RESUMO
Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.
Assuntos
Calcitonina , Linhagem da Célula , Ciona intestinalis , Endoderma , Crista Neural , Células Neuroendócrinas , Animais , Endoderma/metabolismo , Endoderma/citologia , Calcitonina/metabolismo , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/citologia , Ciona intestinalis/metabolismo , Ciona intestinalis/embriologia , Crista Neural/metabolismo , Crista Neural/citologia , Embrião de Galinha , Camundongos , Vertebrados/embriologia , Vertebrados/metabolismo , Peixe-Zebra/embriologia , Anfioxos/embriologia , Anfioxos/metabolismo , Anfioxos/genética , Corpo Ultimobranquial/metabolismoRESUMO
The evolution of a unique craniofacial complex in vertebrates made possible new ways of breathing, eating, communicating and sensing the environment. The head and face develop through interactions of all three germ layers, the endoderm, ectoderm and mesoderm, as well as the so-called fourth germ layer, the cranial neural crest. Over a century of experimental embryology and genetics have revealed an incredible diversity of cell types derived from each germ layer, signaling pathways and genes that coordinate craniofacial development, and how changes to these underlie human disease and vertebrate evolution. Yet for many diseases and congenital anomalies, we have an incomplete picture of the causative genomic changes, in particular how alterations to the non-coding genome might affect craniofacial gene expression. Emerging genomics and single-cell technologies provide an opportunity to obtain a more holistic view of the genes and gene regulatory elements orchestrating craniofacial development across vertebrates. These single-cell studies generate novel hypotheses that can be experimentally validated in vivo. In this Review, we highlight recent advances in single-cell studies of diverse craniofacial structures, as well as potential pitfalls and the need for extensive in vivo validation. We discuss how these studies inform the developmental sources and regulation of head structures, bringing new insights into the etiology of structural birth anomalies that affect the vertebrate head.
Assuntos
Evolução Biológica , Crânio , Animais , Humanos , Vertebrados , Crista Neural/metabolismo , Biologia do Desenvolvimento , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Mammalian hair cells do not functionally regenerate in adulthood but can regenerate at embryonic and neonatal stages in mice by direct transdifferentiation of neighboring supporting cells into new hair cells. Previous work showed loss of transdifferentiation potential of supporting cells is in part due to H3K4me1 enhancer decommissioning of the hair cell gene regulatory network during the first postnatal week. However, inhibiting this decommissioning only partially preserves transdifferentiation potential. Therefore, we explored other repressive epigenetic modifications that may be responsible for this loss of plasticity. We find supporting cells progressively accumulate DNA methylation at promoters of developmentally regulated hair cell genes. Specifically, DNA methylation overlaps with binding sites of Atoh1, a key transcription factor for hair cell fate. We further show that DNA hypermethylation replaces H3K27me3-mediated repression of hair cell genes in mature supporting cells, and is accompanied by progressive loss of chromatin accessibility, suggestive of facultative heterochromatin formation. Another subset of hair cell loci is hypermethylated in supporting cells, but not in hair cells. Ten-eleven translocation (TET) enzyme-mediated demethylation of these hypermethylated sites is necessary for neonatal supporting cells to transdifferentiate into hair cells. We also observe changes in chromatin accessibility of supporting cell subtypes at the single-cell level with increasing age: Gene programs promoting sensory epithelium development loses chromatin accessibility, in favor of gene programs that promote physiological maturation and function of the cochlea. We also find chromatin accessibility is partially recovered in a chronically deafened mouse model, which holds promise for future translational efforts in hearing restoration.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Metilação de DNA , Animais , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cóclea/metabolismo , Regeneração/genética , Cromatina/metabolismo , Mamíferos/genéticaRESUMO
The auditory organ of Corti is comprised of only two major cell types-the mechanosensory hair cells and their associated supporting cells-both specified from a single pool of prosensory progenitors in the cochlear duct. Here, we show that competence to respond to Atoh1, a transcriptional master regulator necessary and sufficient for induction of mechanosensory hair cells, is established in the prosensory progenitors between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11-the two homologous family members transiently expressed in the inner ear at the time of competence establishment-blocks the ability of prosensory progenitors to differentiate as hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Transcrição SOXC , Animais , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Diferenciação Celular , Fatores de Transcrição/metabolismo , Epigênese Genética , Órgão Espiral , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/metabolismoRESUMO
Of all the cell types arising from the neural crest, ectomesenchyme is likely the most unusual. In contrast to the neuroglial cells generated by neural crest throughout the embryo, consistent with its ectodermal origin, cranial neural crest-derived cells (CNCCs) generate many connective tissue and skeletal cell types in common with mesoderm. Whether this ectoderm-derived mesenchyme (ectomesenchyme) potential reflects a distinct developmental origin from other CNCC lineages, and/or epigenetic reprogramming of the ectoderm, remains debated. Whereas decades of lineage tracing studies have defined the potential of CNCC ectomesenchyme, these are being revisited by modern genetic techniques. Recent work is also shedding light on the extent to which intrinsic and extrinsic cues determine ectomesenchyme potential, and whether maintenance or reacquisition of CNCC multipotency influences craniofacial repair.
