ABSTRACT
The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFß signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFß signaling in Gli1+ cell function, the receptor for TGFß, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFß signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFß signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFß controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFß directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.
Subject(s)
Hedgehog Proteins , Transforming Growth Factor beta , Animals , Mice , Hedgehog Proteins/genetics , Zinc Finger Protein GLI1/genetics , Tendons , Signal TransductionABSTRACT
By translating mechanical forces into molecular signals, proprioceptive neurons provide the CNS with information on muscle length and tension, which is necessary to control posture and movement. However, the identities of the molecular players that mediate proprioceptive sensing are largely unknown. Here, we confirm the expression of the mechanosensitive ion channel ASIC2 in proprioceptive sensory neurons. By combining in vivo proprioception-related functional tests with ex vivo electrophysiological analyses of muscle spindles, we showed that mice lacking Asic2 display impairments in muscle spindle responses to stretch and motor coordination tasks. Finally, analysis of skeletons of Asic2 loss-of-function mice revealed a specific effect on spinal alignment. Overall, we identify ASIC2 as a key component in proprioceptive sensing and a regulator of spine alignment.
Subject(s)
Acid Sensing Ion Channels , Proprioception , Animals , Mice , Acid Sensing Ion Channels/metabolism , Muscle Spindles/physiology , Proprioception/physiology , Sensory Receptor Cells/metabolismABSTRACT
Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex, non-consecutive, three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb identified three progenitor states: naive, proximal, and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest an alternative model for limb skeleton development.
Subject(s)
Extremities , Skeleton , Animals , Mice , Cell Differentiation , Extremities/growth & development , Organogenesis , Skeleton/growth & developmentABSTRACT
The proprioceptive system is essential for the control of coordinated movement, posture, and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle isolated from the murine deep masseter muscle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.
Subject(s)
Multiomics , Muscle Spindles , Mice , Animals , Muscle Spindles/physiology , Proteomics , Muscle, Skeletal/physiology , Proprioception/physiologyABSTRACT
The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for force transfer from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the developmental transcriptome of the mouse entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following the prior discovery of the necessity of Gli1-lineage cells for mouse enthesis development and healing, we then examined their transcriptomes at single-cell resolution and demonstrated clonogenicity and multipotency of the Gli1-expressing progenitors. Transplantation of Gli1-lineage cells to mouse enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration.
Subject(s)
Tendons , Wound Healing , Animals , Mice , Zinc Finger Protein GLI1 , Chondrocytes , Transcription FactorsABSTRACT
Wound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.
Subject(s)
Cicatrix , Wound Healing , Animals , Extracellular Matrix , Inflammation , Mice , Tendons , Wound Healing/physiologyABSTRACT
The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process.
Subject(s)
Chondrocytes/physiology , Growth Differentiation Factor 5/metabolism , Growth Plate/growth & development , Animals , Animals, Newborn , Cell Differentiation , Cell Proliferation , Embryo, Mammalian , Female , Growth Differentiation Factor 5/economics , Growth Plate/cytology , Growth Plate/diagnostic imaging , Imaging, Three-Dimensional , Intravital Microscopy , Mice, Knockout , Models, Animal , Tibia/cytology , Tibia/drug effects , Tibia/growth & development , X-Ray MicrotomographyABSTRACT
Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that PDK family is dispensable for murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios but died by postnatal day 4 because of hypoglycemia or ketoacidosis. Moreover, PDC was phosphorylated in these embryos, suggesting that another kinase compensates for PDK family. Bioinformatic analysis implicated branched-chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched-chain amino acids (BCAAs) catabolism. Indeed, knockout of Bckdk and Pdk family led to the loss of PDC phosphorylation, an increase in PDC activity and pyruvate entry into the TCA cycle, and embryonic lethality. These findings reveal a regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.
