Neutral competition for drosophila follicle and cyst stem cell niches requires vesicle trafficking genes

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, we performed a genetic screen through a collection of 126 mutants in essential genes on the X chromosome to identify candidates that increase or decrease competition for the FSC niche. We identified ∼55 and 6% of the mutations screened as putative FSC hypo- or hyper-competitors, respectively. We found that a large majority of mutations in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypo-competition alleles, and we confirmed the hypo-competition phenotype for four of these alleles. We also show that Sec16 and another COPII vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, we demonstrate that, although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells of the testis, there are important tissue-specific differences. Our results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and we identify a number of putative candidates for further exploration

Impact de la sénescence dans la régénération musculaire

Tissue regeneration is a process of replacing lost or damaged cells upon injury. Cellular senescence is a physiological response to stress characterized by a stable cell cycle arrest and is associated with various biological and pathological processes. My Ph.D. project aims to investigate in vivo senescence during muscle regeneration, including its identities, functions, and dynamics. It has been demonstrated that cellular senescence could facilitate optimal wound healing. We previously demonstrated that injury-induced senescence promotes reprogramming in the skeletal muscle, notably through IL-6 (Chiche et al., 2017). Firstly, I showed that senescence was peaking at day 3 post-injury and terminated by day 15 post injury. Then, I found that FAPs is the primary cell type that becomes senescent by RNA sequencing (both bulk population and single-cell). Besides, senescent FAPs enhance myogenesis in vitro in a paracrine manner. Of note, P57 is the major CKI which mediates the FAPs-associated senescence. Importantly, there is a significant reduction of the injury-induced senescence in the P57 null mice. Lastly, we found Mcl-1 is overexpressed in the senescent FAPs, suggesting the potential usage of MCL-1 inhibitors as senolytic drugs.Taken together, my ph.D show that FAPs are senescent upon muscle injury, which is P57-dependent and might be important to muscle regeneration by enhancing myogenesis. These findings strongly support a beneficial role of senescence during muscle regeneration, which has direct implications in muscular degenerative diseases and muscle aging.La régénération tissulaire est un processus de remplacement des cellules perdues ou endommagées lors d'une blessure. La sénescence cellulaire est une réponse physiologique au stress caractérisée par un arrêt du cycle cellulaire stable. Mon doctorat vise à étudier la sénescence in vivo lors de la régénération musculaire, y compris son identité, ses fonctions et sa dynamique. Nous avons précédemment démontré que la sénescence induite par blessure favorise la reprogrammation dans le muscle squelettique. Mon projet vise à étudier la biologie de la sénescence lors de la régénération musculaire de manière systémique. Premièrement, j'ai montré que la sénescence atteignait son maximum au 3ème jour après la blessure et se terminait au 15ème jour après la blessure. Ensuite, j'ai trouvé que les FAPs (Fibro-Adipogenic Progenitors) sont les principales cellules qui deviennent sénescentes. Par ailleurs, les FAPs sénescents améliorent peut-être la myogenèse in vitro de manière paracrine. P57 est le principal inhibiteur de CDK qui permet l’induction de la sénescence associée aux FAPs. En effet, il y a une réduction significative de la sénescence induite par blessure chez les souris P57KO. Enfin, nous avons trouvé que Mcl-1 est surexprimé dans les FAPs sénescents, ce qui suggère l'utilisation potentielle d’inhibiteurs de MCL-1 comme drogues sénolytiques. Dans l’ensemble, mon doctorat montre que les FAPs sont sénescents lors d'une lésion musculaire, via P57 et pourraient être important pour la régénération musculaire en favorisant la myogenèse. Ces découvertes soutiennent fortement le rôle bénéfique de la sénescence lors de la régénération musculaire

Evaluation of Injury-induced Senescence and <em>In Vivo</em> Reprogramming in the Skeletal Muscle

International audienceCellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated β-galactosidase (SA-β-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal β-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry (IHC). The major limitation of the SA-β-Gal assay is the requirement of fresh or frozen samples. Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-β-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming

Neutral Competition for Drosophila Follicle and Cyst Stem Cell Niches Requires Vesicle Trafficking Genes.

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, we performed a genetic screen through a collection of 126 mutants in essential genes on the X chromosome to identify candidates that increase or decrease competition for the FSC niche. We identified ∼55 and 6% of the mutations screened as putative FSC hypo- or hyper-competitors, respectively. We found that a large majority of mutations in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypo-competition alleles, and we confirmed the hypo-competition phenotype for four of these alleles. We also show that Sec16 and another COPII vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, we demonstrate that, although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells of the testis, there are important tissue-specific differences. Our results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and we identify a number of putative candidates for further exploration

Injury-Induced Senescence Enables In Vivo Reprogramming in Skeletal Muscle

International audienceIn vivo reprogramming is a promising approach for tissue regeneration in response to injury. Several examples of in vivo reprogramming have been reported in a variety of lineages, but some including skeletal muscle have so far proven refractory. Here, we show that acute and chronic injury enables transcription-factor-mediated reprogramming in skeletal muscle. Lineage tracing indicates that this response frequently originates from Pax7+ muscle stem cells. Injury is associated with accumulation of senescent cells, and advanced aging or local irradiation further enhanced in vivo reprogramming, while selective elimination of senescent cells reduced reprogramming efficiency. The effect of senescence appears to be, at least in part, due to the release of interleukin 6 (IL-6), suggesting a potential link with the senescence-associated secretory phenotype. Collectively, our findings highlight a beneficial paracrine effect of injury-induced senescence on cellular plasticity, which will be important for devising strategies for reprogramming-based tissue repair