RESUMO
Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury. Whereas reactive gliosis presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish elicit pro-regenerative bridging functions after injury. Here, we perform genetic lineage tracing, assessment of regulatory sequences and inducible cell ablation to define mechanisms that direct the molecular and cellular responses of glial cells after spinal cord injury in adult zebrafish. Using a newly generated CreERT2 transgenic line, we show that the cells directing expression of the bridging glial marker ctgfa give rise to regenerating glia after injury, with negligible contribution to either neuronal or oligodendrocyte lineages. A 1â kb sequence upstream of the ctgfa gene was sufficient to direct expression in early bridging glia after injury. Finally, ablation of ctgfa-expressing cells using a transgenic nitroreductase strategy impaired glial bridging and recovery of swim behavior after injury. This study identifies key regulatory features, cellular progeny, and requirements of glial cells during innate spinal cord regeneration.
Assuntos
Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Neuroglia/metabolismo , Animais Geneticamente Modificados , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Regeneração Nervosa/genética , Mamíferos/metabolismoRESUMO
Sensory neurons located in dorsal root ganglia (DRG) convey sensory information from peripheral tissue to the brain. After peripheral nerve injury, sensory neurons switch to a regenerative state to enable axon regeneration and functional recovery. This process is not cell autonomous and requires glial and immune cells. Macrophages in the DRG (DRGMacs) accumulate in response to nerve injury, but their origin and function remain unclear. Here, we mapped the fate and response of DRGMacs to nerve injury using macrophage depletion, fate-mapping, and single-cell transcriptomics. We identified three subtypes of DRGMacs after nerve injury in addition to a small population of circulating bone-marrow-derived precursors. Self-renewing macrophages, which proliferate from local resident macrophages, represent the largest population of DRGMacs. The other two subtypes include microglia-like cells and macrophage-like satellite glial cells (SGCs) (Imoonglia). We show that self-renewing DRGMacs contribute to promote axon regeneration. Using single-cell transcriptomics data and CellChat to simulate intercellular communication, we reveal that macrophages express the neuroprotective and glioprotective ligand prosaposin and communicate with SGCs via the prosaposin receptor GPR37L1. These data highlight that DRGMacs have the capacity to self-renew, similarly to microglia in the Central nervous system (CNS) and contribute to promote axon regeneration. These data also reveal the heterogeneity of DRGMacs and their potential neuro- and glioprotective roles, which may inform future therapeutic approaches to treat nerve injury.
Assuntos
Axônios , Traumatismos dos Nervos Periféricos , Humanos , Axônios/fisiologia , Regeneração Nervosa/fisiologia , Gânglios Espinais/fisiologia , Macrófagos/fisiologia , Neuroglia , Receptores Acoplados a Proteínas G/genéticaRESUMO
The ability of zebrafish to heal their heart after injury makes them an attractive model for investigating the mechanisms governing the regenerative process. In this study, we show that the gene cellular communication network factor 2a (ccn2a), previously known as ctgfa, is induced in endocardial cells in the injured tissue and regulates CM proliferation and repopulation of the damaged tissue. We find that, whereas in wild-type animals, CMs track along the newly formed blood vessels that revascularize the injured tissue, in ccn2a mutants CM proliferation and repopulation are disrupted, despite apparently unaffected revascularization. In addition, we find that ccn2a overexpression enhances CM proliferation and improves the resolution of transient collagen deposition. Through loss- and gain-of-function as well as pharmacological approaches, we provide evidence that Ccn2a is necessary for and promotes heart regeneration by enhancing the expression of pro-regenerative extracellular matrix genes, and by inhibiting the chemokine receptor gene cxcr3.1 through a mechanism involving Tgfß/pSmad3 signaling. Thus, Ccn2a positively modulates the innate regenerative response of the adult zebrafish heart.