Synovial joint cavitation initiates with microcavities in interzone and is coupled to skeletal flexion and elongation in developing mouse embryo limbs.

The synovial cavity and its fluid are essential for joint function and lubrication, but their developmental biology remains largely obscure. Here, we analyzed E12.5 to E18.5 mouse embryo hindlimbs and discovered that cavitation initiates around E15.0 with emergence of multiple, discrete, µm-wide tissue discontinuities we term microcavities in interzone, evolving into a single joint-wide cavity within 12 h in knees and within 72-84 h in interphalangeal joints. The microcavities were circumscribed by cells as revealed by mTmG imaging and exhibited a carbohydrate and protein content based on infrared spectral imaging at micro and nanoscale. Accounting for differing cavitation kinetics, we found that the growing femur and tibia anlagen progressively flexed at the knee over time, with peak angulation around E15.5 exactly when the full knee cavity consolidated; however, interphalangeal joint geometry changed minimally over time. Indeed, cavitating knee interzone cells were elongated along the flexion angle axis and displayed oblong nuclei, but these traits were marginal in interphalangeal cells. Conditional Gdf5Cre-driven ablation of Has2 - responsible for production of the joint fluid component hyaluronic acid (HA) - delayed the cavitation process. Our data reveal that cavitation is a stepwise process, brought about by sequential action of microcavities, skeletal flexion and elongation, and HA accumulation. This article has an associated First Person interview with the first author of the paper.

Autophagy coordinates chondrocyte development and early joint formation in zebrafish.

Autophagy is a catabolic process responsible for the removal of waste and damaged cellular components by lysosomal degradation. It plays a key role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for cartilage cell (chondrocyte) development and survival, and for the formation of cartilage. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders such as osteoarthritis and osteoporosis. To test the requirement for autophagy during skeletal development in zebrafish, we generated an atg13 CRISPR knockout zebrafish line. This line showed a complete loss of atg13 expression, and restricted autophagic activity in vivo. In the absence of autophagy, chondrocyte maturation was accelerated, with chondrocytes exhibiting signs of premature hypertrophy. Focussing on the jaw element, autophagy disruption affected joint articulation causing restricted mouth opening. This gross behavioural phenotype corresponded with a failure to thrive, and death in homozygote atg13 nulls within 17 days. Taken together, our results are consistent with autophagy contributing to the timely regulation of chondrocyte maturation and for extracellular matrix formation.

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.

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.

Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton.

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP).

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.

A dot-stripe Turing model of joint patterning in the tetrapod limb.

Iterative joints are a hallmark of the tetrapod limb, and their positioning is a key step during limb development. Although the molecular regulation of joint formation is well studied, it remains unclear what controls the location, number and orientation (i.e. the pattern) of joints within each digit. Here, we propose the dot-stripe mechanism for joint patterning, comprising two coupled Turing systems inspired by published gene expression patterns. Our model can explain normal joint morphology in wild-type limbs, hyperphalangy in cetacean flippers, mutant phenotypes with misoriented joints and suggests a reinterpretation of the polydactylous Ichthyosaur fins as a polygonal joint lattice. By formulating a generic dot-stripe model, describing joint patterns rather than molecular joint markers, we demonstrate that the insights from the model should apply regardless of the biological specifics of the underlying mechanism, thus providing a unifying framework to interrogate joint patterning in the tetrapod limb.

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.

BMPs, TGFß, and border security at the interzone.

Synovial joints enable movement and protect the integrity of the articular cartilage. Joints form within skeletal condensations destined to undergo chondrogenesis. The suppression of this chondrogenic program in the interzone is the first morphological sign of joint formation. While we have a fairly good understanding of the essential roles of BMP and TGFß family members in promoting chondrogenic differentiation in developing skeletal elements, we know very little about how BMP activity is suppressed specifically within the interzone, a crucial step in joint development. The function of the BMP ligand Gdf5 has been especially difficult to decipher. On the one hand, Gdf5 is required to promote chondrogenesis of articular elements. On the other hand, Gdf5 is highly expressed in the joint interzone where chondrogenesis must be suppressed for the formation of many joints. Here we review the evidence that BMP signaling must be suppressed within the joint interzone for joint morphogenesis to progress, and consider how Gdf5 exerts its divergent effects on chondrogenesis and joint formation. We also consider how TGFß signaling impacts formation of the interzone. Finally, we propose a model whereby Gdf5 exerts distinct effects in the interzone vs. surrounding cartilage based on the repertoire of BMP receptors available in these tissues. Understanding how BMP antagonists and counteracting TGFß signals intersect with Gdf5 to sculpt the joint interzone is essential for understanding the origin of osteoarthritis and other diseases of joint tissues.

Joints in the appendicular skeleton: Developmental mechanisms and evolutionary influences.

The joints are a diverse group of skeletal structures, and their genesis, morphogenesis, and acquisition of specialized tissues have intrigued biologists for decades. Here we review past and recent studies on important aspects of joint development, including the roles of the interzone and morphogenesis of articular cartilage. Studies have documented the requirement of interzone cells in limb joint initiation and formation of most, if not all, joint tissues. We highlight these studies and also report more detailed interzone dissection experiments in chick embryos. Articular cartilage has always received special attention owing to its complex architecture and phenotype and its importance in long-term joint function. We pay particular attention to mechanisms by which neonatal articular cartilage grows and thickens over time and eventually acquires its multi-zone structure and becomes mechanically fit in adults. These and other studies are placed in the context of evolutionary biology, specifically regarding the dramatic changes in limb joint organization during transition from aquatic to land life. We describe previous studies, and include new data, on the knee joints of aquatic axolotls that unlike those in higher vertebrates, are not cavitated, are filled with rigid fibrous tissues and resemble amphiarthroses. We show that when axolotls metamorph to life on land, their intra-knee fibrous tissue becomes sparse and seemingly more flexible and the articular cartilage becomes distinct and acquires a tidemark. In sum, there have been considerable advances toward a better understanding of limb joint development, biological responsiveness, and evolutionary influences, though much remains unclear. Future progress in these fields should also lead to creation of new developmental biology-based tools to repair and regenerate joint tissues in acute and chronic conditions.

Wnt-signaling in skeletal development.

As the vertebrate skeleton develops it progresses from a solely cartilaginous scaffold to a mineralized bony skeleton. The cells that build up the skeleton, the chondrocytes and osteoblasts, are primarily of mesodermal origin. Yet, some facial bones, as well as the endocranium, are derived from neural crest cells. The differentiation of the mesenchymal cells to skeletal precursors as well as their subsequent differentiation and maturation along the two lineages, chondrogenic and osteogenic, is controlled by various different signaling pathways, among them Wnt-signaling. WNTs comprise a family of 19 secreted cysteine-rich glycoproteins and can signal through a variety of different intracellular pathways. Genetic loss- and gain-of-function experiments of Wnt-signaling pathway genes have helped to uncover their multiple roles in skeletogenesis, which will be discussed in this article primarily focusing on endochondral bone formation.

Bapx1 is required for jaw joint development in amphibians.

The acquisition of a movable jaw and a jaw joint are key events in gnathostome evolution. Jaws are derived from the neural crest derived pharyngeal skeleton and the transition from jawless to jawed vertebrates consists of major morphological changes, which must have a genetic foundation. Recent studies on the effects of bapx1 knockdown in fish and chicken indicate that bapx1 has acquired such a role in primary jaw joint development during vertebrate evolution, but evidence from amphibians is missing so far. In the present study, we use Ambystoma mexicanum, Bombina orientalis, and Xenopus laevis to investigate the effects of bapx1 knockdown on the development of these three different amphibians. Using morpholinos we downregulated the expression of bapx1 and obtain morphants with altered mandibular arch morphology. In the absence of bapx1 Meckels cartilage and the palatoquadrate jaw joint initially develop separately but during further development the joint cavity between both fills with chondrocytes. This results in the fusion of both cartilages and the loss of the jaw joint. Despite this the jaw itself remains usable for feeding and breathing. We show that bapx1 plays a role in jaw joint maintenance during development and that the morphants morphology possibly mirrors the morphology of the jawless ancestors of the gnathostomes.

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'.

A computational model for the joint onset and development.

Joints connect the skeletal components and enable movement. The appearance and development of articulations is due to different genetic, biochemical, and mechanical factors. In the embryonic stage, controlled biochemical processes are critical for organized growth. We developed a computational model, which predicts the appearance, location, and development of joints in the embryonic stage. Biochemical events are modeled with reaction diffusion equations with generic molecules representing molecules that 1) determine the site where the articulation will appear, 2) promote proliferation, and matrix synthesis, and 3) define articular cartilage. Our model accounts for cell differentiation from mesenchymal cells to pre-cartilaginous cells, then cartilaginous cells, and lastly articular cartilage. These reaction-diffusion equations were solved using the finite elements method. From a mesenchymal 'bud' of a phalanx, the model predicts growth, joint cleavage, joint morphology, and articular cartilage formation. Our prediction of the gene expression during development agrees with molecular expression profiles of joint development reported in literature. Our computational model suggests that initial rudiment dimensions affect diffusion profiles result in Turing patterns that dictate sites of cleavage thereby determining the number of joints in a rudiment.

The Role of Bmp2 in the Maturation and Maintenance of the Murine Knee Joint.

Bone morphogenetic proteins (BMPs) are key regulators of skeletal development, growth, and repair. Although BMP signaling is required for synovial joint formation and is also involved in preserving joint function after birth, the role of specific BMP ligands in adult joint homeostasis remains unclear. The purpose of this study was to define the role of Bmp2 in the morphogenesis and maintenance of the knee joint. To do this, we first created Bmp2-LacZ and Gdf5-LacZ knock-in mice and compared their expression patterns in the developing and postnatal murine knee joint. We then generated a knockout mouse model using the Gdf5-cre transgene to specifically delete Bmp2 within synovial joint-forming cells. Joint formation, maturation, and homeostasis were analyzed using histology, immunohistochemistry, qRT-PCR, and atomic force microscopy (AFM)-based nanoindentation to assess the cellular, molecular, and biomechanical changes in meniscus and articular cartilage. Bmp2 is expressed in the articular cartilage and meniscus of the embryonic and adult mouse knee in a pattern distinct from Gdf5. The knee joints of the Bmp2 knockout mice form normally but fail to mature properly. In the absence of Bmp2, the extracellular matrix and shape of the meniscus are altered, resulting in functional deficits in the meniscus and articular cartilage that lead to a progressive osteoarthritis (OA) like knee pathology as the animals age. These findings demonstrate that BMP activity provided by Bmp2 is required for the maturation and maintenance of the murine knee joint and reveal a unique role for Bmp2 that is distinct from Gdf5 in knee joint biology. © 2018 American Society for Bone and Mineral Research.

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.

Wnt signalling controls the response to mechanical loading during zebrafish joint development.

Joint morphogenesis requires mechanical activity during development. Loss of mechanical strain causes abnormal joint development, which can impact long-term joint health. Although cell orientation and proliferation are known to shape the joint, dynamic imaging of developing joints in vivo has not been possible in other species. Using genetic labelling techniques in zebrafish we were able, for the first time, to dynamically track cell behaviours in intact moving joints. We identify that proliferation and migration, which contribute to joint morphogenesis, are mechanically controlled and are significantly reduced in immobilised larvae. By comparison with strain maps of the developing skeleton, we identify canonical Wnt signalling as a candidate for transducing mechanical forces into joint cell behaviours. We show that, in the jaw, Wnt signalling is reduced specifically in regions of high strain in response to loss of muscle activity. By pharmacological manipulation of canonical Wnt signalling, we demonstrate that Wnt acts downstream of mechanical activity and is required for joint patterning and chondrocyte maturation. Wnt16, which is also downstream of muscle activity, controls proliferation and migration, but plays no role in chondrocyte intercalation.

On the Formation of Digits and Joints during Limb Development.

Critical steps in forming the vertebrate limb include the positioning of digits and the positioning of joints within each digit. Recent studies have proposed that the iterative series of digits is established by a Turing-like mechanism generating stripes of chondrogenic domains. However, re-examination of available data suggest that digits are actually patterned as evenly spaced spots, not stripes, which then elongate into rod-shaped digit rays by incorporating new cells at their tips. Moreover, extension of the digit rays and the patterning of the joints occur simultaneously at the distal tip, implying that an integrated model is required to fully understand these processes.

SMOC can act as both an antagonist and an expander of BMP signaling.

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

Articular cartilage and joint development from embryogenesis to adulthood.

Within each synovial joint, the articular cartilage is uniquely adapted to bear dynamic compressive loads and shear forces throughout the joint's range of motion. Injury and age-related degeneration of the articular cartilage often lead to significant pain and disability, as the intrinsic repair capability of the tissue is extremely limited. Current surgical and biological treatment options have been unable to restore cartilage de novo. Before successful clinical cartilage restoration strategies can be developed, a better understanding of how the cartilage forms during normal development is essential. This review focuses on recent progress made towards addressing key questions about articular cartilage morphogenesis, including the origin of synovial joint progenitor cells, postnatal development and growth of the tissue. These advances have provided novel insight into fundamental questions about the developmental biology of articular cartilage, as well as potential cell sources that may participate in joint response to injury.