Subject(s)
Citric Acid Cycle , Embryonic Development , Protein Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/metabolism , Pyruvate Dehydrogenase Complex/metabolism , Animals , Animals, Newborn , Embryo Loss/enzymology , Embryo Loss/pathology , Gene Deletion , Hypoglycemia/complications , Hypoglycemia/enzymology , Hypoglycemia/pathology , Ketosis/complications , Ketosis/enzymology , Ketosis/pathology , Mice, Knockout , Models, Biological , Phosphorylation , Pyruvic Acid/metabolismABSTRACT
The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.
Subject(s)
Chondrocytes/metabolism , Kruppel-Like Transcription Factors/genetics , Tenocytes/metabolism , Transcriptome , Animals , Bone and Bones , Female , Kruppel-Like Factor 4/genetics , Kruppel-Like Factor 4/metabolism , Kruppel-Like Transcription Factors/metabolism , Mice , Regulatory Sequences, Nucleic Acid , TendonsABSTRACT
Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.
Subject(s)
Bone and Bones/ultrastructure , Decalcification Technique/methods , Fluorescent Antibody Technique/methods , Proteins/isolation & purification , Staining and Labeling/methods , Animals , Bone and Bones/diagnostic imaging , Humans , Mice , Paraffin Embedding/methods , Proteins/chemistryABSTRACT
In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.
Subject(s)
Ion Channels/metabolism , Musculoskeletal Abnormalities/metabolism , Musculoskeletal System/metabolism , Neurons/metabolism , Proprioception/physiology , Abnormalities, Multiple , Animals , Bone Remodeling , Core Binding Factor Alpha 3 Subunit/metabolism , Disease Models, Animal , Early Growth Response Protein 3/metabolism , Genetic Predisposition to Disease/genetics , Hip Dislocation/genetics , Hip Dislocation/metabolism , Hip Dislocation/pathology , Hip Joint/anatomy & histology , Hip Joint/metabolism , Hip Joint/pathology , Ion Channels/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Musculoskeletal Abnormalities/genetics , Musculoskeletal Abnormalities/pathology , Musculoskeletal System/pathology , ScoliosisABSTRACT
Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Bone and Bones/anatomy & histology , Bone and Bones/embryology , Genes, Developmental , Homeodomain Proteins/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Development/genetics , Bone and Bones/metabolism , Embryo, Mammalian , Female , Gene Expression Regulation, Developmental/physiology , Genes, Developmental/genetics , Homeodomain Proteins/metabolism , Ligaments/anatomy & histology , Ligaments/embryology , Ligaments/metabolism , Male , Mice , Mice, Transgenic , Organ Specificity/genetics , Pre-B-Cell Leukemia Transcription Factor 1/genetics , Pre-B-Cell Leukemia Transcription Factor 1/metabolism , Pregnancy , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Tendons/anatomy & histology , Tendons/embryology , Tendons/metabolismABSTRACT
Sesamoid bones are small auxiliary bones that form near joints and contribute to their stability and function. Thus far, providing a comprehensive developmental model or classification system for this highly diverse group of bones has been challenging. Here, we compare our previously reported mechanisms of patella development in the mouse with those of two anatomically different sesamoids, namely lateral fabella and digit sesamoids. We show that all three types of sesamoid bones originate from Sox9+ /Scx+ progenitors under the regulation of TGFß and independently of mechanical stimuli from muscles. Whereas BMP2 regulates the growth of all examined sesamoids, the differentiation of lateral fabella or digit sesamoids is regulated redundantly by BMP4 and BMP2. Next, we show that whereas patella and digit sesamoids initially form in juxtaposition to long bones, lateral fabella forms independently and at a distance. Finally, our evidence suggests that, unlike the synovial joint that separates patella from femur, digit sesamoids detach from the phalanx by formation of a fibrocartilaginous joint. These findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, which underscores their evolutionary plasticity.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Muscles/embryology , SOX9 Transcription Factor/genetics , Sesamoid Bones/embryology , Sesamoid Bones/growth & development , Transforming Growth Factor beta/metabolism , Animals , Biological Evolution , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 4/metabolism , Cartilage/metabolism , Cell Lineage , Female , Femur/metabolism , Fibrocartilage/metabolism , Heterozygote , Male , Mice , Mice, Inbred C57BL , Muscles/metabolism , Patella/embryology , Patella/growth & development , Sesamoid Bones/cytology , Signal Transduction , Stress, Mechanical , Synovial Fluid/metabolismABSTRACT