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Coração/fisiopatologia , Regeneração , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/genética , Vasos Coronários/metabolismo , Endocárdio/patologia , Endocárdio/fisiopatologia , Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Transporte Proteico , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
In response to skeletal muscle injury, satellite cells, which function as a myogenic stem cell population, become activated, expand through proliferation, and ultimately fuse with each other and with damaged myofibers to promote muscle regeneration. Here, we show that members of the Myocardin family of transcriptional coactivators, MASTR and MRTF-A, are up-regulated in satellite cells in response to skeletal muscle injury and muscular dystrophy. Global and satellite cell-specific deletion of MASTR in mice impairs skeletal muscle regeneration. This impairment is substantially greater when MRTF-A is also deleted and is due to aberrant differentiation and excessive proliferation of satellite cells. These abnormalities mimic those associated with genetic deletion of MyoD, a master regulator of myogenesis, which is down-regulated in the absence of MASTR and MRTF-A. Consistent with an essential role of MASTR in transcriptional regulation of MyoD expression, MASTR activates a muscle-specific postnatal MyoD enhancer through associations with MEF2 and members of the Myocardin family. Our results provide new insights into the genetic circuitry of muscle regeneration and identify MASTR as a central regulator of this process.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Proteína MyoD/metabolismo , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/citologia , Transativadores/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Elementos Facilitadores Genéticos , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Ligação Proteica , Transativadores/genéticaRESUMO
Histone deacetylases (Hdacs) are transcriptional repressors with crucial roles in mammalian development. Here we provide evidence that Hdac8 specifically controls patterning of the skull by repressing a subset of transcription factors in cranial neural crest cells. Global deletion of Hdac8 in mice leads to perinatal lethality due to skull instability, and this is phenocopied by conditional deletion of Hdac8 in cranial neural crest cells. Hdac8 specifically represses the aberrant expression of homeobox transcription factors such as Otx2 and Lhx1. These findings reveal how the identity and patterning of vertebrate-specific portions of the skull are epigenetically controlled by a histone deacetylase.
Assuntos
Padronização Corporal/genética , Epigênese Genética , Histona Desacetilases/metabolismo , Crânio/embriologia , Animais , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Homeodomínio LIM , Camundongos , Fatores de Transcrição Otx/metabolismo , Crânio/anormalidades , Fatores de TranscriçãoRESUMO
Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the developing or adult heart has not been explored. Through cardiac-specific deletion of MRTF alleles in mice, we show that either MRTF-A or MRTF-B is dispensable for cardiac development and function, whereas deletion of both MRTF-A and MRTF-B causes a spectrum of structural and functional cardiac abnormalities. Defects observed in MRTF-A/B null mice ranged from reduced cardiac contractility and adult onset heart failure to neonatal lethality accompanied by sarcomere disarray. RNA-seq analysis on neonatal hearts identified the most altered pathways in MRTF double knockout hearts as being involved in cytoskeletal organization. Together, these findings demonstrate redundant but essential roles of the MRTFs in maintenance of cardiac structure and function and as indispensible links in cardiac cytoskeletal gene regulatory networks.
