ABSTRACT
Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
Subject(s)
Macrophages/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/injuries , Myoblasts/cytology , Nicotinamide Phosphoribosyltransferase/metabolism , Stem Cell Niche , Zebrafish/metabolism , Animals , Cell Proliferation , Disease Models, Animal , Humans , Macrophages/cytology , Male , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myoblasts/metabolism , Nicotinamide Phosphoribosyltransferase/genetics , PAX7 Transcription Factor/metabolism , RNA-Seq , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Regeneration/physiology , Single-Cell Analysis , Zebrafish/immunologyABSTRACT
The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.
Subject(s)
Animal Fins/embryology , Animal Fins/metabolism , Ectoderm/embryology , Ectoderm/metabolism , Somites/embryology , Somites/metabolism , Zebrafish/embryology , Animals , Biological Evolution , Cell Lineage , Ectoderm/cytology , Female , Limb Buds/cytology , Limb Buds/embryology , Limb Buds/metabolism , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Somites/cytologyABSTRACT
Deficiency of muscle basement membrane (MBM) component laminin-α2 leads to muscular dystrophy congenital type 1A (MDC1A), a currently untreatable myopathy. Laminin--α2 has two main binding partners within the MBM, dystroglycan and integrin. Integrins coordinate both cell adhesion and signalling; however, there is little mechanistic insight into integrin's function at the MBM. In order to study integrin's role in basement membrane development and how this relates to the MBM's capacity to handle force, an itgß1.b-/- zebrafish line was created. Histological examination revealed increased extracellular matrix (ECM) deposition at the MBM in the itgß1.b-/- fish when compared with controls. Surprisingly, both laminin and collagen proteins were found to be increased in expression at the MBM of the itgß1.b-/- larvae when compared with controls. This increase in ECM components resulted in a decrease in myotomal elasticity as determined by novel passive force analyses. To determine if it was possible to control ECM deposition at the MBM by manipulating integrin activity, RGD peptide, a potent inhibitor of integrin-ß1, was injected into a zebrafish model of MDC1A. As postulated an increase in laminin and collagen was observed in the lama2-/- mutant MBM. Importantly, there was also an improvement in fibre stability at the MBM, judged by a reduction in fibre pathology. These results therefore show that blocking ITGß1 signalling increases ECM deposition at the MBM, a process that could be potentially exploited for treatment of MDC1A.
Subject(s)
Integrin beta1/metabolism , Laminin/deficiency , Oligopeptides/pharmacology , Animals , Basement Membrane/metabolism , Biomarkers , Collagen/metabolism , Disease Models, Animal , Disease Susceptibility , Genetic Loci , Immunohistochemistry , Integrin beta1/genetics , Mice, Knockout , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophies/etiology , Muscular Dystrophies/metabolism , Muscular Dystrophies/pathology , Phenotype , Protein Stability/drug effectsABSTRACT
Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.
Subject(s)
Endothelial Cells/cytology , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/metabolism , Somites/cytology , Transcription Factors/metabolism , Zebrafish Proteins/metabolism , Animals , Aorta/cytology , Aorta/embryology , Biomarkers/analysis , Cell Movement , Chemokine CXCL12/analysis , Chemokine CXCL12/metabolism , Chick Embryo , Endothelial Cells/metabolism , Hematopoietic Stem Cells/metabolism , Homeodomain Proteins/analysis , Homeodomain Proteins/genetics , Humans , Mice , Muscles/cytology , Muscles/metabolism , Mutation/genetics , Somites/metabolism , Transcription Factors/analysis , Transcription Factors/genetics , Wnt Proteins/analysis , Wnt Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/analysis , Zebrafish Proteins/geneticsABSTRACT
Bone morphogenetic protein 1 (BMP1) is an astacin metalloprotease with important cellular functions and diverse substrates, including extracellular-matrix proteins and antagonists of some TGFß superfamily members. Combining whole-exome sequencing and filtering for homozygous stretches of identified variants, we found a homozygous causative BMP1 mutation, c.34G>C, in a consanguineous family affected by increased bone mineral density and multiple recurrent fractures. The mutation is located within the BMP1 signal peptide and leads to impaired secretion and an alteration in posttranslational modification. We also characterize a zebrafish bone mutant harboring lesions in bmp1a, demonstrating conservation of BMP1 function in osteogenesis across species. Genetic, biochemical, and histological analyses of this mutant and a comparison to a second, similar locus reveal that Bmp1a is critically required for mature-collagen generation, downstream of osteoblast maturation, in bone. We thus define the molecular and cellular bases of BMP1-dependent osteogenesis and show the importance of this protein for bone formation and stability.
