RESUMO
Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.
Assuntos
Proteínas Morfogenéticas Ósseas , Suturas Cranianas , Mesoderma , Osteogênese , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Suturas Cranianas/metabolismo , Suturas Cranianas/embriologia , Suturas Cranianas/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Mesoderma/metabolismo , Mesoderma/embriologia , Mesoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Crânio/embriologia , Análise de Célula Única , MutaçãoRESUMO
Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.
Assuntos
Receptores Citoplasmáticos e Nucleares , Peixe-Zebra , Camundongos , Animais , Peixe-Zebra/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Diferenciação Celular/fisiologia , Tecido Conjuntivo/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Crista Neural/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Zebrafish (Danio rerio) are aquatic vertebrates with significant homology to their terrestrial counterparts. While zebrafish have a centuries-long track record in developmental and regenerative biology, their utility has grown exponentially with the onset of modern genetics. This is exemplified in studies focused on skeletal development and repair. Herein, the numerous contributions of zebrafish to our understanding of the basic science of cartilage, bone, tendon/ligament, and other skeletal tissues are described, with a particular focus on applications to development and regeneration. We summarize the genetic strengths that have made the zebrafish a powerful model to understand skeletal biology. We also highlight the large body of existing tools and techniques available to understand skeletal development and repair in the zebrafish and introduce emerging methods that will aid in novel discoveries in skeletal biology. Finally, we review the unique contributions of zebrafish to our understanding of regeneration and highlight diverse routes of repair in different contexts of injury. We conclude that zebrafish will continue to fill a niche of increasing breadth and depth in the study of basic cellular mechanisms of skeletal biology.
Assuntos
Tendões , Peixe-Zebra , Animais , Peixe-Zebra/genética , Osso e Ossos , CartilagemRESUMO
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
Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.
Assuntos
Suturas Cranianas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osteogênese/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Acrocefalossindactilia/embriologia , Acrocefalossindactilia/genética , Acrocefalossindactilia/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Suturas Cranianas/citologia , Suturas Cranianas/embriologia , Dura-Máter/citologia , Dura-Máter/embriologia , Dura-Máter/metabolismo , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA-Seq/métodos , Crânio/citologia , Crânio/embriologia , Crânio/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
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
Much of the vertebrate skeleton develops from cartilage templates that are progressively remodeled into bone. Lineage tracing studies in mouse suggest that chondrocytes within these templates persist and become osteoblasts, yet the underlying mechanisms of this process and whether chondrocytes can generate other derivatives remain unclear. We find that zebrafish cartilages undergo extensive remodeling and vascularization during juvenile stages to generate fat-filled bones. Growth plate chondrocytes marked by sox10 and col2a1a contribute to osteoblasts, marrow adipocytes, and mesenchymal cells within adult bones. At the edge of the hypertrophic zone, chondrocytes re-enter the cell cycle and express leptin receptor (lepr), suggesting conversion into progenitors. Further, mutation of matrix metalloproteinase 9 (mmp9) results in delayed growth plate remodeling and fewer marrow adipocytes. Our data support Mmp9-dependent growth plate remodeling and conversion of chondrocytes into osteoblasts and marrow adipocytes as conserved features of bony vertebrates.
Assuntos
Adipócitos/citologia , Diferenciação Celular/genética , Condrócitos/citologia , Osteoblastos/citologia , Animais , Células da Medula Óssea/citologia , Cartilagem/crescimento & desenvolvimento , Colágeno Tipo II/genética , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Fatores de Transcrição SOXE/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.
Assuntos
Acrocefalossindactilia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Craniossinostoses/genética , Proteína 1 Relacionada a Twist/genética , Acrocefalossindactilia/patologia , Animais , Desenvolvimento Ósseo , Craniossinostoses/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Camundongos , Mutação , Crista Neural/crescimento & desenvolvimento , Crista Neural/patologia , Osteogênese/genética , Peixe-Zebra/genéticaRESUMO
While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.
Assuntos
Proteínas Argonautas/metabolismo , MicroRNAs/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/fisiologia , RNA Helicases DEAD-box/metabolismo , Eritroblastos/metabolismo , Estudo de Associação Genômica Ampla , Mamíferos/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Interferência de RNA , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Homologia de Sequência do Ácido NucleicoRESUMO
The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5+ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis.
Assuntos
Linhagem da Célula , Células Epiteliais/citologia , Aparelho Lacrimal/citologia , Aparelho Lacrimal/embriologia , Células Acinares/citologia , Células Acinares/metabolismo , Animais , Biomarcadores/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
The tear film protects the terrestrial animal's ocular surface and the lacrimal gland provides important aqueous secretions necessary for its maintenance. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an important gene for lacrimal gland development. Mice lacking miR-205 fail to properly develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal gland development. Specifically, more than half of knockout lacrimal glands never initiated, suggesting a critical role of miR-205 at the earliest stages of lacrimal gland development. RNA-seq analysis uncovered several up-regulated miR-205 targets that may interfere with signaling to impair lacrimal gland initiation. Supporting this data, combinatorial epistatic deletion of Fgf10, the driver of lacrimal gland initiation, and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways related to Fgf10 in order to regulate glandular development. These data show that a single microRNA is a key regulator for early lacrimal gland development in mice and highlights the important role of microRNAs during organogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Aparelho Lacrimal/metabolismo , MicroRNAs/genética , Organogênese/genética , Animais , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica/métodos , Aparelho Lacrimal/embriologia , Aparelho Lacrimal/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA/métodos , Transdução de Sinais/genéticaRESUMO
The surface ectoderm is the source of ectodermal appendages including hair, teeth, and many glands. The development and function of ectodermal appendages has been researched extensively, but many of the molecular mechanisms that govern the developmental programs of ectodermal appendages remain elusive. While several protein-coding genes are established as key regulators of ectodermal appendage development, the role of noncoding RNAs is an emerging area of investigation. This review highlights recent advances in studies of microRNA-mediated control of ectodermal appendage development using mouse models. We will also discuss future directions and technological advances that will drive the microRNA field forward and expand our understanding of how individual microRNAs control ectodermal appendage development.
Assuntos
Ectoderma/crescimento & desenvolvimento , Cabelo/metabolismo , MicroRNAs/genética , Dente/metabolismo , Animais , Ectoderma/metabolismo , Cabelo/crescimento & desenvolvimento , Humanos , Camundongos , MicroRNAs/metabolismo , Dente/crescimento & desenvolvimentoRESUMO
MicroRNAs are small noncoding RNAs thought to have pivotal roles in numerous diseases and developmental processes. However, a growing body of literature indicates that in vivo elimination of these tiny RNAs usually has little to no observable consequence, suggesting functional redundancy with other microRNAs or cellular pathways. We provide an in-depth analysis of miR-205 expression and define miR-205 as an epithelial-specific microRNA, and for the first time show that ablation of this microRNA knockout exhibits partially penetrant lethality in a constitutive mouse knockout model. Given the role of this microRNA in cancer and development, this mouse model will be an incredible reagent to study the function and mechanisms of miR-205 in epithelial tissue development and disease.