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.

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.

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.

Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Tendons are an essential part of the musculoskeletal system, connecting muscle and skeletal elements to enable force generation. The transcription factor scleraxis marks vertebrate tendons from early specification. Scleraxis-null mice are viable and have a range of tendon and bone defects in the trunk and limbs but no described cranial phenotype. We report the expression of zebrafish scleraxis orthologs: scleraxis homolog (scx)-a and scxb in cranial and intramuscular tendons and in other skeletal elements. Single mutants for either scxa or scxb, generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), are viable and fertile as adult fish. Although scxb mutants show no obvious phenotype, scxa mutant embryos have defects in cranial tendon maturation and muscle misalignment. Mutation of both scleraxis genes results in more severe defects in cranial tendon differentiation, muscle and cartilage dysmorphogenesis and paralysis, and lethality by 2-5 wk, which indicates an essential function of scleraxis for craniofacial development. At juvenile and adult stages, ribs in scxa mutants fail to mineralize and/or are small and heavily fractured. Scxa mutants also have smaller muscle volume, abnormal swim movement, and defects in bone growth and composition. Scleraxis function is therefore essential for normal craniofacial form and function and vital for fish development.-Kague, E., Hughes, S. M., Lawrence, E. A., Cross, S., Martin-Silverstone, E., Hammond, C. L., Hinits, Y. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Giantin-knockout models reveal a feedback loop between Golgi function and glycosyltransferase expression.

The Golgi is the cellular hub for complex glycosylation, controlling accurate processing of complex proteoglycans, receptors, ligands and glycolipids. Its structure and organisation are dependent on golgins, which tether cisternal membranes and incoming transport vesicles. Here, we show that knockout of the largest golgin, giantin, leads to substantial changes in gene expression but only limited effects on Golgi structure. Notably, 22 Golgi-resident glycosyltransferases, but not glycan-processing enzymes or the ER glycosylation machinery, are differentially expressed following giantin ablation. This includes near-complete loss of function of GALNT3 in both mammalian cell and zebrafish models. Giantin-knockout zebrafish exhibit hyperostosis and ectopic calcium deposits, recapitulating phenotypes of hyperphosphatemic familial tumoral calcinosis, a disease caused by mutations in GALNT3. These data reveal a new feature of Golgi homeostasis: the ability to regulate glycosyltransferase expression to generate a functional proteoglycome.

Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures.

During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

BMPs regulate msx gene expression in the dorsal neuroectoderm of Drosophila and vertebrates by distinct mechanisms.

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of "neural identity" gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.

A perspective on muscle phenotyping in musculoskeletal research.

Musculoskeletal research should synergistically investigate bone and muscle to inform approaches for maintaining mobility and to avoid bone fractures. The relationship between sarcopenia and osteoporosis, integrated in the term 'osteosarcopenia', is underscored by the close association shown between these two conditions in many studies, whereby one entity emerges as a predictor of the other. In a recent workshop of Working Group (WG) 2 of the EU Cooperation in Science and Technology (COST) Action 'Genomics of MusculoSkeletal traits Translational Network' (GEMSTONE) consortium (CA18139), muscle characterization was highlighted as being important, but currently under-recognized in the musculoskeletal field. Here, we summarize the opinions of the Consortium and research questions around translational and clinical musculoskeletal research, discussing muscle phenotyping in human experimental research and in two animal models: zebrafish and mouse.

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.

Finding the genes for fragile bones.

Combining transcriptomic data with the analysis of large genome-wide association studies helps identify genes that are likely important for regulating bone mineral density.

Functional Validation of Osteoporosis Genetic Findings Using Small Fish Models.