Subject(s)
Bone Morphogenetic Protein 1/physiology , Osteogenesis/genetics , Osteogenesis/physiology , Animals , Base Sequence , Bone Density Conservation Agents/therapeutic use , Bone Morphogenetic Protein 1/genetics , Bone Morphogenetic Protein 1/metabolism , Bone and Bones/metabolism , Cell Differentiation , Child, Preschool , Collagen/biosynthesis , Diphosphonates/therapeutic use , Exome , Female , Fractures, Bone/drug therapy , Fractures, Bone/prevention & control , Genetic Loci , Heat-Shock Proteins , Humans , Male , Molecular Sequence Data , Mutation , Osteoblasts/drug effects , Osteoblasts/physiology , Osteogenesis/drug effects , Peptide Fragments , Protein Processing, Post-Translational , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
One of the central questions of developmental biology is how cells of equivalent potential-an equivalence group-come to adopt specific cellular fates. In this study we have used a combination of live imaging, single cell lineage analyses, and perturbation of specific signaling pathways to dissect the specification of the adaxial cells of the zebrafish embryo. We show that the adaxial cells are myogenic precursors that form a cell fate equivalence group of approximately 20 cells that consequently give rise to two distinct sub-types of muscle fibers: the superficial slow muscle fibers (SSFs) and muscle pioneer cells (MPs), distinguished by specific gene expression and cell behaviors. Using a combination of live imaging, retrospective and indicative fate mapping, and genetic studies, we show that MP and SSF precursors segregate at the beginning of segmentation and that they arise from distinct regions along the anterior-posterior (AP) and dorsal-ventral (DV) axes of the adaxial cell compartment. FGF signaling restricts MP cell fate in the anterior-most adaxial cells in each somite, while BMP signaling restricts this fate to the middle of the DV axis. Thus our results reveal that the synergistic actions of HH, FGF, and BMP signaling independently create a three-dimensional (3D) signaling milieu that coordinates cell fate within the adaxial cell equivalence group.
Subject(s)
Cell Differentiation , Morphogenesis , Muscle Fibers, Slow-Twitch/cytology , Muscle Fibers, Slow-Twitch/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Animals , Base Sequence , Bone Morphogenetic Proteins/metabolism , Fibroblast Growth Factors/metabolism , Gene Knockdown Techniques , Growth Differentiation Factor 6/metabolism , Hedgehog Proteins/metabolism , Morphogenesis/genetics , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Signal Transduction , Stem Cells/cytology , Stem Cells/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Laminins form essential components of the basement membrane and are integral to forming and maintaining muscle integrity. Mutations in the human Laminin-alpha2 (LAMA2) gene result in the most common form of congenital muscular dystrophy, MDC1A. We have previously identified a zebrafish model of MDC1A called candyfloss (caf), carrying a loss-of-function mutation in the zebrafish lama2 gene. In the skeletal muscle, laminins connect the muscle cell to the extracellular matrix (ECM) by binding either dystroglycan or integrins at the cell membrane. Through epistasis experiments, we have established that both adhesion systems individually contribute to the maintenance of fibre adhesions and exhibit muscle detachment phenotypes. However, larval zebrafish in which both adhesion systems are simultaneously genetically inactivated possess a catastrophic failure of muscle attachment that is far greater than a simple addition of individual phenotypes would predict. We provide evidence that this is due to other crucial laminins present in addition to Lama2, which aid muscle cell attachments and integrity. We have found that lama1 is important for maintaining attachments, whereas lama4 is localized and up-regulated in damaged fibres, which appears to contribute to fibre survival. Importantly, our results show that endogenous secretion of laminins from the surrounding tissues has the potential to reinforce fibre attachments and strengthen laminin-ECM attachments. Collectively these findings provide a better understanding of the cellular pathology of MDC1A and help in designing effective therapies.
