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1.
Immunity ; 56(6): 1204-1219.e8, 2023 06 13.
Article in English | MEDLINE | ID: mdl-37160119

ABSTRACT

During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.


Subject(s)
Organogenesis , Transcription Factors , Cell Differentiation , Lymph Nodes , Lymphoid Tissue
2.
Development ; 151(4)2024 Feb 15.
Article in English | MEDLINE | ID: mdl-38240380

ABSTRACT

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.


Subject(s)
Muscle, Skeletal , Oculomotor Muscles , Mice , Animals , Muscle, Skeletal/metabolism , Oculomotor Muscles/metabolism , Mice, Inbred C57BL , Cell Proliferation , Stem Cells
3.
Cell ; 148(1-2): 112-25, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22265406

ABSTRACT

Satellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFP(Hi) cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFP(Lo) cells. Pax7-nGFP(Hi) can give rise to Pax7-nGFP(Lo) cells after serial transplantations. Proliferating Pax7-nGFP(Hi) cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFP(Lo) cells perform random DNA segregation. Therefore, quiescent Pax7-nGFP(Hi) cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFP(Hi) cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically.


Subject(s)
Adult Stem Cells/cytology , Chromosome Segregation , Muscle, Skeletal/cytology , Satellite Cells, Skeletal Muscle/cytology , Animals , Cell Division , Female , Mice , Mice, Transgenic , PAX7 Transcription Factor/metabolism , Templates, Genetic
4.
Mol Cell ; 74(3): 609-621.e6, 2019 05 02.
Article in English | MEDLINE | ID: mdl-30922843

ABSTRACT

Adult tissue repair and regeneration require stem-progenitor cells that can self-renew and generate differentiated progeny. Skeletal muscle regenerative capacity relies on muscle satellite cells (MuSCs) and their interplay with different cell types within the niche. However, our understanding of skeletal muscle tissue cellular composition is limited. Here, using a combined approach of single-cell RNA sequencing and mass cytometry, we precisely mapped 10 different mononuclear cell types in adult mouse muscle. We also characterized gene signatures and determined key discriminating markers of each cell type. We identified two previously understudied cell populations in the interstitial compartment. One expresses the transcription factor scleraxis and generated tenocytes in vitro. The second expresses markers of smooth muscle and mesenchymal cells (SMMCs) and, while distinct from MuSCs, exhibited myogenic potential and promoted MuSC engraftment following transplantation. The blueprint presented here yields crucial insights into muscle-resident cell-type identities and can be exploited to study muscle diseases.


Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Muscle Fibers, Skeletal/cytology , Satellite Cells, Skeletal Muscle/cytology , Animals , Mice , Muscle Development/genetics , Muscle Fibers, Skeletal/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Single-Cell Analysis , Stem Cells/cytology , Stem Cells/metabolism
5.
PLoS Genet ; 20(6): e1010935, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38875306

ABSTRACT

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.


Subject(s)
Homeobox Protein PITX2 , Homeodomain Proteins , Myogenic Regulatory Factor 5 , Oculomotor Muscles , PAX7 Transcription Factor , Transcription Factors , Animals , Humans , Mice , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice, Inbred mdx , Muscle Development/genetics , Muscle, Skeletal/metabolism , Muscle, Skeletal/growth & development , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/pathology , Myogenic Regulatory Factor 5/genetics , Myogenic Regulatory Factor 5/metabolism , Oculomotor Muscles/metabolism , PAX7 Transcription Factor/metabolism , PAX7 Transcription Factor/genetics , Stem Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
6.
PLoS Genet ; 19(6): e1010781, 2023 06.
Article in English | MEDLINE | ID: mdl-37267426

ABSTRACT

Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells.


