10 research outputs found
Extracellular matrix proteins in regeneration : a study using the zebrafish caudal fin model
A cĂŽtĂ© de leur rĂŽle structural au sein des tissus, les protĂ©ines de la matrice extracellulaire sont impliquĂ©es dans un grand nombre de processus cellulaires au cours de divers Ă©vĂšnements biologiques. En revanche, leur rĂŽle au cours de la rĂ©gĂ©nĂ©ration reste Ă©tonnamment peu Ă©tudiĂ© Ă ce jour. Pourtant, mieux comprendre le rĂŽle de la MEC dans la rĂ©gĂ©nĂ©ration a de nombreuses applications en mĂ©decine rĂ©gĂ©nĂ©rative et reconstructrice. Mon projet de thĂšse vise prĂ©cisĂ©ment Ă rĂ©pondre Ă cette question. Pour cela, nous avons utilisĂ© le modĂšle bien Ă©tabli de la rĂ©gĂ©nĂ©ration de la nageoire caudale du poisson zĂšbre qui prĂ©sente de nombreux avantages tels quâune structure simple, facile dâaccĂšs et un rĂ©gĂ©nĂ©ration rapide, en seulement quelques jours. Une approche globale de transcriptomique sans a priori a permis dâĂ©tablir lâimportance de la matrice extracellulaire au cours de la rĂ©gĂ©nĂ©ration. Une premiĂšre Ă©tape a consistĂ© Ă Ă©tablir la liste des gĂšnes de la matrice extracellulaire du poisson zĂšbre par orthologie, appelĂ© matrisome. Notre Ă©tude a fait Ă©merger le rĂŽle inattendu dâun collagĂšne dans la reconstruction de la membrane basale de lâĂ©piderme, une structure importante pour lâattachement de lâĂ©piderme au derme dans la peau. Cette protĂ©ine, exprimĂ©e uniquement chez lâembryon, est rĂ©-exprimĂ©e dans lâĂ©piderme en rĂ©gĂ©nĂ©ration et dĂ©posĂ©e au niveau de la membrane basale. Par stratĂ©gie anti-sens in vivo, jâai montrĂ© par microscopie Ă force atomique et microscopie Ă©lectronique que lâabsence de ce collagĂšne impacte la structure et les propriĂ©tĂ©s biomĂ©caniques de cette membrane basale en reconstruction. Ces rĂ©sultats ont Ă©tĂ© confirmĂ©s sur une lignĂ©e de poisson, invalidĂ©e pour ce gĂšne que nous avons crĂ©Ă©e par la technologie CRISPR/Cas9. Cette lignĂ©e a permis dâĂ©tablir que ce collagĂšne agit transitoirement comme un « spacer » molĂ©culaire nĂ©cessaire Ă lâorganisation tridimensionnelle des autres composants de la membrane basale pendant la rĂ©gĂ©nĂ©ration.In addition to their role within tissues, extracellular matrix proteins are implicated in a large number of cellular processes. However, their role in regeneration is not well studied at the moment. A better understanding of the extracellular matrix proteins involvement in regeneration can have several future applications for regenerative and reconstructive medicine. The aim of my PhD project is to answer this question.To do this, we used the well-established zebrafish caudal fin model which have many advantages such as a simple structure, easily accessible and a quick regeneration in only few days. A global transcriptomic approach without a priori showed us that extracellular matrix proteins are playing a key role in regeneration. A first step of my work was to use an orthology-based approach to create the first list of extracellular matrix genes in zebrafish, called the matrisome. Our study revealed the unexpected role of a collagen during epidermal basement membrane reconstruction, an importance structure for the dermo-epidermal cohesion in skin. This protein which is expressed only during embryogenesis, is re-expressed in the regenerating epidermis and deposited in the basement membrane. Using an anti-sense strategy in vivo, I have demonstrated by atomic force microscopy and electron microscopy that the absence of this collagen impacts the biomechanics of this reconstructing basement membrane. These results were confirmed on a zebrafish line invalidated for this collagen that I have generated using the genome editing CRISPR/Cas9 technic. We showed that this collagen acts as a molecular spacer needed for the correct tridimensional organization of the other basement membrane components during regeneration
Fishing for collagen function: About development, regeneration and disease
International audienc
Gene profile of zebrafish fin regeneration offers clues to kinetics, organization and biomechanics of basement membrane
International audienceHow some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM
Lack of the myotendinous junction marker col22a1 results in posture and locomotion disabilities in zebrafish