Subject(s)
Epistasis, Genetic , Laminin , Muscle Development/genetics , Muscle, Skeletal , Zebrafish Proteins , Animals , Dystroglycans/metabolism , Extracellular Matrix/metabolism , Humans , Laminin/genetics , Laminin/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , Muscular Dystrophies/genetics , Muscular Dystrophies/metabolism , Muscular Dystrophies/physiopathology , Muscular Dystrophy, Animal/genetics , Muscular Dystrophy, Animal/metabolism , Muscular Dystrophy, Animal/pathology , Protein Binding , Receptors, Laminin/genetics , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.
Subject(s)
CRISPR-Cas Systems , Mutagenesis, Insertional , Plasmids , Zebrafish , Mutagenesis, Insertional/methods , Animals , Plasmids/genetics , Zebrafish/genetics , Genetic Vectors/genetics , Gene Editing/methods , AllelesABSTRACT
Sarcopenia, the age-related decline in muscle function, places a considerable burden on health-care systems. While the stereotypic hallmarks of sarcopenia are well characterized, their contribution to muscle wasting remains elusive, which is partly due to the limited availability of animal models. Here, we have performed cellular and molecular characterization of skeletal muscle from the African killifish-an extremely short-lived vertebrate-revealing that while many characteristics deteriorate with increasing age, supporting the use of killifish as a model for sarcopenia research, some features surprisingly reverse to an "early-life" state in the extremely old stages. This suggests that in extremely old animals, there may be mechanisms that prevent further deterioration of skeletal muscle, contributing to an extension of life span. In line with this, we report a reduction in mortality rates in extremely old killifish. To identify mechanisms for this phenomenon, we used a systems metabolomics approach, which revealed that during aging there is a striking depletion of triglycerides, mimicking a state of calorie restriction. This results in the activation of mitohormesis, increasing Sirt1 levels, which improves lipid metabolism and maintains nutrient homeostasis in extremely old animals. Pharmacological induction of Sirt1 in aged animals was sufficient to induce a late life-like metabolic profile, supporting its role in life span extension in vertebrate populations that are naturally long-lived. Collectively, our results demonstrate that killifish are not only a novel model to study the biological processes that govern sarcopenia, but they also provide a unique vertebrate system to dissect the regulation of longevity.
Subject(s)
Longevity , Sarcopenia , Animals , Sarcopenia/metabolism , Sirtuin 1/metabolism , Aging , Muscle, Skeletal/metabolism , Fundulus heteroclitus , Vertebrates , BiologyABSTRACT
Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1-mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1's essential role in maintaining muscle homeostasis.
Subject(s)
Disease Models, Animal , Endoplasmic Reticulum Chaperone BiP , Homeostasis , Muscle Proteins , Muscle, Skeletal , Muscular Dystrophy, Duchenne , SKP Cullin F-Box Protein Ligases , Zebrafish , Animals , SKP Cullin F-Box Protein Ligases/metabolism , SKP Cullin F-Box Protein Ligases/genetics , Muscle Proteins/metabolism , Muscle Proteins/genetics , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophy, Duchenne/genetics , Humans , Endoplasmic Reticulum Chaperone BiP/metabolism , Heat-Shock Proteins/metabolism , Heat-Shock Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Endoplasmic Reticulum/metabolism , Mitochondrial DynamicsABSTRACT
The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.