Osteocytes, cells forming an elaborate network within the bones of most vertebrate taxa, are thought to be the master regulators of bone modeling, a process of coordinated, local bone-tissue deposition and removal that keeps bone strains at safe levels throughout life. Neoteleost fish, however, lack osteocytes and yet are known to be capable of bone modeling, although no osteocyte-independent modeling regulatory mechanism has so far been described. Here, we characterize a novel, to our knowledge, bone-modeling regulatory mechanism in a fish species (medaka), showing that although lacking osteocytes (i.e., internal mechanosensors), when loaded, medaka bones model in mechanically directed ways, successfully reducing high tissue strains. We establish that as in mammals, modeling in medaka is regulated by the SOST gene, demonstrating a mechanistic link between skeletal loading, SOST down-regulation, and intense bone deposition. However, whereas mammalian SOST is expressed almost exclusively by osteocytes, in both medaka and zebrafish (a species with osteocytic bones), SOST is expressed by a variety of nonosteocytic cells, none of which reside within the bone bulk. These findings argue that in fishes (and perhaps other vertebrates), nonosteocytic skeletal cells are both sensors and responders, shouldering duties believed exclusive to osteocytes. This previously unrecognized, SOST-dependent, osteocyte-independent mechanism challenges current paradigms of osteocyte exclusivity in bone-modeling regulation, suggesting the existence of multivariate feedback networks in bone modeling-perhaps also in mammalian bones-and thus arguing for the possibility of untapped potential for cell targets in bone therapeutics.
Subject(s)
Feedback, Physiological , Fish Proteins/genetics , Glycoproteins/genetics , Mechanotransduction, Cellular/genetics , Oryzias/genetics , Osteogenesis/genetics , Zebrafish Proteins/genetics , Animals , Biomechanical Phenomena , Bone Remodeling/genetics , Bone and Bones/cytology , Bone and Bones/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Collagen Type I/genetics , Collagen Type I/metabolism , Fish Proteins/metabolism , Gene Expression Regulation , Glycoproteins/metabolism , Humans , Oryzias/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Osteocytes , Protein Isoforms/genetics , Protein Isoforms/metabolism , Species Specificity , Swimming/physiology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Tendon-bone attachment sites, called entheses, are essential for musculoskeletal function. They are formed embryonically by Sox9+ progenitors and continue to develop postnatally, utilizing Gli1 lineage cells. Despite their importance, we lack information on the transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and the Gli1 lineage. Here, by performing a series of lineage tracing experiments in mice, we identify the onset of Gli1 lineage contribution to different entheses. We show that Gli1 expression is regulated embryonically by SHH signaling, whereas postnatally it is maintained by IHH signaling. During bone elongation, some entheses migrate along the bone shaft, whereas others remain stationary. Interestingly, in stationary entheses Sox9+ cells differentiate into the Gli1 lineage, but in migrating entheses this lineage is replaced by Gli1 lineage. These Gli1+ progenitors are defined embryonically to occupy the different domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple embryonic tissue, whereas another population contributes to its maturation. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.
Subject(s)
Bone and Bones/physiology , Movement , Stem Cells/cytology , Tendons/physiology , Animals , Animals, Newborn , Cell Compartmentation , Cell Death , Cell Lineage , Embryo, Mammalian/cytology , Embryonic Development , Female , Hedgehog Proteins/metabolism , Male , Mice, Inbred C57BL , Models, Biological , Osteoclasts/cytology , Osteoclasts/metabolism , Phagocytes/cytology , Phagocytes/metabolism , SOX9 Transcription Factor/metabolism , Stem Cells/metabolism , Zinc Finger Protein GLI1/metabolismABSTRACT
Muscle spindles and Golgi tendon organs (GTOs) are two types of sensory receptors that respond to changes in length or tension of skeletal muscles. These mechanosensors have long been known to participate in both proprioception and stretch reflex. Here, we present recent findings implicating these organs in maintenance of spine alignment as well as in realignment of fractured bones. These discoveries have been made in several mouse lines lacking functional mechanosensors in part or completely. In both studies, the absence of functional spindles and GTOs produced a more severe phenotype than that of spindles alone. Interestingly, the spinal curve phenotype, which appeared during peripubertal development, bears resemblance to the human condition adolescent idiopathic scoliosis. This similarity may contribute to the study of the disease by offering both an animal model and a clue as to its aetiology. Moreover, it raises the possibility that impaired proprioceptive signalling may be involved in the aetiology of other conditions. Overall, these new findings expand considerably the scope of involvement of proprioception in musculoskeletal development and function.This article is part of the Theo Murphy meeting issue 'Mechanics of development'.