Subject(s)
Homeodomain Proteins , Somites , Mice , Animals , Homeodomain Proteins/metabolism , Cell Differentiation/genetics , Somites/metabolism , Muscle Development/genetics , Gene Expression Regulation, Developmental , Muscle, Skeletal/metabolism
7.
Exp Cell Res ; 424(1): 113484, 2023 03 01.
Article in English | MEDLINE | ID: mdl-36693490

ABSTRACT

A major challenge in the study of living systems is understanding how tissues and organs are established, maintained during homeostasis, reconstituted following injury or deteriorated during disease. Most of the studies that interrogate in vivo cell biological properties of cell populations within tissues are obtained through static imaging approaches. However, in vertebrates, little is known about which, when, and how extracellular and intracellular signals are dynamically integrated to regulate cell behaviour and fates, due largely to technical challenges. Intravital imaging of cellular dynamics in mammalian models has exposed surprising properties that have been missed by conventional static imaging approaches. Here we highlight some selected examples of intravital imaging in mouse intestinal stem cells, hematopoietic stem cells, hair follicle stem cells, and neural stem cells in the brain, each of which have distinct features from an anatomical and niche-architecture perspective. Intravital imaging of mouse skeletal muscles is comparatively less advanced due to several technical constraints that will be discussed, yet this approach holds great promise as a complementary investigative method to validate findings obtained by static imaging, as well as a method for discovery.


Subject(s)
Muscle, Skeletal , Neural Stem Cells , Mice , Animals , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/physiology , Hair Follicle , Hematopoietic Stem Cells , Mammals
8.
Nature ; 557(7707): 714-718, 2018 05.
Article in English | MEDLINE | ID: mdl-29795344

ABSTRACT

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix1-3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells4-9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.


Subject(s)
Calcitonin Receptor-Like Protein/metabolism , Collagen/metabolism , Muscle, Skeletal/cytology , Receptors, Notch/metabolism , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism , Signal Transduction , Stem Cell Niche , Animals , Cell Differentiation , Cell Proliferation , Cell Self Renewal , Collagen/genetics , Gene Expression Regulation , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Mice , Transcription, Genetic
9.
EMBO J ; 38(24): e103924, 2019 12 16.
Article in English | MEDLINE | ID: mdl-31797391

ABSTRACT

Ageing is a multi-factorial condition that results in a gradual decline in tissue and organ function. Systemic, local and intrinsic factors play major roles in this process that also results in a decline in stem cell number and function. In this issue of The EMBO Journal, Li et al (2019) show that a subpopulation of mouse muscle stem cells is depleted in aged mice through loss of niche-derived granulocyte colony-stimulating factor (G-CSF).


Subject(s)
Cellular Senescence/physiology , Granulocyte Colony-Stimulating Factor/metabolism , Satellite Cells, Skeletal Muscle/physiology , Animals , Genetic Heterogeneity , Mice , Signal Transduction
10.
Development ; 147(19)2020 10 09.
Article in English | MEDLINE | ID: mdl-32591430

ABSTRACT

Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.


Subject(s)
Homeodomain Proteins/metabolism , PAX7 Transcription Factor/metabolism , Trans-Activators/metabolism , Animals , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Muscle Development/genetics , Muscle Development/physiology , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , PAX7 Transcription Factor/genetics , Stem Cells/cytology , Stem Cells/metabolism , Trans-Activators/genetics
11.
PLoS Biol ; 18(11): e3000902, 2020 11.
Article in English | MEDLINE | ID: mdl-33201874

ABSTRACT

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.


Subject(s)
Oculomotor Muscles/embryology , Oculomotor Muscles/growth & development , Tretinoin/metabolism , Animals , Connective Tissue/physiology , Embryonic Development , Eye , Imaging, Three-Dimensional/methods , Mice/embryology , Mice, Inbred C57BL , Mice, Inbred DBA , Morphogenesis , Signal Transduction , Tendons/physiology , Tretinoin/physiology
12.
Annu Rev Cell Dev Biol ; 25: 671-99, 2009.
Article in English | MEDLINE | ID: mdl-19575640

ABSTRACT

The regulation of self-renewal, cell diversity, and differentiation can occur by modulating symmetric and asymmetric cell divisions. Remarkably, asymmetric cell divisions can arise through multiple processes in which molecules in the cytoplasm and nucleus, as well as template "immortal" DNA strands, can segregate to one daughter cell during cell division. Explaining how these events direct distinct daughter cell fates is a major challenge to understanding how the organism is assembled and maintained for a lifetime. Numerous technical issues that are associated with assessing how distinct cell fates are executed in vivo have resulted in divergent interpretations of experimental findings. This review addresses some of these points and considers different developmental model systems that attempt to investigate how cell fate decisions are determined, as well as the molecules that guide these choices.


Subject(s)
Cell Division , Animals , Drosophila/cytology , Humans , Stem Cells/cytology
13.
PLoS Genet ; 16(10): e1009022, 2020 10.
Article in English | MEDLINE | ID: mdl-33125370

ABSTRACT

Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.


Subject(s)
Cell Plasticity/genetics , Epigenome/genetics , Stem Cell Transplantation , Transcriptome/genetics , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Proliferation/genetics , Extremities/growth & development , Genetic Variation/genetics , Humans , Mice , Mice, Inbred C57BL , Muscle Cells/cytology , Muscle Fibers, Skeletal , Muscle, Skeletal/cytology , Myoblasts/cytology , Regeneration/genetics , Stem Cells/cytology , Stem Cells/metabolism
14.
Development ; 146(20)2019 10 24.
Article in English | MEDLINE | ID: mdl-31575648

ABSTRACT

The control of all our motor outputs requires constant monitoring by proprioceptive sensory neurons (PSNs) that convey continuous muscle sensory inputs to the spinal motor network. Yet the molecular programs that control the establishment of this sensorimotor circuit remain largely unknown. The transcription factor RUNX3 is essential for the early steps of PSNs differentiation, making it difficult to study its role during later aspects of PSNs specification. Here, we conditionally inactivate Runx3 in PSNs after peripheral innervation and identify that RUNX3 is necessary for maintenance of cell identity of only a subgroup of PSNs, without discernable cell death. RUNX3 also controls the sensorimotor connection between PSNs and motor neurons at limb level, with muscle-by-muscle variable sensitivities to the loss of Runx3 that correlate with levels of RUNX3 in PSNs. Finally, we find that muscles and neurotrophin 3 signaling are necessary for maintenance of RUNX3 expression in PSNs. Hence, a transcriptional regulator that is crucial for specifying a generic PSN type identity after neurogenesis is later regulated by target muscle-derived signals to contribute to the specialized aspects of the sensorimotor connection selectivity.


Subject(s)
Core Binding Factor Alpha 3 Subunit/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Core Binding Factor Alpha 3 Subunit/genetics , Female , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Motor Neurons/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Sensory Receptor Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
15.
Development ; 145(23)2018 11 26.
Article in English | MEDLINE | ID: mdl-30478226

ABSTRACT

Cell fate decisions occur through the action of multiple factors, including signalling molecules and transcription factors. Recently, the regulation of translation has emerged as an important step for modulating cellular function and fate, as exemplified by ribosomes that play distinct roles in regulating cell behaviour. Notchless (Nle) is a conserved nuclear protein that is involved in a crucial step in ribosome biogenesis, and is required for the maintenance of adult haematopoietic and intestinal stem/progenitor cells. Here, we show that activated skeletal muscle satellite cells in conditional Nle mutant mice are arrested in proliferation; however, deletion of Nle in myofibres does not impair myogenesis. Furthermore, conditional deletion of Nle in satellite cells during homeostasis did not impact on their fate for up to 3 months. In contrast, loss of Nle function in primary myogenic cells blocked proliferation because of major defects in ribosome formation. Taken together, we show that muscle stem cells undergo a stage-specific regulation of ribosome biogenesis, thereby underscoring the importance of differential modulation of mRNA translation for controlling cell fate decisions.


