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After tissue injury, inflammatory cells are rapidly recruited to the wound where they clear microbes and other debris, and coordinate the behaviour of other cell lineages at the repair site in both positive and negative ways. In this study, we take advantage of the translucency and genetic tractability of zebrafish to evaluate the feasibility of reprogramming innate immune cells in vivo with cargo-loaded protocells and investigate how this alters the inflammatory response in the context of skin and skeletal repair. Using live imaging we show that protocells loaded with R848 cargo (which targets TLR7/8 signalling), are engulfed by macrophages resulting in their switching to a pro-inflammatory phenotype and altering their regulation of angiogenesis, collagen deposition and re-epithelialization during skin wound healing, as well as dampening osteoblast and osteoclast recruitment and bone mineralization during fracture repair. For infected skin wounds, R848-reprogrammed macrophages exhibited enhanced bactericidal activities leading to improved healing. We replicated our zebrafish studies in cultured human macrophages, and showed that R848-loaded protocells similarly reprogramme human cells, indicating how this strategy might be used to modulate wound inflammation in the clinic.
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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.
Assuntos
Peixe-Zebra , Animais , Anisotropia , Estresse MecânicoRESUMO
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.
Assuntos
Comportamento Animal/fisiologia , Modelos Biológicos , Comportamento Espacial/fisiologia , Peixe-Zebra/fisiologia , Animais , Biologia Computacional , Imageamento Tridimensional , Natação/fisiologiaRESUMO
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.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Perfilação da Expressão Gênica , Humanos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
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Cartilagem , Peixe-Zebra , Animais , Arcada Osseodentária , Larva , Morfogênese , Articulação TemporomandibularRESUMO
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.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Condrócitos/citologia , Condrogênese , Articulações/embriologia , Peixe-Zebra/embriologia , Animais , Autofagia , Diferenciação CelularRESUMO
Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Proteínas Musculares/metabolismo , Animais , Centríolos/genética , Cílios/genética , Técnicas de Inativação de Genes , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/metabolismo , Humanos , Proteínas Musculares/genética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Peixe-ZebraRESUMO
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.
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Articulações/metabolismo , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Condrogênese/genética , Condrogênese/fisiologia , Análise de Elementos Finitos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Arcada Osseodentária/embriologia , Arcada Osseodentária/metabolismo , Articulações/embriologia , Morfogênese/genética , Morfogênese/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
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Cartilagem Articular/metabolismo , Modelos Animais de Doenças , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Peixe-Zebra/genética , Animais , Homeostase , MicroRNAs/genética , Osteoartrite/genéticaRESUMO
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.
Assuntos
Autofagia , Modelos Animais de Doenças , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Peixe-Zebra , Animais , Homeostase , Doenças Musculares/patologiaRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Desenvolvimento Musculoesquelético/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Desenvolvimento Ósseo/genética , Calcificação Fisiológica/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Costelas/anormalidades , Costelas/crescimento & desenvolvimento , Costelas/metabolismo , Tendões/anormalidades , Tendões/crescimento & desenvolvimento , Tendões/metabolismo , Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Glicosiltransferases/genética , Complexo de Golgi/genética , Proteínas de Membrana/genética , N-Acetilgalactosaminiltransferases/genética , Animais , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Complexo de Golgi/enzimologia , Proteínas da Matriz do Complexo de Golgi , Humanos , Mutação , Peixe-Zebra , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization.
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Calcificação Fisiológica , Homeostase , Fosfatos/metabolismo , Pirofosfatases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Embrião não Mamífero/metabolismo , Fator de Crescimento de Fibroblastos 23 , Humanos , Dados de Sequência Molecular , Mutação/genética , Especificidade de Órgãos , Osteoblastos/enzimologia , Fenótipo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/química , Pirofosfatases/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The zebrafish is an important developmental model. Surprisingly, there are few studies that describe the glycosaminoglycan composition of its extracellular matrix during skeletogenesis. Glycosaminoglycans on proteoglycans contribute to the material properties of musculo skeletal connective tissues, and are important in regulating signalling events during morphogenesis. Sulfation motifs within the chain structure of glycosaminoglycans on cell-associated and extracellular matrix proteoglycans allow them to bind and regulate the sequestration/presentation of bioactive signalling molecules important in musculo-skeletal development. RESULTS: We describe the spatio-temporal expression of different glycosaminoglycan moieties during zebrafish skeletogenesis with antibodies recognising (1) native sulfation motifs within chondroitin and keratan sulfate chains, and (2) enzyme-generated neoepitope sequences within the chain structure of chondroitin sulfate (i.e., 0-, 4-, and 6-sulfated isoforms) and heparan sulfate glycosaminoglycans. We show that all the glycosaminoglycan moieties investigated are expressed within the developing skeletal tissues of larval zebrafish. However, subtle changes in their patterns of spatio-temporal expression over the period examined suggest that their expression is tightly and dynamically controlled during development. CONCLUSIONS: The subtle differences observed in the domains of expression between different glycosaminoglycan moieties suggest differences in their functional roles during establishment of the primitive analogues of the skeleton.
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Osso e Ossos/embriologia , Epitopos/metabolismo , Glicosaminoglicanos/metabolismo , Peixe-Zebra/embriologia , Animais , Condroitina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Heparitina Sulfato/metabolismo , Imuno-Histoquímica , Sulfato de Queratano/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
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.
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Osteoartrite , Peixe-Zebra , Masculino , Animais , Camundongos , Regulação para Baixo , Esclerose , Proteoglicanas , Osteoartrite/genéticaRESUMO
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.
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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.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Proteína Smad8/fisiologia , Células-Tronco/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Biomarcadores/metabolismo , Cartilagem/crescimento & desenvolvimento , Desenvolvimento Embrionário , Peixe-Zebra/embriologiaRESUMO
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.
Assuntos
Cordoma , Disco Intervertebral , Adulto , Animais , Homeostase , Humanos , Inflamação/metabolismo , Notocorda , Peixe-ZebraRESUMO
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.
Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Pró-Colágeno/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas da Matriz do Complexo de Golgi/antagonistas & inibidores , Proteínas da Matriz do Complexo de Golgi/genética , Humanos , Peixe-ZebraRESUMO
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).