The advancement of human genomics has revolutionized our understanding of the genetic architecture of many skeletal diseases, including osteoporosis. However, interpreting results from human association studies remains a challenge, since index variants often reside in non-coding regions of the genome and do not possess an obvious regulatory function. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary, such as the one offered by animal models. These models enable us to identify causal mechanisms, clarify the underlying biology, and apply interventions. Over the past several decades, small teleost fishes, mostly zebrafish and medaka, have emerged as powerful systems for modeling the genetics of human diseases. Due to their amenability to genetic intervention and the highly conserved genetic and physiological features, fish have become indispensable for skeletal genomic studies. The goal of this review is to summarize the evidence supporting the utility of Zebrafish (Danio rerio) for accelerating our understanding of human skeletal genomics and outlining the remaining gaps in knowledge. We provide an overview of zebrafish skeletal morphophysiology and gene homology, shedding light on the advantages of human skeletal genomic exploration and validation. Knowledge of the biology underlying osteoporosis through animal models will lead to the translation into new, better and more effective therapeutic approaches.

The genetic overlap between osteoporosis and craniosynostosis.

Osteoporosis is the most prevalent bone condition in the ageing population. This systemic disease is characterized by microarchitectural deterioration of bone, leading to increased fracture risk. In the past 15 years, genome-wide association studies (GWAS), have pinpointed hundreds of loci associated with bone mineral density (BMD), helping elucidate the underlying molecular mechanisms and genetic architecture of fracture risk. However, the challenge remains in pinpointing causative genes driving GWAS signals as a pivotal step to drawing the translational therapeutic roadmap. Recently, a skull BMD-GWAS uncovered an intriguing intersection with craniosynostosis, a congenital anomaly due to premature suture fusion in the skull. Here, we recapitulate the genetic contribution to both osteoporosis and craniosynostosis, describing the biological underpinnings of this overlap and using zebrafish models to leverage the functional investigation of genes associated with skull development and systemic skeletal homeostasis.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis.

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short.

Welfare Assessment of Adult Laboratory Zebrafish: A Practical Guide.

Teleost fish such as Danio rerio (zebrafish) have been successfully used in biomedical research since decades. Genetically altered fish lines obtained by state-of-the-art genetic technologies are serving as well-known model organisms. In Europe, following Directive 2010/63/EU, generation, breeding, and husbandry of new genetically altered lines of laboratory animals require governmental state approval in case pain, suffering, distress, or long-lasting harm to the offspring derived by breeding of these lines cannot be excluded. The identification and assessment of pain, distress, or harm, according to a severity classification of mild, moderate, severe, or humane endpoint, became a new challenging task for all scientists, animal technicians, and veterinarians for daily work with laboratory zebrafish. In this study, we describe the performance of the assessment of welfare parameters of selected pathologic phenotypes and abnormalities frequently found in laboratory fish facilities based on veterinary, biological, and physiological aspects by using a dedicated score sheet. In a colony of zebrafish, we evaluated the frequency of genotype-independent abnormalities observed within 3 years. We give examples for severity classification and measures once an abnormality has been identified according to the 3Rs (Replacement, Reduction and Refinement).

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.

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.

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.

Bone Phenotyping Approaches in Human, Mice and Zebrafish - Expert Overview of the EU Cost Action GEMSTONE ("GEnomics of MusculoSkeletal traits TranslatiOnal NEtwork").

A synoptic overview of scientific methods applied in bone and associated research fields across species has yet to be published. Experts from the EU Cost Action GEMSTONE ("GEnomics of MusculoSkeletal Traits translational Network") Working Group 2 present an overview of the routine techniques as well as clinical and research approaches employed to characterize bone phenotypes in humans and selected animal models (mice and zebrafish) of health and disease. The goal is consolidation of knowledge and a map for future research. This expert paper provides a comprehensive overview of state-of-the-art technologies to investigate bone properties in humans and animals - including their strengths and weaknesses. New research methodologies are outlined and future strategies are discussed to combine phenotypic with rapidly developing -omics data in order to advance musculoskeletal research and move towards "personalised medicine".

Perspective of the GEMSTONE Consortium on Current and Future Approaches to Functional Validation for Skeletal Genetic Disease Using Cellular, Molecular and Animal-Modeling Techniques.

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits ("endophenotypes"), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative in vivo platforms need to be used and developed in parallel with the current -omics and traditional in vivo resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.