Subject(s)
Cell Lineage , Membrane Proteins/metabolism , Muscle Development , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , Organelle Biogenesis , Ribosomes/metabolism , Animals , Cell Cycle , Cell Differentiation , Cells, Cultured , Cyclin E/metabolism , Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Mice, Knockout , Mutation/genetics , Myoblasts/cytology , Myoblasts/metabolism , Regeneration , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism
16.
Development ; 145(21)2018 10 29.
Article in English | MEDLINE | ID: mdl-30266829

ABSTRACT

The transcription factor Nfix belongs to the nuclear factor one family and has an essential role in prenatal skeletal muscle development, where it is a master regulator of the transition from embryonic to foetal myogenesis. Recently, Nfix was shown to be involved in adult muscle regeneration and in muscular dystrophies. Here, we have investigated the signalling that regulates Nfix expression, and show that JunB, a member of the AP-1 family, is an activator of Nfix, which then leads to foetal myogenesis. Moreover, we demonstrate that their expression is regulated through the RhoA/ROCK axis, which maintains embryonic myogenesis. Specifically, RhoA and ROCK repress ERK kinase activity, which promotes JunB and Nfix expression. Notably, the role of ERK in the activation of Nfix is conserved postnatally in satellite cells, which represent the canonical myogenic stem cells of adult muscle. As lack of Nfix in muscular dystrophies rescues the dystrophic phenotype, the identification of this pathway provides an opportunity to pharmacologically target Nfix in muscular dystrophies.


Subject(s)
MAP Kinase Signaling System , Muscle Development , Myoblasts/metabolism , NFI Transcription Factors/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Animals, Newborn , Embryo, Mammalian/metabolism , Female , Fetus/metabolism , Gene Expression Regulation, Developmental , Gene Silencing , Male , Mice , NFI Transcription Factors/genetics , Stem Cells/metabolism , Transcription Factors/metabolism , rho-Associated Kinases/metabolism
17.
Development ; 145(6)2018 03 19.
Article in English | MEDLINE | ID: mdl-29555813

ABSTRACT

Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.


Subject(s)
Cell Differentiation/genetics , Mesoderm/cytology , Muscle Development/genetics , Muscle, Skeletal/cytology , Pluripotent Stem Cells/cytology , Animals , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/physiology , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Humans , Immunohistochemistry , Immunophenotyping , In Situ Hybridization , In Vitro Techniques , Mesoderm/metabolism , Mesoderm/physiology , Mice , Muscle Development/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/physiology , Real-Time Polymerase Chain Reaction , Tissue Array Analysis , Wnt Signaling Pathway/genetics
18.
Nat Rev Mol Cell Biol ; 10(11): 804-10, 2009 11.
Article in English | MEDLINE | ID: mdl-19851338

ABSTRACT

Old and newly synthesized centrosomes have different microtubule nucleating abilities and they contribute to cell polarity when they migrate to opposite poles during cell division. The asymmetric localization of epigenetic marks and kinetochore proteins could lead to the differential recognition of sister chromatids and the biased segregation of DNA strands to daughter cells during cell division. We propose that this asymmetric localization is linked to biased chromatid segregation, which might also be related to the acquisition of distinct cell fates after mitosis.


Subject(s)
Centrosome , DNA/genetics , Mitosis/physiology , Animals , Humans
19.
Genes Dev ; 27(9): 1059-71, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23651858

ABSTRACT

Notch signaling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighboring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and, together with the transcription factor moiety of Notch (NICD), regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole-genome binding profiles were generated for RBPJ; its coactivator, p300; NICD; and the histone H3 modifications H3 Lys 4 trimethylation (H3K4me3), H3 Lys 4 monomethylation (H3K4me1), and histone H3 Lys 27 acetylation (H3K27ac) in myogenic cells under active or inhibitory Notch signaling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300 and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signaling mediate their activities and consequently impact on cell fate decisions.


Subject(s)
Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Receptors, Notch/metabolism , Signal Transduction , Animals , Binding Sites , Cell Line , Chromatin/genetics , Genome-Wide Association Study , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Mice , Protein Binding , Regulatory Elements, Transcriptional/genetics
20.
J Cell Sci ; 131(14)2018 07 27.
Article in English | MEDLINE | ID: mdl-30054310

ABSTRACT

During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process.


Subject(s)
Muscle Development , Muscle, Skeletal/growth & development , Stem Cells/cytology , Stem Cells/metabolism , Animals , Cell Proliferation , Fatty Acids/metabolism , Mice , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Oxidation-Reduction , Peroxisomes/metabolism , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/metabolism
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