Subject(s)
Calcium-Binding Proteins/genetics , Embryo, Nonmammalian/metabolism , Extracellular Matrix Proteins/genetics , Hedgehog Proteins/genetics , Signal Transduction/genetics , Zebrafish Proteins/genetics , Animals , Blotting, Western , COS Cells , Calcium-Binding Proteins/metabolism , Chlorocebus aethiops , DNA, Complementary/chemistry , DNA, Complementary/genetics , Embryo, Nonmammalian/embryology , Extracellular Matrix Proteins/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Hedgehog Proteins/metabolism , In Situ Hybridization , Molecular Sequence Data , Multigene Family , Mutation , Phenotype , Sequence Analysis, DNA , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.
Subject(s)
Embryo, Nonmammalian/metabolism , Extracellular Matrix Proteins/genetics , Frasier Syndrome/genetics , Furin/genetics , Mutation , Proprotein Convertases/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Amino Acid Sequence , Animals , Base Sequence , Extracellular Matrix Proteins/metabolism , Furin/metabolism , Gene Expression Regulation, Developmental , Mice , Molecular Sequence Data , Proprotein Convertases/metabolism , Zebrafish Proteins/metabolismABSTRACT
Variants in UBA5 have been reported to cause neurological disease with impaired motor function, developmental delay, intellectual disability and brain pathology as recurrent clinical manifestations. UBA5 encodes a ubiquitin-activating-like enzyme that activates ufmylation, a post-translational ubiquitin-like modification pathway, which has been implicated in neurodevelopment and neuronal survival. The reason behind the variation in severity and clinical manifestations in affected individuals and the signal transduction pathways regulated by ufmylation that compromise the nervous system remains unknown. Zebrafish have emerged as a powerful model to study neurodegenerative disease due to its amenability for in vivo analysis of muscle and neuronal tissues, high-throughput examination of motor function and rapid embryonic development allowing an examination of disease progression. Using clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing, we developed and characterized zebrafish mutant models to investigate disease pathophysiology. uba5 mutant zebrafish showed a significantly impaired motor function accompanied by delayed growth and reduced lifespan, reproducing key phenotypes observed in affected individuals. Our study demonstrates the suitability of zebrafish to study the pathophysiology of UBA5-related disease and as a powerful tool to identify pathways that could reduce disease progression. Furthermore, uba5 mutants exhibited widespread mitochondrial damage in both the nervous system and the skeletal muscle, suggesting that a perturbation of mitochondrial function may contribute to disease pathology.
ABSTRACT
The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin beta2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.
Subject(s)
Laminin/genetics , Laminin/physiology , Muscular Dystrophy, Animal/embryology , Muscular Dystrophy, Animal/genetics , Mutation , Zebrafish Proteins/genetics , Zebrafish Proteins/physiology , Zebrafish/embryology , Zebrafish/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Basement Membrane/pathology , Cell Survival , DNA Primers/genetics , Eye/embryology , Homozygote , Molecular Sequence Data , Muscle Fibers, Skeletal/pathology , Muscular Dystrophy, Animal/pathology , Sarcolemma/pathology , Sequence Homology, Amino AcidABSTRACT
Muscle fiber detachment from myoseptal boundaries is a common finding in zebrafish models of muscular dystrophies. In some instances, there is a weakening of the interaction between muscle fiber and myosepta, which is yet to manifest as a fiber detachment phenotype. Therefore, to push the fiber detachment of muscle, mutant fish but not their wild-type siblings, beyond their binding threshold, a series of small electrical pulses can be applied to the larvae to create a maximal force contraction and ultimately fiber detachment. To do this, we built a digital pulse generator which delivers four 8 ms 30 V pulses in quick succession, and it has the advantage over older analog approaches to pulse generation because it improves accuracy and is appreciably less expensive. Our pulse generator significantly increases fiber detachment in the laminin-α2 deficient, congenital muscular dystrophy type 1a (MDC1a) model lama2-/- fish when compared with controls.
