Biology subjects

Le Grand, F.

Publications and source records attributed to Le Grand, F..

14 recordsLinked to original sources

bioRxiv2026

Postnatal Pax7-expressing limb cells are multipotent and generate non-myogenic lineages that persist into adulthood.

Organs are composed of a complex arrangement of diverse cell types that can originate from independent cell lineages or shared progenitors. Skeletal muscle is derived from mesodermal Pax7+ stem/progenitor cells that can differentiate into myoblasts to form muscle fibers. During embryogenesis, however, somitic Pax7+ cells can also give rise to non-muscle cell types, including dermis and adipocytes. Here, we asked whether Pax7+ cells retain such multipotency during early postnatal growth of limb muscles. Using lineage tracing, we uncovered unexpected plasticity at early postnatal days, leading to the generation of multiple non-myogenic lineages, including a previously unrecognized Pax7-derived subpopulation of fibro-adipogenic progenitors that we termed Pax7FAPs and further investigated. Using mouse models, we further show that Notch signaling primes neonatal Pax7+ cells toward a fibrogenic molecular identity at the expense of myogenic differentiation, thereby biasing their trajectory toward a fibrogenic fate. In the adult muscle, long-term tracing revealed that neonatally produced Pax7FAPs persist into adulthood. In addition, injury in adult muscle triggered de novo generation of Pax7FAPs, which displayed higher proliferative capacity than resident stromal cells. This newfound multipotency of postnatal Pax7+ cells adds a new dimension to our understanding of cellular contributions during postnatal muscle development and regeneration.

developmental biology↗

bioRxiv2024

Early lineage segregation of primary myotubes from secondary myotubes and adult muscle stem cells.

Myogenesis in amniotes unfolds through two consecutive waves. The primary myotube lineage is characterized by the expression of slow myosin, sometimes in combination with fast myosin, and may serve as a scaffold for the secondary lineage, which expresses exclusively fast myosin. The embryonic origin of these two lineages, their relationship, and their connection to adult muscle stem cells are unknown. Here, we employed innovative strategies, combining novel TCF-LEF/{beta}-catenin signaling reporters with the precise spatiotemporal control of in vivo electroporation in avian embryos, to track limb muscle progenitors from early migration to late fetal stages. Strikingly, we uncovered two distinct progenitor populations co-existing from the earliest stages of limb myogenesis, with specific developmental fates: reporter-positive progenitors exclusively form primary myotubes, while reporter-negative progenitors generate secondary myotubes and adult muscle stem cells. Furthermore, we uncovered a novel function of TCF-LEF/{beta}-catenin signaling in regulating the spatial organization of the primary myotube lineage via CXCR4-mediated control of myoblast migration, likely contributing to its proposed organizing function. By redefining the embryonic origins of these myogenic populations, our findings not only resolve a longstanding question in muscle biology but also provide a crucial molecular entry point for understanding the cellular and molecular underpinnings of muscle fiber type diversity and function.

developmental biology↗

bioRxiv2024

Immune aging impairs muscle regeneration via macrophage-derived anti-oxidant selenoprotein P

Muscle regeneration is impaired in the aged organism, due to both intrinsic defects of muscle stem cells (MuSCs) and alterations of their environmental niche. However, the latter has still been poorly explored. Here, we compared and analyzed the time course of the various cell types constituting the MuSC niche during muscle generation in young and old mice. Aging altered the amplification of all niche cells with particularly prominent phenotypes in macrophages that impaired the resolution of inflammation in the old regenerating muscle. RNAsequencing of FACs-isolated MuSCs and non-myogenic niche cells during regeneration uncovered specific profiles and kinetics of genes and molecular pathways differentially regulated in old versus young regenerating muscle, indicating that each cell type responded to aging in a specific manner. Through this, we discovered that macrophages have a strong signature of aging with altered the activation of Selenoprotein P (Sepp1) expression in macrophages during the resolution of inflammation in regenerating muscle. Macrophage-specific deletion of Sepp1 gene was sufficient to impair the acquisition of the repair inflammatory profile, perturbed the support of macrophages to MuSCs in vitro and in vivo, and to cause inefficient skeletal muscle regeneration. When transplanted in aged mice, bone marrow from young WT mice, but not Sepp1 KOs, restored muscle regeneration to youthful levels. Altogether this work provides a unique resource to study the aging of the MuSC niche, reveals that aging of niche cells is asynchronous and establishes impaired macrophage dynamics/polarization and the anti-oxidant Selenoprotein P expression as drivers of age-related decline of muscle regeneration. Teaser: Cell profiling reveals asynchronicity of aging in the muscle stem cell niche and age-dependent macrophage/stem cell interactions through anti-oxidant selenoprotein P

physiology↗

bioRxiv2024

Integrating in silico predictions with an engineered tissue assay identifies Perlecan as an age-perturbed re-quiescence cue for muscle stem cells

Skeletal muscle regeneration is mediated by resident muscle stem cells that produce progeny to repair or recreate muscle. Critical to this function is the ability to transition between states of proliferation and quiescence. This balancing act is disrupted with age, leading to eroded regenerative capacity. Notwistanding, mechanisms by which the regenerating niche directs MuSCs return to the dormant state are largely unknown. Since single-cell RNAseq methods exclude the analysis of multinucleated cells, we generated single-nuclei RNAseq datasets of regenerating muscle to capture the full breadth of myogenic progression. With this, we uncovered new transition states between differentiating myocytes and syncytial multinucleated cells. Using cell communication inference tools, we highlighted receptor-ligand interactions between MuSCs and fusing nuclei. We leveraged a bespoke biomimetic 3D niche that induces MuSC quiescence, to filter the predicted interactions using a Cas9-based functional genomics approach. We found the proteoglycan Perlecan (Hspg2) promotes MuSC re-quiescence. Hspg2 silencing in vivo, during muscle regeneration, induced an aging-like phenotype and perturbed MuSC re-quiescence. Notably, the temporal profile and overall levels of Perlecan were altered with age. Exogenous supplementation with Endorepellin (truncated Perlecan) rescued MuSC decline following aged muscle regeneration, offering a new therapeutic target. Thus, coupling in silico predictions with a 3D in vitro assay followed by in vivo investigations revealed a previously inaccessible window of biology; that spatiotemporal coordination of MuSC re-quiescence is directed by fusing myonuclei.

cell biology↗

bioRxiv2023

Setdb1 safeguards genome integrity in muscle stem cells to allow for regenerative myogenesis and inflammation

Modulations in chromatin structure orchestrate gene expression and direct stem cell fate. More specifically, the histone 3 lysine 9 Methyltransferase Setdb1 controls transcriptional repression to regulate pluripotency and self-renewal. While Setdb1 functions have been extensively studied in embryonic stem cells and in cancer cells, less is known on its role in adult stem cells in vivo. Here, we show that Setdb1 expression by adult muscle stem cells (MuSCs) is required for muscle tissue regeneration following acute injury. We find that SETDB1 represses the expression of the endogenous retroviruses (ERVs) family of transposable elements in MuSCs. ERV de-expression in Setdb1-null MuSCs prevents their amplification following exit from quiescence and promotes cell death. Multi-omics profiling further shows that the absence of SETDB1 in MuSCs leads to the activation of the DNA-sensing cGAS-STING pathway, entailing increased cytokine expression. In vivo, conditional disruption of Setdb1 in MuSCs provokes aberrant infiltration of inflammatory cells including the appearance of a pathological macrophage lineage. The ensuing histiocytosis is accompanied by necrosis of the newly formed muscle fibers which, in addition with the progressive loss of MuSCs, completely abolish skeletal muscle tissue repair. In contrast, disruption of Setdb1 gene in another muscle-resident cell type, the fibro-adipogenic progenitors (FAPs), does not impact regenerative inflammation. In conclusion, the control of genome stability by SETDB1 in an adult somatic stem cell is necessary for both its regenerative potential and an adequate inflammation regulating tissue repair.

cell biology↗

bioRxiv2022

Odd skipped-related 1 controls the pro-regenerative response of Fibro-Adipogenic Progenitors

Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGF{beta} signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.

developmental biology↗

bioRxiv2021

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for muscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which is problematic in the context of limited patient sample. Here we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments. Spontaneous contractions in myotubes derived from primary human myoblasts were observed. Analysis of nuclear morphology confirmed a similar organization between obtained myotubes and in vivo myofibers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modelled with successful development of mutant 3D myotubes displaying contractile dysfunction. This technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should allow to substantially improve drug screening capability.

bioengineering↗

bioRxiv2020

GLI3 Processing by the Primary Cilium Regulates Muscle Stem Cell Entry into GAlert

