Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales.

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new therapeutics that could ease the burden on healthcare systems. As these diseases are complex, it is difficult and expensive to accurately model bone pathophysiology in a lab setting. The challenge for the field is to establish a cost-effective, biologically relevant platform for modeling bone disease that can be used to test potential therapeutic compounds. Such a platform should ideally allow dynamic visualization of cell behaviors of bone-building osteoblasts and bone-degrading osteoclasts acting in their mineralized matrix environment. Zebrafish are increasingly used as models due to the availability of genetic tools, including transgenic reporter lines, and the fact that some skeletal tissues (including the scales) remain translucent to adulthood, allowing dynamic imaging options. Since zebrafish scales have both osteoblasts and osteoclasts and are highly abundant, they provide an easily accessible and abundantly available resource of independent bone units. Moreover, once removed, adult zebrafish scales fully regenerate, therefore offering a way to study the spatiotemporal growth of mineralized tissue in vivo. Here, we detail protocols for harvesting and tracking the regeneration of the scales. Lastly, a protocol for stable culture of scales ex vivo for a week and following the healing response after controlled damage to the mineralized matrix of the scale over time is also presented.

Compressive stress gradients direct mechanoregulation of anisotropic growth in the zebrafish jaw joint.

Mechanical stimuli arising from fetal movements are critical factors underlying joint growth. Abnormal fetal movements negatively affect joint shape features with important implications for joint health, but the mechanisms by which mechanical forces from fetal movements influence joint growth are still unclear. In this research, we quantify zebrafish jaw joint growth in 3D in free-to-move and immobilised fish larvae between four and five days post fertilisation. We found that the main changes in size and shape in normally moving fish were in the ventrodorsal axis, while growth anisotropy was lost in the immobilised larvae. We next sought to determine the cell level activities underlying mechanoregulated growth anisotropy by tracking individual cells in the presence or absence of jaw movements, finding that the most dramatic changes in growth rates due to jaw immobility were in the ventrodorsal axis. Finally, we implemented mechanobiological simulations of joint growth with which we tested hypotheses relating specific mechanical stimuli to mechanoregulated growth anisotropy. Different types of mechanical stimulation were incorporated into the simulation to provide the mechanoregulated component of growth, in addition to the baseline (non-mechanoregulated) growth which occurs in the immobilised animals. We found that when average tissue stress over the opening and closing cycle of the joint was used as the stimulus for mechanoregulated growth, joint morphogenesis was not accurately predicted. Predictions were improved when using the stress gradients along the rudiment axes (i.e., the variation in magnitude of compression to magnitude of tension between local regions). However, the most accurate predictions were obtained when using the compressive stress gradients (i.e., the variation in compressive stress magnitude) along the rudiment axes. We conclude therefore that the dominant biophysical stimulus contributing to growth anisotropy during early joint development is the gradient of compressive stress experienced along the growth axes under cyclical loading.

Osteomodulin downregulation is associated with osteoarthritis development.

Abnormal subchondral bone remodeling leading to sclerosis is a main feature of osteoarthritis (OA), and osteomodulin (OMD), a proteoglycan involved in extracellular matrix mineralization, is associated with the sclerotic phenotype. However, the functions of OMD remain poorly understood, specifically in vivo. We used Omd knockout and overexpressing male mice and mutant zebrafish to study its roles in bone and cartilage metabolism and in the development of OA. The expression of Omd is deeply correlated with bone and cartilage microarchitectures affecting the bone volume and the onset of subchondral bone sclerosis and spontaneous cartilage lesions. Mechanistically, OMD binds to RANKL and inhibits osteoclastogenesis, thus controlling the balance of bone remodeling. In conclusion, OMD is a key factor in subchondral bone sclerosis associated with OA. It participates in bone and cartilage homeostasis by acting on the regulation of osteoclastogenesis. Targeting OMD may be a promising new and personalized approach for OA.

Growth orientations, rather than heterogeneous growth rates, dominate jaw joint morphogenesis in the larval zebrafish.

