RESUMO
The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.
Assuntos
Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Proteínas de Homeodomínio/metabolismo , Osteocondrodisplasias/embriologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Cartilagem/embriologia , Cartilagem/patologia , Condrócitos/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/anormalidades , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Arcada Osseodentária/embriologia , Arcada Osseodentária/patologia , Articulações/anormalidades , Articulações/embriologia , Articulações/patologia , Mitose/genética , Morfolinos/farmacologia , Mutação/genética , RNA-Seq , Análise de Célula Única , Crânio/anormalidades , Crânio/embriologia , Crânio/patologia , Coluna Vertebral/anormalidades , Coluna Vertebral/embriologia , Coluna Vertebral/patologia , Estresse Fisiológico/genética , Regulação para Cima/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The vertebrate jaw is a versatile feeding apparatus. To function, it requires a joint between the upper and lower jaws, so jaw joint defects are often highly disruptive and difficult to study. To describe the consequences of jaw joint dysfunction, we engineered two independent null alleles of a single jaw joint marker gene, nkx3.2, in zebrafish. These mutations caused zebrafish to become functionally jawless via fusion of the upper and lower jaw cartilages (ankylosis). Despite lacking jaw joints, nkx3.2 mutants survived to adulthood and accommodated this defect by: (a) having a remodeled skull with a fixed open gape, reduced snout and enlarged branchial region; and (b) performing ram feeding in the absence of jaw-generated suction. The late onset and broad extent of phenotypic changes in the mutants suggest that modifications to the skull are induced by functional agnathia, secondarily to nkx3.2 loss of function. Interestingly, nkx3.2 mutants superficially resemble ancient jawless vertebrates (anaspids and furcacaudiid thelodonts) in overall head shape. Because no homology exists in individual skull elements between these taxa, the adult nkx3.2 phenotype is not a reversal but rather a convergence due to similar functional requirements of feeding without moveable jaws. This remarkable analogy strongly suggests that jaw movements themselves dramatically influence the development of jawed vertebrate skulls. Thus, these mutants provide a unique model with which to: (a) investigate adaptive responses to perturbation in skeletal development; (b) re-evaluate evolutionarily inspired interpretations of phenocopies generated by gene knockdowns and knockouts; and (c) gain insight into feeding mechanics of the extinct agnathans.
Assuntos
Arcada Osseodentária , Peixe-Zebra , Animais , Evolução Biológica , Cabeça , Proteínas de Homeodomínio , Masculino , Fenótipo , Ovinos , Crânio , Fatores de Transcrição , Peixe-Zebra/genética , Proteínas de Peixe-ZebraRESUMO
The poor intrinsic repair capacity of mammalian joint cartilage likely contributes to the high incidence of arthritis worldwide. Adult zebrafish can regenerate many structures that show limited or no healing capacity in mammals, including the jawbone. To test whether zebrafish can also regenerate damaged joints, we developed a surgical injury model in which the zebrafish jaw joint is destabilized via transection of the major jaw joint ligament, the interopercular-mandibular (IOM). Unilateral transection of the IOM ligament in 1-year-old fish resulted in an initial reduction of jaw joint cartilage by 14 days, with full regeneration of joint cartilage by 28 days. Joint cartilage regeneration involves the re-entry of articular chondrocytes into the cell cycle and the upregulated expression of sox10, a marker of developing chondrocytes in the embryo that becomes restricted to a subset of joint chondrocytes in adults. Genetic ablation of these sox10-expressing chondrocytes shows that they are essential for joint cartilage regeneration. To uncover the potential source of new chondrocytes during joint regeneration, we performed single-cell RNA sequencing of the uninjured adult jaw joint and identified multiple skeletal, connective tissue, and fibroblast subtypes. In particular, we uncovered a joint-specific periosteal population expressing coch and grem1a, with the jaw joint chondrocytes marked by grem1a expression during regeneration. Our findings demonstrate the capacity of zebrafish to regenerate adult joint cartilage and identify candidate cell types that can be tested for their roles in regenerative response.