Subject(s)
Mechanoreceptors/physiology , Muscle, Skeletal/physiology , Proprioception/physiology , Spine/embryology , Animals , Disease Models, Animal , Humans , Mice/embryology , Muscle Spindles/physiology , Scoliosis/etiology , Scoliosis/pathologyABSTRACT
During embryogenesis, the musculoskeletal system develops while containing within itself a force generator in the form of the musculature. This generator becomes functional relatively early in development, exerting an increasing mechanical load on neighboring tissues as development proceeds. A growing body of evidence indicates that such mechanical forces can be translated into signals that combine with the genetic program of organogenesis. This unique situation presents both a major challenge and an opportunity to the other tissues of the musculoskeletal system, namely bones, joints, tendons, ligaments and the tissues connecting them. Here, we summarize the involvement of muscle-induced mechanical forces in the development of various vertebrate musculoskeletal components and their integration into one functional unit.
Subject(s)
Musculoskeletal Development/physiology , Animals , Biomechanical Phenomena , Bone Development/physiology , Chondrogenesis/physiology , Humans , Mice , Models, Biological , Muscle Development/physiology , Signal TransductionABSTRACT
Maintaining posture requires tight regulation of the position and orientation of numerous spinal components. Yet, surprisingly little is known about this regulatory mechanism, whose failure may result in spinal deformity as in adolescent idiopathic scoliosis. Here, we use genetic mouse models to demonstrate the involvement of proprioception in regulating spine alignment. Null mutants for Runx3 transcription factor, which lack TrkC neurons connecting between proprioceptive mechanoreceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking muscle spindles, but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types may be required for this regulatory mechanism. These findings uncover a central role for the proprioceptive system in maintaining spinal alignment.
Subject(s)
Core Binding Factor Alpha 3 Subunit/genetics , Early Growth Response Protein 3/genetics , Mechanoreceptors/metabolism , Proprioception , Scoliosis/genetics , Animals , Enhancer Elements, Genetic , Mechanoreceptors/physiology , Mice , Mice, Inbred C57BL , Muscle, Skeletal/growth & development , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Phenotype , Spinal Cord/growth & development , Spinal Cord/metabolism , Spinal Cord/physiologyABSTRACT
Successful fracture repair requires restoration of bone morphology and mechanical integrity. Recent evidence shows that fractured bones of neonatal mice undergo spontaneous realignment, dubbed "natural reduction." Here, we show that natural reduction is regulated by the proprioceptive system and improves with age. Comparison among mice of different ages revealed, surprisingly, that 3-month-old mice exhibited more rapid and effective natural reduction than newborns. Fractured bones of null mutants for transcription factor Runx3, lacking functional proprioceptors, failed to realign properly. Blocking Runx3 expression in the peripheral nervous system, but not in limb mesenchyme, recapitulated the null phenotype, as did inactivation of muscles flanking the fracture site. Egr3 knockout mice, which lack muscle spindles but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types, as well as muscle contraction, are required for this regulatory mechanism. These findings uncover a physiological role for proprioception in non-autonomous regulation of skeletal integrity.
Subject(s)
Bone and Bones/pathology , Fractures, Bone/etiology , Animals , Fractures, Bone/pathology , Humans , Mice , ProprioceptionABSTRACT
Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix.