Building a Co-ordinated Musculoskeletal System: The Plasticity of the Developing Skeleton in Response to Muscle Contractions.

The skeletal musculature and the cartilage, bone and other connective tissues of the skeleton are intimately co-ordinated. The shape, size and structure of each bone in the body is sculpted through dynamic physical stimuli generated by muscle contraction, from early development, with onset of the first embryo movements, and through repair and remodelling in later life. The importance of muscle movement during development is shown by congenital abnormalities where infants that experience reduced movement in the uterus present a sequence of skeletal issues including temporary brittle bones and joint dysplasia. A variety of animal models, utilising different immobilisation scenarios, have demonstrated the precise timing and events that are dependent on mechanical stimulation from movement. This chapter lays out the evidence for skeletal system dependence on muscle movement, gleaned largely from mouse and chick immobilised embryos, showing the many aspects of skeletal development affected. Effects are seen in joint development, ossification, the size and shape of skeletal rudiments and tendons, including compromised mechanical function. The enormous plasticity of the skeletal system in response to muscle contraction is a key factor in building a responsive, functional system. Insights from this work have implications for our understanding of morphological evolution, particularly the challenging concept of emergence of new structures. It is also providing insight for the potential of physical therapy for infants suffering the effects of reduced uterine movement and is enhancing our understanding of the cellular and molecular mechanisms involved in skeletal tissue differentiation, with potential for informing regenerative therapies.

Geometric analysis of chondrogenic self-organisation of embryonic limb bud cells in micromass culture.

Spatial and temporal control of chondrogenesis generates precise, species-specific patterns of skeletal structures in the developing vertebrate limb. The pattern-template is laid down when mesenchymal cells at the core of the early limb bud condense and undergo chondrogenic differentiation. Although the mechanisms involved in organising such complex patterns are not fully understood, the interplay between BMP and Wnt signalling pathways is fundamental. Primary embryonic limb bud cells grown under high-density micromass culture conditions spontaneously create a simple cartilage nodule pattern, presenting a model to investigate pattern generation. We describe a novel analytical approach to quantify geometric properties and spatial relationships between chondrogenic condensations, utilizing the micromass model. We follow the emergence of pattern in live cultures with nodules forming at regular distances, growing and changing shape over time. Gene expression profiling supports rapid chondrogenesis and transition to hypertrophy, mimicking the process of endochondral ossification within the limb bud. Manipulating the signalling environment through addition of BMP or Wnt ligands, as well as the BMP pathway antagonist Noggin, altered the differentiation profile and nodule pattern. BMP2 addition increased chondrogenesis while WNT3A or Noggin had the opposite effect, but with distinct pattern outcomes. Titrating these pro- and anti-chondrogenic factors and examining the resulting patterns support the hypothesis that regularly spaced cartilage nodules formed by primary limb bud cells in micromass culture are influenced by the balance of Wnt and BMP signalling under a Turing-like mechanism. This study demonstrates an approach for investigating the mechanisms governing chondrogenic spatial organization using simple micromass culture.

Mechanical Stimulation via Muscle Activity Is Necessary for the Maturation of Tendon Multiscale Mechanics During Embryonic Development.

During embryonic development, tendons transform into a hypocellular tissue with robust tensile load-bearing capabilities. Previous work suggests that this mechanical transformation is due to increases in collagen fibril length and is dependent on mechanical stimulation via muscle activity. However, the relationship between changes in the microscale tissue structure and changes in macroscale tendon mechanics is still unclear. Additionally, the specific effect of mechanical stimulation on the multiscale structure-function relationships of developing tendons is also unknown. Therefore, the objective of this study was to measure the changes in tendon mechanics and structure at multiple length scales during embryonic development with and without skeletal muscle paralysis. Tensile testing of tendons from chick embryos was performed to determine the macroscale tensile modulus as well as the magnitude of the fibril strains and interfibrillar sliding with applied tissue strain. Embryos were also treated with either decamethonium bromide or pancuronium bromide to produce rigid or flaccid paralysis. Histology was performed to assess changes in tendon size, spacing between tendon subunits, and collagen fiber diameter. We found that the increase in the macroscale modulus observed with development is accompanied by an increase in the fibril:tissue strain ratio, which is consistent with an increase in collagen fibril length. Additionally, we found that flaccid paralysis reduced the macroscale tendon modulus and the fibril:tissue strain ratio, whereas less pronounced effects that were not statistically significant were observed with rigid paralysis. Finally, skeletal paralysis also reduced the size of collagen fibril bundles (i.e., fibers). Together, these data suggest that more of the applied tissue strain is transmitted to the collagen fibrils at later embryonic ages, which leads to an increase in the tendon macroscale tensile mechanics. Furthermore, our data suggest that mechanical stimulation during development is necessary to induce structural and mechanical changes at multiple physical length scales. This information provides valuable insight into the multiscale structure-function relationships of developing tendons and the importance of mechanical stimulation in producing a robust tensile load-bearing soft tissue.

Joint development recovery on resumption of embryonic movement following paralysis.

Fetal activity in utero is a normal part of pregnancy and reduced or absent movement can lead to long-term skeletal defects, such as Fetal Akinesia Deformation Sequence, joint dysplasia and arthrogryposis. A variety of animal models with decreased or absent embryonic movements show a consistent set of developmental defects, providing insight into the aetiology of congenital skeletal abnormalities. At developing joints, defects include reduced joint interzones with frequent fusion of cartilaginous skeletal rudiments across the joint. At the spine, defects include shortening and a spectrum of curvature deformations. An important question, with relevance to possible therapeutic interventions for human conditions, is the capacity for recovery with resumption of movement following short-term immobilisation. Here, we use the well-established chick model to compare the effects of sustained immobilisation from embryonic day (E)4-10 to two different recovery scenarios: (1) natural recovery from E6 until E10 and (2) the addition of hyperactive movement stimulation during the recovery period. We demonstrate partial recovery of movement and partial recovery of joint development under both recovery conditions, but no improvement in spine defects. The joints examined (elbow, hip and knee) showed better recovery in hindlimb than forelimb, with hyperactive mobility leading to greater recovery in the knee and hip. The hip joint showed the best recovery with improved rudiment separation, tissue organisation and commencement of cavitation. This work demonstrates that movement post paralysis can partially recover specific aspects of joint development, which could inform therapeutic approaches to ameliorate the effects of human fetal immobility. This article has an associated First Person interview with the first author of the paper.

Quantifying the tolerance of chick hip joint development to temporary paralysis and the potential for recovery.

BACKGROUND: Abnormal fetal movements are implicated in joint pathologies such as arthrogryposis and developmental dysplasia of the hip (DDH). Experimentally induced paralysis disrupts joint cavitation and morphogenesis leading to postnatal abnormalities. However, the developmental window(s) most sensitive to immobility-and therefore the best time for intervention-have never been identified. Here, we systematically vary the timing and duration of paralysis during early chick hip joint development. We then test whether external manipulation of immobilized limbs can mitigate the effects of immobility. RESULTS: Timing of paralysis affected the level of disruption to joints, with paralysis periods between embryonic days 4 and 7 most detrimental. Longer paralysis periods produced greater disruption in terms of failed cavitation and abnormal femoral and acetabular geometry. External manipulation of an immobilized limb led to more normal morphogenesis and cavitation compared to un-manipulated limbs. CONCLUSIONS: Temporary paralysis is detrimental to joint development, particularly during days 4 to 7. Developmental processes in the very early stages of joint development may be critical to DDH, arthrogryposis, and other joint pathologies. The developing limb has the potential to recover from periods of immobility, and external manipulation provides an innovative avenue for prevention and treatment of developmental joint pathologies.

Localization of YAP activity in developing skeletal rudiments is responsive to mechanical stimulation.

BACKGROUND: Normal skeletal development, in particular ossification, joint formation and shape features of condyles, depends on appropriate mechanical input from embryonic movement but it is unknown how such physical stimuli are transduced to alter gene regulation. Hippo/Yes-Associated Protein (YAP) signalling has been shown to respond to the physical environment of the cell and here we specifically investigate the YAP effector of the pathway as a potential mechanoresponsive mediator in the developing limb skeleton. RESULTS: We show spatial localization of YAP protein and of pathway target gene expression within developing skeletal rudiments where predicted biophysical stimuli patterns and shape are affected in immobilization models, coincident with the period of sensitivity to movement, but not coincident with the expression of the Hippo receptor Fat4. Furthermore, we show that under reduced mechanical stimulation, in immobile, muscle-less mouse embryos, this spatial localization is lost. In culture blocking YAP reduces chondrogenesis but the effect differs depending on the timing and/or level of YAP reduction. CONCLUSIONS: These findings implicate YAP signalling, independent of Fat4, in the transduction of mechanical signals during key stages of skeletal patterning in the developing limb, in particular endochondral ossification and shape emergence, as well as patterning of tissues at the developing synovial joint.

Effects of Abnormal Muscle Forces on Prenatal Joint Morphogenesis in Mice.

Fetal movements are essential for normal development of the human skeleton. When fetal movements are reduced or restricted, infants are at higher risk of developmental dysplasia of the hip and arthrogryposis (multiple joint contractures). Joint shape abnormalities have been reported in mouse models with abnormal or absent musculature, but the effects on joint shape in such models have not been quantified or characterized in detail. In this study, embryonic mouse forelimbs and hindlimbs at a single developmental stage (Theiler Stage 23) with normal, reduced, or absent muscle were imaged in three-dimensions. Skeletal rudiments were virtually segmented and rigid image registration was used to reliably align rudiments with each other, enabling repeatable assessment and measurement of joint shape differences between normal, reduced-muscle and absent-muscle groups. We demonstrate qualitatively and quantitatively that joint shapes are differentially affected by a lack of, or reduction in, skeletal muscle, with the elbow joint being the most affected of the major limb joints. Surprisingly, the effects of reduced muscle were often more pronounced than those of absent skeletal muscle, indicating a complex relationship between muscle mass and joint morphogenesis. These findings have relevance for human developmental disorders of the skeleton in which abnormal fetal movements are implicated, particularly developmental dysplasia of the hip and arthrogryposis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2287-2296, 2019.

Investigating the mechanistic basis of biomechanical input controlling skeletal development: exploring the interplay with Wnt signalling at the joint.

Embryo movement is essential to the formation of a functional skeleton. Using mouse and chick models, we previously showed that mechanical forces influence gene regulation and tissue patterning, particularly at developing limb joints. However, the molecular mechanisms that underpin the influence of mechanical signals are poorly understood. Wnt signalling is required during skeletal development and is altered under reduced mechanical stimulation. Here, to explore Wnt signalling as a mediator of mechanical input, the expression of Wnt ligand and Fzd receptor genes in the developing skeletal rudiments was profiled. Canonical Wnt activity restricted to the developing joint was shown to be reduced under immobilization, while overexpression of activated ß-catenin following electroporation of chick embryo limbs led to joint expansion, supporting the proposed role for Wnt signalling in mechanoresponsive joint patterning. Two key findings advance our understanding of the interplay between Wnt signalling and mechanical stimuli: first, loss of canonical Wnt activity at the joint shows reciprocal, coordinated misregulation of BMP signalling under altered mechanical influence. Second, this occurs simultaneously with increased expression of several Wnt pathway component genes in a territory peripheral to the joint, indicating the importance of mechanical stimulation for a population of potential joint progenitor cells.This article is part of the Theo Murphy meeting issue 'Mechanics of Development'.

Precise spatial restriction of BMP signaling in developing joints is perturbed upon loss of embryo movement.

Dynamic mechanical loading of synovial joints is necessary for normal joint development, as evidenced in certain clinical conditions, congenital disorders and animal models where dynamic muscle contractions are reduced or absent. Although the importance of mechanical forces on joint development is unequivocal, little is known about the molecular mechanisms involved. Here, using chick and mouse embryos, we observed that molecular changes in expression of multiple genes analyzed in the absence of mechanical stimulation are consistent across species. Our results suggest that abnormal joint development in immobilized embryos involves inappropriate regulation of Wnt and BMP signaling during definition of the emerging joint territories, i.e. reduced ß-catenin activation and concomitant upregulation of pSMAD1/5/8 signaling. Moreover, dynamic mechanical loading of the developing knee joint activates Smurf1 expression; our data suggest that Smurf1 insulates the joint region from pSMAD1/5/8 signaling and is essential for maintenance of joint progenitor cell fate.

Abnormal fetal muscle forces result in defects in spinal curvature and alterations in vertebral segmentation and shape.