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3, specifically, plays a critical role in satellite cell activation. Primary cilia-mediated processing of GLI3 is required to maintain satellite cells in a G0 dormant state. Strikingly, satellite cells lacking GLI3 enter GAlert in the absence of injury. Furthermore, GLI3 depletion or inhibition of its processing stimulates symmetrical division in satellite cells and expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced long-term regenerative capacity. Therefore, our results reveal an essential role for primary cilia processing of GLI3 in regulating muscle stem cell activation and fate.

cell biology↗

bioRxiv2020

Trophic niche of the invasive gregarious species Crepidula fornicata, in relation to ontogenic changes

AO_SCPLOWBSTRACTC_SCPLOWThe slipper limpet Crepidula fornicata is a common and widespread invasive gregarious species along the European coast. Among its life-history traits, well documented ontogenic changes in behavior (i.e., motile male to sessile female) suggests a potential shift in feeding strategy across its life stages. Considering the ecological significance of this species in colonized areas, understanding how conspecifics share the trophic resource is crucial. Using fatty acids (FA) and stable isotopes (SI) as complementary trophic markers, we conducted a field survey between late winter and spring to investigate the trophic niche of three ontogenic stages of C. fornicata that bear different sexual (male/female) and motility (motile/sessile) traits. Potential trophic sources were characterized by their pigment, FA and SI compositions and discriminated well over the study period. We showed that the biofilm covering C. fornicata shells harbored a higher biomass of primary producers (i.e., chlorophytes and diatoms) than the surrounding sediment surface. Over the study period, we observed a covariation between the three ontogenic stages for both FA and SI compositions which suggest that the trophic niche of C. fornicata does not change significantly across its benthic life. During periods of low food availability, slipper limpets displayed an opportunistic suspension-feeding behaviour, relying on both fresh and detrital organic matter, likely coming from superficial sedimentary organic matter. During high food availability (i.e., spring phytoplankton bloom), all ontogenic stages largely benefited from this fresh supply of organic matter (pelagic diatoms in this case). However, the three ontogenic stages showed consistent differences in FA composition, and to a lesser extent in SI composition. These differences persist over time, as they originate more likely from ontogenic physiological changes (e.g., differential growth rates, metabolic rate or gametogenesis) rather than diet discrepancies.

ecology↗

bioRxiv2020

Differential myoblast and tenoblast affinity to collagen, fibrin and mixed threads in the prospect of muscle-tendon junction modelisation

The myotendinous junction transfers forces from muscle to tendon. As such, it must hold two tissues of completely different biological and cellular compositions as well as mechanical properties (kPa-MPa to MPa-GPa) and is subject to frequent stresses of high amplitude. This region remains a weak point of the muscle-tendon unit and is involved in frequent injuries. We here produce fibrin (40 mg/mL, E0 =0.10 {+/-} 0.02 MPa) and collagen (60 mg/mL, E0=0.57 {+/-} 0.05 MPa) threads as well as mixed collagen:fibrin threads (3:2 in mass, E0 = 0.33 {+/-} 0.05 MPa) and investigate the difference of affinity between primary murine myoblasts and tenoblasts. We demonstrate a similar behavior of cells on mixed and fibrin threads with high adherence of tenoblasts and myoblasts, in comparison to collagen threads that promote high adherence and proliferation of tenoblasts but not of myoblasts. Besides, we show that myoblasts on threads differentiate but do not fuse, on the contrary to 2D control substrates, raising the question of the effect of substrate curvature on the ability of myoblasts to fuse in vitro.

bioengineering↗

bioRxiv2020

GREM1 is epigenetically reprogrammed in muscle cells after exercise training and controls myogenesis and metabolism

Exercise training improves skeletal muscle function, notably through tissue regeneration by muscle stem cells. Here, we hypothesized that exercise training reprograms the epigenome of muscle cell, which could account for better muscle function. Genome-wide DNA methylation of myotube cultures established from middle-aged obese men before and after endurance exercise training identified a differentially methylated region (DMR) located downstream of Gremlin 1 (GREM1), which was associated with increased GREM1 expression. GREM1 expression was lower in muscle satellite cells from obese, compared to lean mice, and exercise training restored GREM1 levels to those of control animals. We show that GREM1 regulates muscle differentiation through the negative control of satellite cell self-renewal, and that GREM1 controls muscle lineage commitment and lipid oxidation through the AMPK pathway. Our study identifies novel functions of GREM1 and reveals an epigenetic mechanism by which exercise training reprograms muscle stem cells to improve skeletal muscle function.

cell biology↗

bioRxiv2020

Myofiber stretch induces tensile and shear deformation of muscle stem cells in their native niche

