30 research outputs found

    Mise en place de l'identité des muscles au cours de la spécification des myoblastes chez la drosophile

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    La formation des muscles squelettiques au cours de l'embryogenèse de la drosophile est un modèle d'étude du contrôle génétique de la différentiation cellulaire. La formation de chaque muscle comprend quatre étapes successives: spécification d'un groupe promusculaire, sélection d'un progéniteur (PC) à partir de ce groupe, division asymétrique de ce progéniteur pour donner des cellules fondatrices de muscles (FC) ; fusion de chaque FC avec des myoblastes compétents (FCM), suivie de la différenciation musculaire. Chaque muscle squelettique est composé d'une fibre. Chaque muscle présente des propriétés spécifiques de taille, forme, position, attachement, et patron d'innervation. Ces propriétés sont groupées sous le terme d'identité musculaire. Cette identité est conférée par l'expression dans chaque PC/FC d'une combinatoire de Facteurs de Transcription identitaires (FTi). Notre laboratoire étudie ce processus, en utilisant comme point d'entrée l'expression et les rôles du FTi Collier (Col) au cours du développement d'un muscle dorso-latéral, le muscle DA3 (Dorsal Acute 3). Au cours de la première partie de ma thèse, j'ai étudié la régulation transcriptionnelle de col durant les phases de spécification des groupes promusculaires et de sélection du PC à l'origine du muscle DA3. Partant de prédictions bioinformatiques j'ai caractérisé le module cis régulateur (CRM) de col actif durant ces phases (CRM précoce). Un CRM " tardif ", actif du stade progéniteur à la complétion de la formation du muscle DA3, avait été préalablement caractérisé dans l'équipe. Afin de déterminer plus précisément les fenêtres temporelles d'activité des deux CRM mésodermiques de col, j'ai mis au point un nouveau gène rapporteur comportant un intron permettant de détecter les transcrits primaires. Ceci m'a permis de montrer que les CRM précoce et tardif reproduisent ensemble l'expression endogène de col. La caractérisation du CRM précoce de col m'a aussi permis de suivre le destin des FCM du groupe promusculaire Col dans les embryons tardifs et de montrer que ces FCM contribuent uniquement à des muscles dorsaux-latéraux. Au cours de la deuxième partie de ma thèse, j'ai caractérisé le rôle, inconnu jusqu'alors, du FT à domaine LIM-Homeodomaine Tailup (Tup)/Islet1 dans la myogenèse. J'ai d'abord montré que Tup est spécifiquement exprimé dans les 4 muscles les plus dorsaux. L'analyse de mutants m'a permis de montrer qu'en absence de Tup, le muscle dorsal DA2 exprime Col et est transformé en muscle dorso-latéral de type DA3. J'ai ensuite montré que le PC du DA2 est à l'origine de la FC DA2 et d'un précurseur musculaire adulte (AMP). Ce PC est sélectionné à partir du groupe promusculaire Col quand les cellules de ce groupe expriment encore le FT à homéodomaine Tinman/NKx2.5. Tin active tup dans le PC DA2. Tup, en retour, réprime col et cette répression permet de distinguer les identités musculaires DA2 et DA3. En conclusion, mes travaux de thèse m'ont permis de proposer un nouveau modèle permettant de relier le processus de spécification des progéniteurs au contrôle temporel et spatial de l'expression des FTi. Une vision dynamique de ce processus de spécification permet de mieux comprendre le programme identitaire propre à chaque muscle. L'analyse des interactions entre Tin, Tup, et Col au cours de la formation des muscles dorsaux révèle de nouveaux parallèles avec les interactions entre Nkx2.5, Islet, EBF au cours de la formation des muscles pharyngaux chez les chordés.The somatic musculature of the Drosophila embryo is a classical model to study the regulatory processes that generate cellular diversity. Muscle formation is a multistep process: the first step is the specification, within the mesoderm, of a group of competent cells, called promuscular cluster. The second step is the selection of a progenitor cell (PC) from this cluster. Asymmetric division of each PC then generates muscle founder cells (FC). Finally, each FC undergoes a fusion process with fusion competent myoblasts (FCM) to generate a muscle fiber. Each muscle is formed of a single multinucleate fiber. Each Drosophila muscle has a specific identity, as it can be distinguished by its position, shape, orientation, attachment, and innervation pattern. Muscle identity reflects the expression by each PC/FC of a specific combination of identity Transcription Factors (iTF). In the laboratory, we study the control of muscle identity, using as entry point, the expression and requirement of the iTF Collier (Col) during development of a dorso-lateral (DA3) muscle. I started my PhD by characterizing col transcriptional regulation during early steps of DA3 muscle formation. Starting from computational predictions, I identified an early col cis regulatory module (Early CRM) responsible for col activation in a promuscular cluster. A late col CRM, active from the PC stage, had previously been characterized in the laboratory. To determine with more precision the temporal windows of activity of each of these CRM, I designed a novel intron-containing reporter gene in order to detect primary transcripts. This allowed me to show that the late and the early CRMs together reproduce precisely the endogenous col expression pattern. Characterization of the early mesodermal col CRM also allowed to do lineage experiments and determine the fate of FCMs that transiently express Col at the promuscular stage. I found that these myoblasts contribute mostly to dorso-lateral muscles. During the second part of my thesis, I described a new role of the LIM-homeodomain TF Tailup/Islet1 (Tup) in specifying dorsal muscles. I first showed that Tup is specifically expressed in the four dorsal muscles. In tup null mutants, on one hand, the dorsal musculature is severely disorganized and, on the other hand, the dorsal DA2 muscle ectopically expresses Col and is transformed into a dorso-lateral DA3-like muscle. I showed that the DA2 PC is singled out from the Col promuscular cluster when cells of this cluster still express (transitorily) the homeodomain TF Tinman/Nkx2.5 (Tin). The DA2 PC gives rise to the DA2 FC and a (dorso-lateral) adult muscle precursor (AMP). tup activation by Tin in the DA2 PC is required to repress col and establish a DA2 instead of DA3 identity. In conclusion, my work allowed to propose a model which connects a temporal sequence of transcriptional regulation of iTFs to the specification of muscle PC identity and final muscle pattern. It provides a novel, dynamic view of how muscle identity is specified. These findings also provide novel parallels with the specification of pharyngeal muscles in vertebrates

    Notch directly regulates the cell morphogenesis genes Reck, talin and trio in adult muscle progenitors.

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    There is growing evidence that activation of the Notch pathway can result in consequences on cell morphogenesis and behaviour, both during embryonic development and cancer progression. In general, Notch is proposed to coordinate these processes by regulating expression of key transcription factors. However, many Notch-regulated genes identified in genome-wide studies are involved in fundamental aspects of cell behaviour, suggesting a more direct influence on cellular properties. By testing the functions of 25 such genes we confirmed that 12 are required in developing adult muscles, consistent with roles downstream of Notch. Focusing on three, Reck, rhea/talin and trio, we verify their expression in adult muscle progenitors and identify Notch-regulated enhancers in each. Full activity of these enhancers requires functional binding sites for Su(H), the DNA-binding transcription factor in the Notch pathway, validating their direct regulation. Thus, besides its well-known roles in regulating the expression of cell-fate-determining transcription factors, Notch signalling also has the potential to directly affect cell morphology and behaviour by modulating expression of genes such as Reck, rhea/talin and trio. This sheds new light on the functional outputs of Notch activation in morphogenetic processes.This is the author's accepted manuscript. The final version is available from the Journal of Cell Science at http://dx.doi.org/10.1242/​jcs.15178

    Characterization of Drosophila ATPsynC mutants as a new model of mitochondrial ATP synthase disorders