International audienceThe myotendinous junction (MTJ) is essential for the integrity of the musculoskeletal unit. Here, we show that gene ablation of the MTJ marker col22a1 in zebrafish results in MTJ dysfunction but with variable degrees of expression and distinct phenotypic classes. While most individuals reach adulthood with no overt muscle phenotype (class 1), a subset of the progeny displays severe movement impairment and die before metamorphosis (class 2). Yet all mutants display muscle weakness due to ineffective muscle force transmission that is ultimately detrimental for class-specific locomotion-related functions. Movement impairment at the critical stage of swimming postural learning causes class 2 larval death by compromising food intake. In class 1 adults, intensive exercise is required to uncover a decline in muscle performance, accompanied by higher energy demand and mitochondrial adaptation. This study underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease
The myotendinous junction marker collagen XXII enables zebrafish postural control learning and optimal swimming performance through its force transmission activity
Abstract Although the myotendinous junction (MTJ) is essential for skeletal muscle integrity, its contribution to skeletal muscle function remains largely unknown. Here, we show that CRISPR-Cas9-mediated gene ablation of the MTJ marker col22a1 in zebrafish identifies two distinctive phenotypic classes: class 1 individuals reach adulthood with no overt muscle phenotype while class 2 display severe movement impairment and eventually dye before metamorphosis. Yet mutants that are unequally affected are all found to display defective force transmission attributed to a loss of ultrastructural integrity of the MTJ and myosepta, though with distinct degrees of severity. The behavior-related consequences of the resulting muscle weakness similarly reveal variable phenotypic expressivity. Movement impairment at the critical stage of swimming postural learning eventually causes class 2 larval death by compromising food intake while intensive exercise is required to uncover a decline in muscle performance in class 1 adults. By confronting MTJ gene expression compensation and structural, functional and behavioral insights of MTJ dysfunction, our work unravels variable expressivity of col22a1 mutant phenotype. This study also underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease
Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption
International audienceBACKGROUND: Bone and joint infection, mainly caused by Staphylococcus aureus, is associated with significant morbidity and mortality, characterized by severe inflammation and progressive bone destruction. Studies mostly focused on the interaction between S. aureus and osteoblasts, the bone matrix-forming cells, while interactions between S. aureus and osteoclasts, the only cells known to be able to degrade bone, have been poorly explored. METHODS: We developed an in vitro infection model of primary murine osteoclasts to study the direct impact of live S. aureus on osteoclastogenesis and osteoclast resorption activity. RESULTS: Staphylococcal infection of bone marrow-derived osteoclast precursors induced their differentiation into activated macrophages that actively secreted proinflammatory cytokines. These cytokines enhanced the bone resorption capacity of uninfected mature osteoclasts and promoted osteoclastogenesis of the uninfected precursors at the site of infection. Moreover, infection of mature osteoclasts by live S. aureus directly enhanced their ability to resorb bone by promoting cellular fusion. CONCLUSIONS: Our results highlighted two complementary mechanisms involved in bone loss during bone and joint infection, suggesting that osteoclasts could be a pivotal target for limiting bone destruction
Dual Impact of Live Staphylococcus aureus on the Osteoclast Lineage, Leading to Increased Bone Resorption
Proâinflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Angâ(1â7)
Abstract Recessive dystrophic epidermolysis bullosa (RDEB), a genetic skin blistering disease, is a paradigmatic condition of tissue fragilityâdriven multiâorgan fibrosis. Here, longitudinal analyses of the tissue proteome through the course of naturally developing disease in RDEB mice revealed that increased proâinflammatory immunity associates with fibrosis evolution. Mechanistically, this fibrosis is a consequence of altered extracellular matrix organization rather than that of increased abundance of major structural proteins. In a humanized system of disease progression, we targeted inflammatory cell fibroblast communication with Angâ(1â7)âan antiâinflammatory heptapeptide of the reninâangiotensin system, which reduced the fibrosisâevoking aptitude of RDEB cells. In vivo, systemic administration of Angâ(1â7) efficiently attenuated progression of multiâorgan fibrosis and increased survival of RDEB mice. Collectively, our study shows that selective downâmodulation of proâinflammatory immunity may mitigate injuryâinduced fibrosis. Furthermore, together with published data, our data highlight molecular diversity among fibrotic conditions. Both findings have direct implications for the design of therapies addressing skin fragility and fibrosis