The incidence of congenital spine deformities, including congenital scoliosis, kyphosis, and lordosis, may be influenced by the in utero mechanical environment, and particularly by fetal movements at critical time-points. There is a limited understanding of the influence of fetal movements on spinal development, despite the fact that mechanical forces have been shown to play an essential role in skeletal development of the limb. This study investigates the effects of muscle forces on spinal curvature, vertebral segmentation, and vertebral shape by inducing rigid or flaccid paralysis in the embryonic chick. The critical time-points for the influence of fetal movements on spinal development were identified by varying the time of onset of paralysis. Prolonged rigid paralysis induced severe defects in the spine, including curvature abnormalities, posterior and anterior vertebral fusions, and altered vertebral shape, while flaccid paralysis did not affect spinal curvature or vertebral segmentation. Early rigid paralysis resulted in more severe abnormalities in the spine than later rigid paralysis. The findings of this study support the hypothesis that the timing and nature of fetal muscle activity are critical influences on the normal development of the spine, with implications for the understanding of congenital spine deformities. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2135-2144, 2017.

Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure.

Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.

Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways.

BACKGROUND: Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. RESULTS: We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus into a transcriptional response. CONCLUSIONS: This work identifies key developmental regulatory genes impacted by altered mechanical stimulation, sheds light on the molecular mechanisms that interpret mechanical stimulation during skeletal development and provides valuable resources for further investigation of the mechanistic basis of mechanoregulation. In particular it highlights the Wnt signalling pathway as a potential point of integration of mechanical and molecular signalling and cytoskeletal components as mediators of the response.

Transcriptome analysis of the mouse E14.5 (TS23) developing humerus and differential expression in muscle-less mutant embryos lacking mechanical stimulation.

Mechanical stimulation is important for the correct formation of the skeleton. Splotch-delayed mutant embryos (Pax3 (Spd/Spd) ) that develop with no limb muscle and therefore no limb movement experience an altered mechanical environment resulting in specific defects in ossification and joint formation, particularly in the forelimb. To test the hypothesis that mechanical stimuli influence the regulation of genes important in skeletal development we generated a transcriptome profile of the developing humerus at Theiler stage 23 (TS23), and then identified differentially expressed genes in muscle-less mutant embryos compared to control littermates. Here we describe the experimental methods and analysis of the resulting data, publically available in the ArrayExpress database under E-MTAB-1745 (Transcriptome of control humerus), E-MTAB-1744 (Microarray; differential expression) and E-MTAB-1746 (RNA-sequencing; differential expression). Our data provide a resource for exploring the transcriptome that underlies skeletal development at TS23 in the mouse humerus. The interpretation and description of this data can be found in a recent publication in BMC Genomics [1]. This is a resource for exploring the molecular mechanisms that are involved in skeletal development and mechanotransduction.

Mechanical regulation of skeletal development.

Development of the various components of a normal skeleton requires highly regulated signalling systems that co-ordinate spatial and temporal patterns of cell division, cell differentiation, and morphogenesis. Much work in recent decades has revealed cascades of molecular signalling, acting through key transcription factors to regulate, for example, organized chondrogenic and osteogenic differentiation. It is now clear that mechanical stimuli are also required for aspects of skeletogenesis but very little is known about how the mechanical signals are integrated with classic biochemical signalling. Spatially organized differentiation is vital to the production of functionally appropriate tissues contributing to precise, region specific morphologies, for example transient chondrogenesis of long bone skeletal rudiments, which prefigures osteogenic replacement of the cartilage template, compared with the production of permanent cartilage at the sites of articulation. Currently a lack of understanding of how these tissues are differentially regulated hampers efforts to specifically regenerate stable bone and cartilage. Here, we review current research revealing the influence of mechanical stimuli on specific aspects of skeletal development and refer to other developing systems to set the scene for current and future work to uncover the molecular mechanisms involved. We integrate this with a brief overview of the effects of mechanical stimulation on stem cells in culture bringing together developmental and tissue engineering aspects of mechanoregulation of cell behavior. A better understanding of the molecular mechanisms that link mechanical stimuli to transcriptional control guiding cell differentiation will lead to new ideas about how to effectively prime stem cells for tissue engineering and regenerative therapies.

Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells.

Hydrostatic pressure (HP) is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-ß3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs), synovial membrane derived stem cells (SDSCs) and infrapatellar fat pad derived stem cells (FPSCs) were subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-ß3 (0, 1 and 10 ng/mL) for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-ß3 stimulation. HP in the absence of TGF-ß3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-ß3 (1 ng/mL), HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-ß3 (10 ng/mL). Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.