine dans le muscle. Mais aussi l'étude de l'expression de Gasp1 au cours de l'embryogenèse et de la myogenèse.Genetic mechanisms of myogenesis concern many research projects. Among recent progresses, the identification of the role of myostatin has opened a new way to create new therapeutics for several myopathies. Variants of Gdf8, gene coding for myostatin, were discovered. Myostatin amount are controlled by several mechanisms. Thus Gasp1 is able to link with the myostatin or the myostatin propeptide. It has been shown that Gasp1 controls the proteolytic cleavage necessary to obtain the active form of myostatin. During my thesis, I knock-out the Gasp1 gene in the mouse, and to get better inside into the molecular mechanisms controlling myostatin rates in muscles. Moreover, Gasp1 expression has been studied during embryogenesis and myogenesis.LIMOGES-BU Sciences (870852109) / SudocSudocFranceF

Nouvelles données sur les expansions croisées d'Icosium tomentosum Lucas, 1854 et de Lucasianus levaillantii (Lucas, 1846) (Coleoptera, Cerambycidae)

De nouvelles investigations sur les distributions d'Icosium totemtosum et de Lucasianus levaillantii ont été menées dans la basse vallée duVidourle, en limite des deux départements français du Gard et de l'Hérault. I. totemtosum a été trouvé pour la première fois dans l'Hérault et L. levaillantii pour la première fois dans le Gard, ce qui montre le croisement de leur expansion aux confins des deux départements ; des hypothèses d'évolution de ces expansions sont émises

Nouvelles données sur les expansions croisées d'Icosium tomentosum Lucas, 1854 et de Lucasianus levaillantii (Lucas, 1846) (Coleoptera, Cerambycidae)

De nouvelles investigations sur les distributions d'Icosium totemtosum et de Lucasianus levaillantii ont été menées dans la basse vallée duVidourle, en limite des deux départements français du Gard et de l'Hérault. I. totemtosum a été trouvé pour la première fois dans l'Hérault et L. levaillantii pour la première fois dans le Gard, ce qui montre le croisement de leur expansion aux confins des deux départements ; des hypothèses d'évolution de ces expansions sont émises

GASP/WFIKKN proteins: evolutionary aspects of their functions

Chantier qualité GAGrowth and differentiation factor Associated Serum Protein (GASP) 1 and 2 are proteins known to be involved in the control of myostatin activity at least in vitro. Most deuterostome GASPs share a modular organization including WAP, follistatin/kazal, IGc2, two kunitz, and NTR domains. Based on an exon shuffling model, we performed independent phylogenetic analyses on these modules and assessed that papilin is probably a sister sequence to GASP with a divergence date estimated from the last common ancestor to bilateria. The final organization was acquired by the addition of the FS domain in early deuterostomes. Our study revealed that Gasp genes diverged during the first round of genome duplication in early vertebrates. By evaluating the substitution rate at different sites on the proteins, we showed a better conservation of the follistatin/kazal domain of GASP1 than GASP2 in mammals, suggesting a stronger interaction with myostatin. We also observed a progressive increase in the conservation of follistatin and kunitz domains from the ancestor of Ciona to early vertebrates. In situ hybridization performed on mouse embryos showed a weak Gasp1 expression in the formed somites at 10.5 dpc and in limb buds from embryonic E10.0 to E12.5. Similar results were obtained for zebrafish embryos. We propose a synthetic view showing possible interactions between GASP1 and myostatin and highlighting the role of the second kunitz domain in preventing myostatin proteolysis

Fork Stalling and Template Switching As a Mechanism for Polyalanine Tract Expansion Affecting the DYC Mutant of HOXD13, a New Murine Model of Synpolydactyly

Polyalanine expansion diseases are proposed to result from unequal crossover of sister chromatids that increases the number of repeats. In this report we suggest an alternative mechanism we put forward while we investigated a new spontaneous mutant that we named “Dyc” for “Digit in Y and Carpe” phenotype. Phenotypic analysis revealed an abnormal limb patterning similar to that of the human inherited congenital disease synpolydactyly (SPD) and to the mouse mutant model Spdh. Both human SPD and mouse Spdh mutations affect the Hoxd13 gene within a 15-residue polyalanine-encoding repeat in the first exon of the gene, leading to a dominant negative HOXD13. Genetic analysis of the Dyc mutant revealed a trinucleotide expansion in the polyalanine-encoding region of the Hoxd13 gene resulting in a 7-alanine expansion. However, unlike the Spdh mutation, this expansion cannot result from a simple duplication of a short segment. Instead, we propose the fork stalling and template switching (FosTeS) described for generation of nonrecurrent genomic rearrangements as a possible mechanism for the Dyc polyalanine extension, as well as for other polyalanine expansions described in the literature and that could not be explained by unequal crossing over

Substitution rate of GASP proteins in the different branches of the evolutionary tree.

<p>For each site the substitution rate was calculated from a moving average using a window size of 20 amino acids. The site position from start methionine is on the abscissa and the mean substitution rate on the ordinate. The black curves correspond to the ancestor of GASP1 and GASP2 proteins. The red and blue curves correspond to GASP1 and GASP2 respectively. Red asterisks represent gaps in sequence alignment due to one or two species. In the last two parts, the dotted lines correspond to fish and the solid lines to tetrapods. Abbreviations: SP: signal peptide, W: WAP, F: follistatin, K: kazal, Ku: kunitz, NTR: netrin domains.</p

Global view of GASP/Papilin evolution.

<p>This representation derived from the phylogenies conducted on the separate modules (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s001" target="_blank">Fig. S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s003" target="_blank">S3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s004" target="_blank">S4</a>). From a protein close to the actual <i>N. vectensis</i> 292 aa protein (bottom right), an intermediate protein probably arose through a duplication of the kunitz domain. This form would represent a common ancestor to papilin (PPN, arrow 1) and GASP (arrow 2). The schematized papilin modular organization corresponds to that found in <i>Drosophila melanogaster</i> isoforms. The phylogeny of the IGc2 modules allowed us to situate ADAMTS protein close to papilin. The GASP kazal domain came from a donor polypeptide which could be agrin, FSTL1, 4, or 5 proteins. The FST and FSTL3 kazal domains are unlikely related to GASP module but very close to the units present in <i>N. vectensis</i> 4887 aa protein. Eppin, EXPI, and WAP8C are examples of proteins that probably evolved from the intermediate form. Domains related to <i>N. vectensis 292</i> aa protein are outlined in red, the new kunitz domain B (intermediate step) is outlined in yellow. The closest kazal domain between agrin, FSTL1, 4, 5 proteins and GASP is outlined in orange.</p