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    Mitochondrial disorders associated with genetic defects of the ATP synthase are among the most deleterious diseases of the neuromuscular system that primarily manifest in newborns. Nevertheless, the number of established animal models for the elucidation of the molecular mechanisms behind such pathologies is limited. In this paper, we target the Drosophila melanogaster gene encoding for the ATP synthase subunit c, ATPsynC, in order to create a fruit fly model for investigating defects in mitochondrial bioenergetics and to better understand the comprehensive pathological spectrum associated with mitochondrial ATP synthase dysfunctions. Using P-element and EMS mutagenesis, we isolated a set of mutations showing a wide range of effects, from larval lethality to complex pleiotropic phenotypes encompassing developmental delay, early adult lethality, hypoactivity, sterility, hypofertility, aberrant male courtship behavior, locomotor defects and aberrant gonadogenesis. ATPsynC mutations impair ATP synthesis and mitochondrial morphology, and represent a powerful toolkit for the screening of genetic modifiers that can lead to potential therapeutic solutions. Furthermore, the molecular characterization of ATPsynC mutations allowed us to better understand the genetics of the ATPsynC locus and to define three broad pathological consequences of mutations affecting the mitochondrial ATP synthase functionality in Drosophila: i) pre-adult lethality; ii) multi-trait pathology accompanied by early adult lethality; iii) multi-trait adult pathology. We finally predict plausible parallelisms with genetic defects of mitochondrial ATP synthase in humans.This work was supported by grants from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) to C.C. and University of Bari D.R. n. 12939 to D.P

    N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

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    Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. The most common protein modification in eukaryotes is N-terminal acetylation, but its functional impact has remained enigmatic. Here, the authors find that a key role for N-terminal acetylation is shielding proteins from ubiquitin ligase-mediated degradation, mediating motility and longevity.Association Francaise contre les Myopathies 261981, Canadian Institutes of Health Research (CIHR) 249843, United States Department of Health & Human Services National Institutes of Health (NIH) - USA F-12540, Portuguese national funding through Fundaco para a Ciencia e a Tecnologia (FCT) 171752-PR-2009-0222, National Funds through Fundaco para a Ciencia e a Tecnologia (FCT) G008018N, G002721N, University of Bergen MC_UU_00028/6, FDN-143264, FDN-143265, PJT-180285, PJT-463531, R01HG005853, R01HG005084, DL 57/2016/CP1361/CT0019, 2022.01782.PTDC,PTDC/BIA-BID/28441/2017,PTDC/BIA-BID/1606/2020, ALG-01-0145-FEDER-028441, PPBI-POCI-01-0145-FEDER-022122, LISBOA-01-0145-FEDER-022170info:eu-repo/semantics/publishedVersio

    Drosophila, an Integrative Model to Study the Features of Muscle Stem Cells in Development and Regeneration

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    International audienceMuscle stem cells (MuSCs) are essential for muscle growth, maintenance and repair. Over the past decade, experiments in have been instrumental in understanding the molecular and cellular mechanisms regulating MuSCs (also known as adult muscle precursors, AMPs) during development. A large number of genetic tools available in fruit flies provides an ideal framework to address new questions which could not be addressed with other model organisms. This review reports the main findings revealed by the study of AMPs, with a specific focus on how AMPs are specified and properly positioned, how they acquire their identity and which are the environmental cues controlling their behavior and fate. The review also describes the recent identification of the adult MuSCs that have similar characteristics to vertebrates MuSCs. Integration of the different levels of MuSCs analysis in flies is likely to provide new fundamental knowledge in muscle stem cell biology largely applicable to other systems

    Insights and perspectives on the enigmatic alary muscles of arthropods

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    International audienceThree types of muscles, cardiac, smooth and skeletal muscles are classically distinguished in eubilaterian animals. The skeletal, striated muscles are innervated multinucleated syncytia, which, together with bones and tendons, carry out voluntary and reflex body movements. Alary muscles (AMs) are another type of striated syncytial muscles, which connect the exoskeleton to the heart in adult arthropods and were proposed to control hemolymph flux. Developmental studies in Drosophila showed that larval AMs are specified in embryos under control of conserved myogenic transcription factors and interact with excretory, respiratory and hematopoietic tissues in addition to the heart. They also revealed the existence of thoracic AMs (TARMs) connecting to specific gut regions. Their asymmetric attachment sites, deformation properties in crawling larvae and ablation-induced phenotypes, suggest that AMs and TARMs could play both architectural and signalling functions. During metamorphosis, and heart remodelling, some AMs trans-differentiate into another type of muscles. Remaining critical questions include the enigmatic modes and roles of AM innervation, mechanical properties of AMs and TARMS and their evolutionary origin. The purpose of this review is to consolidate facts and hypotheses surrounding AMs/TARMs and underscore the need for further detailed investigation into these atypical muscles

    Studying Muscle Transcriptional Dynamics at Single-molecule Scales in <em>Drosophila</em>

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    International audienceSkeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing singlemolecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells. © 2023 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License
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