Assuntos
Redes Reguladoras de Genes/fisiologia , Coração/embriologia , Morfogênese/fisiologia , Sarcômeros/fisiologia , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Citoesqueleto/fisiologia , Ecocardiografia , Coração/fisiologia , Técnicas Histológicas , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Sarcômeros/metabolismo , Análise de Sequência de RNA , Transativadores/deficiência , Fatores de Transcrição/deficiênciaRESUMO
Striated muscle development requires the coordinated expression of genes involved in sarcomere formation and contractility, as well as genes that determine muscle morphology. However, relatively little is known about the molecular mechanisms that control the early stages of muscle morphogenesis. To explore this facet of myogenesis, we performed a genetic screen for regulators of somatic muscle morphology in Drosophila, and identified the putative RNA-binding protein (RBP) Hoi Polloi (Hoip). Hoip is expressed in striated muscle precursors within the muscle lineage and controls two genetically separable events: myotube elongation and sarcomeric protein expression. Myotubes fail to elongate in hoip mutant embryos, even though the known regulators of somatic muscle elongation, target recognition and muscle attachment are expressed normally. In addition, a majority of sarcomeric proteins, including Myosin Heavy Chain (MHC) and Tropomyosin, require Hoip for their expression. A transgenic MHC construct that contains the endogenous MHC promoter and a spliced open reading frame rescues MHC protein expression in hoip embryos, demonstrating the involvement of Hoip in pre-mRNA splicing, but not in transcription, of muscle structural genes. In addition, the human Hoip ortholog NHP2L1 rescues muscle defects in hoip embryos, and knockdown of endogenous nhp2l1 in zebrafish disrupts skeletal muscle development. We conclude that Hoip is a conserved, post-transcriptional regulator of muscle morphogenesis and structural gene expression.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Estriado/embriologia , Proteínas de Ligação a RNA/metabolismo , Sarcômeros/metabolismo , Animais , Sequência de Bases , Western Blotting , Primers do DNA/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Desenvolvimento Muscular/genética , Músculo Estriado/metabolismo , Mutagênese Sítio-Dirigida , Cadeias Pesadas de Miosina/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Análise de Sequência de RNA , Tropomiosina/metabolismo , Peixe-Zebra/embriologiaRESUMO
Intercellular signal transduction pathways regulate the NK-2 family of transcription factors in a conserved gene regulatory network that directs cardiogenesis in both flies and mammals. The Drosophila NK-2 protein Tinman (Tin) was recently shown to regulate Stat92E, the Janus kinase (JAK) and Signal transducer and activator of transcription (Stat) pathway effector, in the developing mesoderm. To understand whether the JAK/Stat pathway also regulates cardiogenesis, we performed a systematic characterization of JAK/Stat signaling during mesoderm development. Drosophila embryos with mutations in the JAK/Stat ligand upd or in Stat92E have non-functional hearts with luminal defects and inappropriate cell aggregations. Using strong Stat92E loss-of-function alleles, we show that the JAK/Stat pathway regulates tin expression prior to heart precursor cell diversification. tin expression can be subdivided into four phases and, in Stat92E mutant embryos, the broad phase 2 expression pattern in the dorsal mesoderm does not restrict to the constrained phase 3 pattern. These embryos also have an expanded pericardial cell domain. We show the E(spl)-C gene HLHm5 is expressed in a pattern complementary to tin during phase 3 and that this expression is JAK/Stat dependent. In addition, E(spl)-C mutant embryos phenocopy the cardiac defects of Stat92E embryos. Mechanistically, JAK/Stat signals activate E(spl)-C genes to restrict Tin expression and the subsequent expression of the T-box transcription factor H15 to direct heart precursor diversification. This study is the first to characterize a role for the JAK/Stat pathway during cardiogenesis and identifies an autoregulatory circuit in which tin limits its own expression domain.
Assuntos
Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Janus Quinases/metabolismo , Organogênese/fisiologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Coração/anatomia & histologia , Coração/embriologia , Janus Quinases/genética , Masculino , Mesoderma/citologia , Mesoderma/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição STAT/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, a transient population of injury-responsive neurons (iNeurons) show elevated plasticity 1 week post-injury. We found iNeurons are injury-surviving neurons that acquire a neuroblast-like gene expression signature after injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.
Assuntos
Neurogênese , Plasticidade Neuronal , Análise de Célula Única , Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Medula Espinal , Peixe-Zebra , Animais , Traumatismos da Medula Espinal/metabolismo , Plasticidade Neuronal/fisiologia , Neurogênese/genética , Medula Espinal/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Sistemas CRISPR-Cas , Neurônios GABAérgicos/metabolismo , Recuperação de Função Fisiológica , Modelos Animais de Doenças , Regeneração Nervosa/fisiologia , Animais Geneticamente ModificadosRESUMO
Immune cells elicit a continuum of transcriptional and functional states after spinal cord injury (SCI). In mammals, inefficient debris clearance and chronic inflammation impede recovery and overshadow pro-regenerative immune functions. We found that, unlike mammals, zebrafish SCI elicits transient immune activation and efficient debris clearance, without causing chronic inflammation. Single-cell transcriptomics and inducible genetic ablation showed zebrafish macrophages are highly phagocytic and required for regeneration. Cross-species comparisons between zebrafish and mammalian macrophages identified transcription and immune response regulator ( tcim ) as a macrophage-enriched zebrafish gene. Genetic deletion of zebrafish tcim impairs phagocytosis and regeneration, causes aberrant and chronic immune activation, and can be rescued by transplanting wild-type immune precursors into tcim mutants. Conversely, genetic expression of human TCIM accelerates debris clearance and regeneration by reprogramming myeloid precursors into activated phagocytes. This study establishes a central requirement for elevated phagocytic capacity to achieve innate spinal cord repair.