BackgroundMuscle stem cells (MuSCs) are requisite for skeletal muscle regeneration and homeostasis. Proper functioning of MuSCs, including activation, proliferation, and fate decision, is determined by an orchestrated series of events and communication between MuSCs and their niche consisting of the host myofiber and neighbouring cells. A multitude of biochemical stimuli are known to regulate fate and function of MuSCs. However, in addition to biochemical factors, it is conceivable that MuSCs residing between basal lamina and sarcolemma of myofibers are subjected to mechanical forces during muscle stretch-shortening cycles due to myofascial connections between MuSCs and myofibers. MuSCs have been shown to respond to mechanical forces in vitro but it remains to be proven whether physical forces are also exerted on MuSCs in their native niche and whether they contribute to the functioning and fate of MuSCs. MethodsMuSCs deformation in their native niche resulting from mechanical loading of ex vivo myofiber bundles were visualized utilizing mT/mG double-fluorescent Cre-reporter mouse and multiphoton microscopy. MuSCs were subjected to 1 hour pulsating fluid shear stress with a peak shear stress rate of 8.8 Pa/s. After treatment, nitric oxide and mRNA expression levels of genes involved in regulation of MuSC proliferation and differentiation were determined. ResultsEx vivo stretching of extensor digitorum longus and soleus myofiber bundles caused compression as well as tensile and shear deformation of MuSCs in their niche. MuSCs responded to pulsating fluid shear stress in vitro with increased nitric oxide production and an upward trend in iNOS mRNA levels, while nNOS expression was unaltered. Pulsating fluid shear stress enhanced gene expression of c-Fos, Cdk4, and IL-6, while expression of Wnt1, MyoD, Myog, Wnt5a, COX2, Rspo1, Vangl2, Wnt10b, and MGF remained unchanged. ConclusionsWe conclude that MuSCs in their native niche are subjected to force-induced deformations due to myofiber stretch-shortening. Moreover, MuSCs are mechanoresponsive as evident by pulsating fluid shear stress-mediated expression of factors by MuSCs known to promote proliferation.

cell biology↗

bioRxiv2019

TGFβ signaling curbs cell fusion and muscle regeneration

Fusion of muscle progenitor cells is necessary for skeletal muscle development and repair. Cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. While the cellular and molecular mechanisms promoting muscle cell fusion have been intensely investigated in recent years, molecular brakes restraining cell-cell fusion events to control syncytia formation have remained elusive. Here, we show that transforming growth factor beta (TGF{beta}) signaling is active in adult muscle cells throughout the fusion process and reduce muscle cell fusion independently of the differentiation step. In contrast, inhibition of TGF{beta} signaling enhances cell fusion and promotes branching between myotubes. Pharmacological modulation of the pathway in vivo perturbs muscle regeneration after injury. Exogenous addition of TGF{beta} protein results in a loss of muscle function while inhibition of the TGF{beta} pathway induces the formation of giant myofibres. Transcriptome analyses and functional assays revealed that TGF{beta} acts on actin dynamics and reduce cell spreading through modulation of actin-based protrusions. Together our results reveal a signaling pathway that limits mammalian myoblast fusion and add a new level of understanding to the molecular regulation of myogenesis.

cell biology↗

bioRxiv2018

High-dimensional single-cell cartography reveals novel skeletal muscle resident cell populations

Adult tissue repair and regeneration require the activation of resident stem/progenitor cells that can self-renew and generate differentiated progeny. The regenerative capacity of skeletal muscle relies on muscle satellite cells (MuSCs) and their interplay with different cell types within the niche. Yet, our understanding of the cells that compose the skeletal muscle tissue is limited and molecular definitions of the principal cell types are lacking. Using a combined approach of single-cell RNA-sequencing and mass cytometry, we precisely mapped the different cell types in adult skeletal muscle tissue and highlighted previously overlooked populations. We identified known functional populations, characterized their gene signatures, and determined key markers. Among the ten main cell populations present in skeletal muscle, we found an unexpected complexity in the interstitial compartment and identified two new cell populations. One express the transcription factor Scleraxis and generate tenocyte-like cells. The second express smooth muscle and mesenchymal cell markers (SMMCs). While distinct from MuSCs, SMMCs are endowed with myogenic potential and promote MuSC engraftment following transplantation. Our high-dimensional single-cell atlas uncovers principles of an adult tissue composition and can be exploited to reveal unknown cellular sub-fractions that contribute to tissue regeneration.

cell biology↗