Assuntos
Mesoderma , Crista Neural , Crista Neural/metabolismo , Ectoderma/metabolismo , Embrião de MamíferosRESUMO
A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in either of the bHLH transcription factors TWIST1 and TCF12. Although compound heterozygous Tcf12; Twist1 mice display severe coronal synostosis, the individual role of Tcf12 had remained unexplored. Here, we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement for Tcf12 in suture formation, as combined deletion of Tcf12 in embryonic neural crest and mesoderm, but not in postnatal suture mesenchyme, disrupts the coronal suture. We also detected asymmetric distribution of mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. In Tcf12 mutants, reduced asymmetry is associated with bones meeting end-on-end, possibly contributing to synostosis. Our results support embryonic requirements of Tcf12 in proper formation of the overlapping coronal suture.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Craniossinostoses/metabolismo , Osteogênese , Crânio/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Craniossinostoses/embriologia , Craniossinostoses/genética , Células-Tronco Mesenquimais/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Crista Neural/metabolismo , Crânio/metabolismoRESUMO
Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.
Assuntos
Consolidação da Fratura , Peixe-Zebra , Animais , Regeneração Óssea , Calo Ósseo/metabolismo , Consolidação da Fratura/fisiologia , Mamíferos , MandíbulaRESUMO
Proper function of the vertebrate skeleton requires the development of distinct articulating embryonic cartilages. Irx transcription factors are arranged in co-regulated clusters that are expressed in the developing skeletons of the face and appendages. IrxB cluster genes are required for the separation of toes in mice and formation of the hyoid joint in zebrafish, yet whether Irx genes have broader roles in skeletal development remains unclear. Here, we perform a comprehensive loss-of-function analysis of all 11 Irx genes in zebrafish. We uncover conserved requirements for IrxB genes in formation of the fish and mouse scapula. In the face, we find a requirement for IrxAb genes and irx7 in formation of anterior neural crest precursors of the jaw, and for IrxBa genes in formation of endodermal pouches and gill cartilages. We also observe extensive joint loss and cartilage fusions in animals with combinatorial losses of Irx clusters, with in vivo imaging revealing that at least some of these fusions arise through inappropriate chondrogenesis. Our analysis reveals diverse roles for Irx genes in the formation and later segmentation of the facial skeleton.
Assuntos
Cartilagem/embriologia , Condrogênese/genética , Proteínas de Homeodomínio/metabolismo , Família Multigênica , Proteínas Mutantes/metabolismo , Crânio/embriologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Alelos , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mutação , Crista Neural/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Tendons and ligaments are fibrous connective tissues vital to the transmission of force and stabilization of the musculoskeletal system. Arising in precise regions of the embryo, tendons and ligaments share many properties and little is known about the molecular differences that differentiate them. Recent studies have revealed heterogeneity and plasticity within tendon and ligament cells, raising questions regarding the developmental mechanisms regulating tendon and ligament identity. Here, we discuss recent findings that contribute to our understanding of the mechanisms that establish and maintain tendon progenitors and their differentiated progeny in the head, trunk and limb. We also review the extent to which these findings are specific to certain anatomical regions and model organisms, and indicate which findings similarly apply to ligaments. Finally, we address current research regarding the cellular lineages that contribute to tendon and ligament repair, and to what extent their regulation is conserved within tendon and ligament development.
Assuntos
Diferenciação Celular , Ligamentos/embriologia , Desenvolvimento Musculoesquelético , Células-Tronco/metabolismo , Tendões/embriologia , Animais , Humanos , Ligamentos/citologia , Células-Tronco/citologia , Tendões/citologiaRESUMO
Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.