In early limb embryogenesis, synovial joints acquire specific shapes which determine joint motion and function. The process by which the opposing cartilaginous joint surfaces are moulded into reciprocal and interlocking shapes, called joint morphogenesis, is one of the least understood aspects of joint formation and the cell-level dynamics underlying it are yet to be unravelled. In this research, we quantified key cellular dynamics involved in growth and morphogenesis of the zebrafish jaw joint and synthesised them in a predictive computational simulation of joint development. Cells in larval zebrafish jaw joints labelled with cartilage markers were tracked over a 48-h time window using confocal imaging. Changes in distance and angle between adjacent cell centroids resulting from cell rearrangement, volume expansion and extracellular matrix (ECM) deposition were measured and used to calculate the rate and direction of local tissue deformations. We observed spatially and temporally heterogeneous growth patterns with marked anisotropy over the developmental period assessed. There was notably elevated growth at the level of the retroarticular process of the Meckel's cartilage, a feature known to undergo pronounced shape changes during zebrafish development. Analysis of cell dynamics indicated a dominant role for cell volume expansion in growth, with minor influences from ECM volume increases and cell intercalation. Cell proliferation in the joint was minimal over the timeframe of interest. Synthesising the dynamic cell data into a finite element model of jaw joint development resulted in accurate shape predictions. Our biofidelic computational simulation demonstrated that zebrafish jaw joint growth can be reasonably approximated based on cell positional information over time, where cell positional information derives mainly from cell orientation and cell volume expansion. By modifying the input parameters of the simulation, we were able to assess the relative contributions of heterogeneous growth rates and of growth orientation. The use of uniform rather than heterogeneous growth rates only minorly impacted the shape predictions, whereas isotropic growth fields resulted in altered shape predictions. The simulation results suggest that growth anisotropy is the dominant influence on joint growth and morphogenesis. This study addresses the gap of the cellular processes underlying joint morphogenesis, with implications for understanding the aetiology of developmental joint disorders such as developmental dysplasia of the hip and arthrogryposis.

Dominating lengthscales of zebrafish collective behaviour.

Collective behaviour in living systems is observed across many scales, from bacteria to insects, to fish shoals. Zebrafish have emerged as a model system amenable to laboratory study. Here we report a three-dimensional study of the collective dynamics of fifty zebrafish. We observed the emergence of collective behaviour changing between ordered to randomised, upon adaptation to new environmental conditions. We quantify the spatial and temporal correlation functions of the fish and identify two length scales, the persistence length and the nearest neighbour distance, that capture the essence of the behavioural changes. The ratio of the two length scales correlates robustly with the polarisation of collective motion that we explain with a reductionist model of self-propelled particles with alignment interactions.

Regenerating zebrafish scales express a subset of evolutionary conserved genes involved in human skeletal disease.

BACKGROUND: Scales are mineralised exoskeletal structures that are part of the dermal skeleton. Scales have been mostly lost during evolution of terrestrial vertebrates whilst bony fish have retained a mineralised dermal skeleton in the form of fin rays and scales. Each scale is a mineralised collagen plate that is decorated with both matrix-building and resorbing cells. When removed, an ontogenetic scale is quickly replaced following differentiation of the scale pocket-lining cells that regenerate a scale. Processes promoting de novo matrix formation and mineralisation initiated during scale regeneration are poorly understood. Therefore, we performed transcriptomic analysis to determine gene networks and their pathways involved in dermal scale regeneration. RESULTS: We defined the transcriptomic profiles of ontogenetic and regenerating scales of zebrafish and identified 604 differentially expressed genes (DEGs). These were enriched for extracellular matrix, ossification, and cell adhesion pathways, but not in enamel or dentin formation processes indicating that scales are reminiscent to bone. Hypergeometric tests involving monogenetic skeletal disorders showed that DEGs were strongly enriched for human orthologues that are mutated in low bone mass and abnormal bone mineralisation diseases (P< 2× 10-3). The DEGs were also enriched for human orthologues associated with polygenetic skeletal traits, including height (P< 6× 10-4), and estimated bone mineral density (eBMD, P< 2× 10-5). Zebrafish mutants of two human orthologues that were robustly associated with height (COL11A2, P=6× 10-24) or eBMD (SPP1, P=6× 10-20) showed both exo- and endo- skeletal abnormalities as predicted by our genetic association analyses; col11a2Y228X/Y228X mutants showed exoskeletal and endoskeletal features consistent with abnormal growth, whereas spp1P160X/P160X mutants predominantly showed mineralisation defects. CONCLUSION: We show that scales have a strong osteogenic expression profile comparable to other elements of the dermal skeleton, enriched in genes that favour collagen matrix growth. Despite the many differences between scale and endoskeletal developmental processes, we also show that zebrafish scales express an evolutionarily conserved sub-population of genes that are relevant to human skeletal disease.

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.

3D assessment of intervertebral disc degeneration in zebrafish identifies changes in bone density that prime disc disease.

Back pain is a common condition with a high social impact and represents a global health burden. Intervertebral disc disease (IVDD) is one of the major causes of back pain; no therapeutics are currently available to reverse this disease. The impact of bone mineral density (BMD) on IVDD has been controversial, with some studies suggesting osteoporosis as causative for IVDD and others suggesting it as protective for IVDD. Functional studies to evaluate the influence of genetic components of BMD in IVDD could highlight opportunities for drug development and repurposing. By taking a holistic 3D approach, we established an aging zebrafish model for spontaneous IVDD. Increased BMD in aging, detected by automated computational analysis, is caused by bone deformities at the endplates. However, aged zebrafish spines showed changes in bone morphology, microstructure, mineral heterogeneity, and increased fragility that resembled osteoporosis. Elements of the discs recapitulated IVDD symptoms found in humans: the intervertebral ligament (equivalent to the annulus fibrosus) showed disorganized collagen fibers and herniation, while the disc center (nucleus pulposus equivalent) showed dehydration and cellular abnormalities. We manipulated BMD in young zebrafish by mutating sp7 and cathepsin K, leading to low and high BMD, respectively. Remarkably, we detected IVDD in both groups, demonstrating that low BMD does not protect against IVDD, and we found a strong correlation between high BMD and IVDD. Deep learning was applied to high-resolution synchrotron µCT image data to analyze osteocyte 3D lacunar distribution and morphology, revealing a role of sp7 in controlling the osteocyte lacunar 3D profile. Our findings suggest potential avenues through which bone quality can be targeted to identify beneficial therapeutics for IVDD.

Giantin is required for intracellular N-terminal processing of type I procollagen.

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations.

Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in SMAD9 has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9+ cells are proliferative, and that the Smad9+ pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9+ cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.

Wnt16 Elicits a Protective Effect Against Fractures and Supports Bone Repair in Zebrafish.

Bone homeostasis is a dynamic, multicellular process that is required throughout life to maintain bone integrity, prevent fracture, and respond to skeletal damage. WNT16 has been linked to bone fragility and osteoporosis in human genome wide-association studies, as well as the functional hematopoiesis of leukocytes in vivo. However, the mechanisms by which WNT16 promotes bone health and repair are not fully understood. In this study, CRISPR-Cas9 was used to generate mutant zebrafish lacking Wnt16 (wnt16 -/- ) to study its effect on bone dynamically. The wnt16 mutants displayed variable tissue mineral density (TMD) and were susceptible to spontaneous fractures and the accumulation of bone calluses at an early age. Fractures were induced in the lepidotrichia of the caudal fins of wnt16 -/- and WT zebrafish; this model was used to probe the mechanisms by which Wnt16 regulates skeletal and immune cell dynamics in vivo. In WT fins, wnt16 expression increased significantly during the early stages for bone repair. Mineralization of bone during fracture repair was significantly delayed in wnt16 mutants compared with WT zebrafish. Surprisingly, there was no evidence that the recruitment of innate immune cells to fractures or soft callus formation was altered in wnt16 mutants. However, osteoblast recruitment was significantly delayed in wnt16 mutants postfracture, coinciding with precocious activation of the canonical Wnt signaling pathway. In situ hybridization suggests that canonical Wnt-responsive cells within fractures are osteoblast progenitors, and that osteoblast differentiation during bone repair is coordinated by the dynamic expression of runx2a and wnt16. This study highlights zebrafish as an emerging model for functionally validating osteoporosis-associated genes and investigating fracture repair dynamically in vivo. Using this model, it was found that Wnt16 protects against fracture and supports bone repair, likely by modulating canonical Wnt activity via runx2a to facilitate osteoblast differentiation and bone matrix deposition. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Exposure to hypergravity during zebrafish development alters cartilage material properties and strain distribution.

AIMS: Vertebrates have adapted to life on Earth and its constant gravitational field, which exerts load on the body and influences the structure and function of tissues. While the effects of microgravity on muscle and bone homeostasis are well described, with sarcopenia and osteoporosis observed in astronauts returning from space, the effects of shorter exposures to increased gravitational fields are less well characterized. We aimed to test how hypergravity affects early cartilage and skeletal development in a zebrafish model. METHODS: We exposed zebrafish to 3 g and 6 g hypergravity from three to five days post-fertilization, when key events in jaw cartilage morphogenesis occur. Following this exposure, we performed immunostaining along with a range of histological stains and transmission electron microscopy (TEM) to examine cartilage morphology and structure, atomic force microscopy (AFM) and nanoindentation experiments to investigate the cartilage material properties, and finite element modelling to map the pattern of strain and stress in the skeletal rudiments. RESULTS: We did not observe changes to larval growth, or morphology of cartilage or muscle. However, we observed altered mechanical properties of jaw cartilages, and in these regions we saw changes to chondrocyte morphology and extracellular matrix (ECM) composition. These areas also correspond to places where strain and stress distribution are predicted to be most different following hypergravity exposure. CONCLUSION: Our results suggest that altered mechanical loading, through hypergravity exposure, affects chondrocyte maturation and ECM components, ultimately leading to changes to cartilage structure and function. Cite this article: Bone Joint Res 2021;10(2):137-148.

Transformed notochordal cells trigger chronic wounds in zebrafish, destabilizing the vertebral column and bone homeostasis.

Notochordal cells play a pivotal role in vertebral column patterning, contributing to the formation of the inner architecture of intervertebral discs (IVDs). Their disappearance during development has been associated with reduced repair capacity and IVD degeneration. Notochord cells can give rise to chordomas, a highly invasive bone cancer associated with late diagnosis. Understanding the impact of neoplastic cells during development and on the surrounding vertebral column could open avenues for earlier intervention and therapeutics. We investigated the impact of transformed notochord cells in the zebrafish skeleton using a line expressing RAS in the notochord under the control of the kita promoter, with the advantage of adulthood endurance. Transformed cells caused damage in the notochord and destabilised the sheath layer, triggering a wound repair mechanism, with enrolment of sheath cells (col9a2+) and expression of wt1b, similar to induced notochord wounds. Moreover, increased recruitment of neutrophils and macrophages, displaying abnormal behaviour in proximity to the notochord sheath and transformed cells, supported parallels between chordomas, wound and inflammation. Cancerous notochordal cells interfere with differentiation of sheath cells to form chordacentra domains, leading to fusions and vertebral clefts during development. Adults displayed IVD irregularities reminiscent of degeneration, including reduced bone mineral density and increased osteoclast activity, along with disorganised osteoblasts and collagen, indicating impaired bone homeostasis. By depleting inflammatory cells, we abrogated chordoma development and rescued the skeletal features of the vertebral column. Therefore, we showed that transformed notochord cells alter the skeleton during life, causing a wound-like phenotype and activating chronic wound response, suggesting parallels between chordoma, wound, IVD degeneration and inflammation, highlighting inflammation as a promising target for future therapeutics. This article has an associated First Person interview with the first author of the paper.

Skeletal Biology and Disease Modeling in Zebrafish.

Zebrafish are teleosts (bony fish) that share with mammals a common ancestor belonging to the phylum Osteichthyes, from which their endoskeletal systems have been inherited. Indeed, teleosts and mammals have numerous genetically conserved features in terms of skeletal elements, ossification mechanisms, and bone matrix components in common. Yet differences related to bone morphology and function need to be considered when investigating zebrafish in skeletal research. In this review, we focus on zebrafish skeletal architecture with emphasis on the morphology of the vertebral column and associated anatomical structures. We provide an overview of the different ossification types and osseous cells in zebrafish and describe bone matrix composition at the microscopic tissue level with a focus on assessing mineralization. Processes of bone formation also strongly depend on loading in zebrafish, as we elaborate here. Furthermore, we illustrate the high regenerative capacity of zebrafish bones and present some of the technological advantages of using zebrafish as a model. We highlight zebrafish axial and fin skeleton patterning mechanisms, metabolic bone disease such as after immunosuppressive glucocorticoid treatment, as well as osteogenesis imperfecta (OI) and osteopetrosis research in zebrafish. We conclude with a view of why larval zebrafish xenografts are a powerful tool to study bone metastasis. © 2021 American Society for Bone and Mineral Research (ASBMR).

NCOA3 identified as a new candidate to explain autosomal dominant progressive hearing loss.

Hearing loss is a frequent sensory impairment in humans and genetic factors account for an elevated fraction of the cases. We have investigated a large family of five generations, with 15 reported individuals presenting non-syndromic, sensorineural, bilateral and progressive hearing loss, segregating as an autosomal dominant condition. Linkage analysis, using SNP-array and selected microsatellites, identified a region of near 13 cM in chromosome 20 as the best candidate to harbour the causative mutation. After exome sequencing and filtering of variants, only one predicted deleterious variant in the NCOA3 gene (NM_181659, c.2810C > G; p.Ser937Cys) fit in with our linkage data. RT-PCR, immunostaining and in situ hybridization showed expression of ncoa3 in the inner ear of mice and zebrafish. We generated a stable homozygous zebrafish mutant line using the CRISPR/Cas9 system. ncoa3-/- did not display any major morphological abnormalities in the ear, however, anterior macular hair cells showed altered orientation. Surprisingly, chondrocytes forming the ear cartilage showed abnormal behaviour in ncoa3-/-, detaching from their location, invading the ear canal and blocking the cristae. Adult mutants displayed accumulation of denser material wrapping the otoliths of ncoa3-/- and increased bone mineral density. Altered zebrafish swimming behaviour corroborates a potential role of ncoa3 in hearing loss. In conclusion, we identified a potential candidate gene to explain hereditary hearing loss, and our functional analyses suggest subtle and abnormal skeletal behaviour as mechanisms involved in the pathogenesis of progressive sensory function impairment.

Potential of zebrafish as a model to characterise MicroRNA profiles in mechanically mediated joint degeneration.

Mechanically mediated joint degeneration and cartilage dyshomeostasis is implicated in highly prevalent diseases such as osteoarthritis. Increasingly, MicroRNAs are being associated with maintaining the normal state of cartilage, making them an exciting and potentially key contributor to joint health and disease onset. Here, we present a summary of current in vitro and in vivo models which can be used to study the role of mechanical load and MicroRNAs in joint degeneration, including: non-invasive murine models of PTOA, surgical models which involve ligament transection, and unloading models based around immobilisation of joints or removal of load from the joint through suspension. We also discuss how zebrafish could be used to advance this field, namely through the availability of transgenic lines relevant to cartilage homeostasis and the ability to accurately map strain through the cartilage, enabling the response of downstream MicroRNA targets to be followed dynamically at a cellular level in areas of high and low strain.

Muscle defects due to perturbed somite segmentation contribute to late adult scoliosis.

Scoliosis is an abnormal bending of the body axis. Truncated vertebrae or a debilitated ability to control the musculature in the back can cause this condition, but in most cases the causative reason for scoliosis is unknown (idiopathic). Using mutants for somite clock genes with mild defects in the vertebral column, we here show that early defects in somitogenesis are not overcome during development and have long lasting and profound consequences for muscle fiber organization, structure and whole muscle volume. These mutants present only mild alterations in the vertebral column, and muscle shortcomings are uncoupled from skeletal defects. None of the mutants presents an overt musculoskeletal phenotype at larval or early adult stages, presumably due to compensatory growth mechanisms. Scoliosis becomes only apparent during aging. We conclude that adult degenerative scoliosis is due to disturbed crosstalk between vertebrae and muscles during early development, resulting in subsequent adult muscle weakness and bending of the body axis.

Zebrafish as a model to study autophagy and its role in skeletal development and disease.

In the last twenty years, research using zebrafish as a model organism has increased immensely. With the many advantages that zebrafish offer such as high fecundity, optical transparency, ex vivo development, and genetic tractability, they are well suited to studying developmental processes and the effect of genetic mutations. More recently, zebrafish models have been used to study autophagy. This important protein degradation pathway is needed for cell and tissue homeostasis in a variety of contexts. Correspondingly, its dysregulation has been implicated in multiple diseases including skeletal disorders. In this review, we explore how zebrafish are being used to study autophagy in the context of skeletal development and disease, and the ways these areas are intersecting to help identify potential therapeutic targets for skeletal disorders.