Subject(s)
Electric Stimulation/adverse effects , Muscle Fibers, Skeletal/pathology , Muscular Dystrophy, Animal/pathology , Zebrafish/physiology , Animals , Animals, Genetically Modified/growth & development , Bioelectric Energy Sources , Laminin/physiology , Larva/physiology , Larva/radiation effects , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/radiation effects , Muscular Dystrophy, Animal/metabolism , Mutation , Phenotype , Zebrafish/growth & development , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.
Subject(s)
Cell Lineage/physiology , G2 Phase Cell Cycle Checkpoints/physiology , Homeodomain Proteins/metabolism , Myoblasts/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Cell Line , Cyclin B1/genetics , Cyclin B1/metabolism , Homeodomain Proteins/genetics , Mice , Myoblasts/cytology , Transcription Factors , Zebrafish Proteins/geneticsABSTRACT
Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare "immortal" stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.
Subject(s)
Cell Division/physiology , Cell Tracking/methods , Muscle, Skeletal/physiology , Regeneration/physiology , Satellite Cells, Skeletal Muscle/physiology , Animals , Animals, Genetically Modified , Cell Division/genetics , Clone Cells , Muscle Development/genetics , Muscle Development/physiology , Muscle, Skeletal/embryology , Muscle, Skeletal/injuries , Mutation , Myogenic Regulatory Factor 5/genetics , Myogenin/genetics , Regeneration/genetics , Satellite Cells, Skeletal Muscle/cytology , Transgenes , ZebrafishABSTRACT
The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.
Subject(s)
Cell Differentiation/genetics , DNA-Binding Proteins/metabolism , Dwarfism/genetics , Pituitary Gland, Anterior/embryology , Transcription Factors/metabolism , Animals , Cell Lineage/genetics , Embryo, Nonmammalian/cytology , Gene Expression Regulation, Developmental/physiology , Genetic Techniques , Homeodomain Proteins/metabolism , Mutation/genetics , Transcription Factor Pit-1 , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
The adenohypophysis consists of at least six different cell types, somatotropes, lactotropes, thyrotropes, melanotropes, corticotropes, and gonadotropes. In mouse, cloning of spontaneous mutations and gene targeting has revealed multiple genes required for different steps of adenohypophysis development. Here, we report the results of a systematic search for genes required for adenohypophysis formation and patterning in zebrafish. By screening F3 offspring of N-ethyl-N-nitrosourea-mutagenized founder fish, we isolated eleven mutants with absent or reduced expression of GH, the product of somatotropes, but a normally developing hypothalamus. Of such mutants, eight were further analyzed and mapped. They define four genes essential for different steps of adenohypophysis development. Two of them, lia and pia, affect the entire adenohypophysis, whereas the other two are required for a subset of adenohypophyseal cell types only. The third gene is zebrafish pit1 and is required for lactotropes, thyrotropes, and somatotropes, similar to its mouse ortholog, whereas the fourth, aal, is required for corticotropes, melanotropes, thyrotropes, and somatotropes, but not lactotropes. In conclusion, the isolated zebrafish mutants confirm principles of adenohypophysis development revealed in mouse, thereby demonstrating the high degree of molecular and mechanistic conservation among the different vertebrate species. In addition, they point to thus far unknown features of adenohypophysis development, such as the existence of a new lineage of pituitary cells, which partially overlaps with the Pit1 lineage. Positional cloning of the lia, pia, and aal genes might reveal novel regulators of vertebrate pituitary development.
Subject(s)
Cell Lineage/genetics , Mutation/genetics , Pituitary Gland, Anterior/growth & development , Zebrafish/metabolism , Animals , Cloning, Molecular , Gene Expression Regulation, Developmental , Morphogenesis/genetics , Pituitary Gland, Anterior/metabolism , Zebrafish/geneticsABSTRACT
Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.