Assuntos
Diferenciação Celular/fisiologia , Mesoderma/metabolismo , Mesoderma/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rim/metabolismo , Rim/fisiologia , Organogênese/fisiologia , Fatores de Transcrição/metabolismoRESUMO
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
Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance and repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with inconsistent characteristics. However, recent in vivo tracking experiments have revealed the presence of SSCs not only within the bone marrow but also within the periosteum and growth plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to lineage potential. It has also been revealed that, during digit tip regeneration and in some non-mammalian vertebrates, the dedifferentiation of osteoblasts may contribute to skeletal regeneration. Here, we examine how these research findings have furthered our understanding of the diversity and plasticity of SSCs that mediate skeletal maintenance and repair.
Assuntos
Desenvolvimento Ósseo/fisiologia , Regeneração Óssea/fisiologia , Osteogênese/fisiologia , Periósteo/citologia , Células-Tronco/citologia , Animais , Células da Medula Óssea/citologia , Condrócitos/citologia , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Humanos , Camundongos , Osteoblastos/citologia , Peixe-ZebraRESUMO
Whereas the gill chambers of jawless vertebrates open directly into the environment, jawed vertebrates evolved skeletal appendages that drive oxygenated water unidirectionally over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here, we find that these divergent patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a deeply conserved Pou3f3 arch enhancer present in humans through sharks but undetectable in jawless fish. Minor differences between the bony and cartilaginous fish enhancers account for their restricted versus pan-arch expression patterns. In zebrafish, mutation of Pou3f3 or the conserved enhancer disrupts gill cover formation, whereas ectopic pan-arch Pou3f3b expression generates ectopic skeletal elements resembling the multimeric covers of cartilaginous fishes. Emergence of this Pou3f3 arch enhancer >430 Mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.
Assuntos
Elementos Facilitadores Genéticos , Evolução Molecular , Brânquias/crescimento & desenvolvimento , Fatores do Domínio POU/genética , Vertebrados/genética , Animais , Peixes/classificação , Peixes/genética , Peixes/crescimento & desenvolvimento , Mutação , Filogenia , Tubarões/classificação , Tubarões/genética , Tubarões/crescimento & desenvolvimento , Vertebrados/classificação , Vertebrados/crescimento & desenvolvimentoRESUMO
Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrblow mesenchymal cells. The emergence of peri-arterial pdgfrbhigh MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrbhigh MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrbhigh MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.
Assuntos
Artérias/citologia , Artérias/embriologia , Padronização Corporal , Mesoderma/embriologia , Receptores Notch/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Animais , Biomarcadores/metabolismo , Endotélio Vascular/metabolismo , Mesoderma/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Imagem com Lapso de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.
Assuntos
Fator II de Transcrição COUP/metabolismo , Proteínas de Ligação a DNA/metabolismo , Miócitos Cardíacos/metabolismo , Músculos Faríngeos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Fator II de Transcrição COUP/genética , Linhagem da Célula/genética , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Ventrículos do Coração/citologia , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Modelos Animais , Mutação , Miócitos Cardíacos/citologia , Músculos Faríngeos/citologia , Músculos Faríngeos/embriologia , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/genética , Tretinoína/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Facial form depends on the precise positioning of cartilage, bone, and tooth fields in the embryonic pharyngeal arches. How complex signaling information is integrated to specify these cell types remains a mystery. We find that modular expression of Forkhead domain transcription factors (Fox proteins) in the zebrafish face arises through integration of Hh, Fgf, Bmp, Edn1 and Jagged-Notch pathways. Whereas loss of C-class Fox proteins results in reduced upper facial cartilages, loss of F-class Fox proteins results in distal jaw truncations and absent midline cartilages and teeth. We show that Fox proteins are required for Sox9a to promote chondrogenic gene expression. Fox proteins are sufficient in neural crest-derived cells for cartilage development, and neural crest-specific misexpression of Fox proteins expands the cartilage domain but inhibits bone. These results support a modular role for Fox proteins in establishing the competency of progenitors to form cartilage and teeth in the face.
Assuntos
Padronização Corporal , Cartilagem/embriologia , Cartilagem/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Dente/embriologia , Dente/metabolismo , Animais , Padronização Corporal/genética , Osso e Ossos/metabolismo , Região Branquial/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Condrogênese/genética , Face , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação/genética , Crista Neural/citologia , Transdução de Sinais , Crânio/citologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.
Assuntos
Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Região Branquial/embriologia , Região Branquial/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Proteínas de Peixe-Zebra/genéticaRESUMO
OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.
Assuntos
Osteoartrite/patologia , Osteófito/patologia , Periósteo/patologia , Células-Tronco/patologia , Membrana Sinovial/patologia , Animais , Linhagem da Célula , CamundongosRESUMO
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.