RESUMO
Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. Here, we show that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development.
Assuntos
Movimento Celular/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Movimento Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neuritos/metabolismo , Neurogênese , Proteínas Nucleares , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transporte Proteico/genética , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de Sinais/genética , Transativadores , Fatores de Transcrição/genéticaRESUMO
Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, transient populations of injury-responsive neurons (iNeurons) show elevated plasticity between 1 and 3 weeks post-injury. Using cross-species transcriptomics and CRISPR/Cas9 mutagenesis, we found iNeurons are injury-surviving neurons that share transcriptional similarities with a rare population of spontaneously plastic mouse neurons. iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.
RESUMO
Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Axon growth, glial bridging and adult neurogenesis are hallmarks of cellular regeneration during spinal cord repair. However, the correlation between these cellular regenerative processes and functional recovery remains to be elucidated. Whereas the majority of established functional regeneration metrics measure swim capacity, we hypothesize that gait quality is more directly related to neurological health. Here, we performed a longitudinal swim tracking study for sixty individual zebrafish spanning eight weeks of spinal cord regeneration. Multiple swim parameters as well as axonal and glial bridging were integrated. We established rostral compensation as a new gait quality metric that highly correlates with functional recovery. Tensor component analysis of longitudinal data supports a correspondence between functional recovery trajectories and neurological outcomes. Moreover, our studies predicted and validated that a subset of functional regeneration parameters measured 1 to 2 weeks post-injury is sufficient to predict the regenerative outcomes of individual animals at 8 weeks post-injury. Our findings established new functional regeneration parameters and generated a comprehensive correlative database between various functional and cellular regeneration outputs.
RESUMO
Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Axon growth, glial bridging and adult neurogenesis are hallmarks of cellular regeneration during spinal cord repair. However, the correlation between these cellular regenerative processes and functional recovery remains to be elucidated. Whereas the majority of established functional regeneration metrics measure swim capacity, we hypothesize that gait quality is more directly related to neurological health. Here, we performed a longitudinal swim tracking study for 60 individual zebrafish spanning 8 weeks of spinal cord regeneration. Multiple swim parameters as well as axonal and glial bridging were integrated. We established rostral compensation as a new gait quality metric that highly correlates with functional recovery. Tensor component analysis of longitudinal data supports a correspondence between functional recovery trajectories and neurological outcomes. Moreover, our studies predicted and validated that a subset of functional regeneration parameters measured 1 to 2 weeks post-injury is sufficient to predict the regenerative outcomes of individual animals at 8 weeks post-injury. Our findings established new functional regeneration parameters and generated a comprehensive correlative database between various functional and cellular regeneration outputs.
RESUMO
Histone deacetylase inhibitors (HDACi) represent a new group of drugs currently being tested in a wide variety of clinical applications. They are especially effective in preclinical models of cancer where they show antiproliferative action in many different types of cancer cells. Recently, the first HDACi was approved for the treatment of cutaneous T cell lymphomas. Most HDACi currently in clinical development act by unspecifically interfering with the enzymatic activity of all class I HDACs (HDAC1, 2, 3, and 8), and it is widely believed that the development of isoform-specific HDACi could lead to better therapeutic efficacy. The contribution of the individual class I HDACs to different disease states, however, has so far not been fully elucidated. Here, we use a genetic approach to dissect the involvement of the different class I HDACs in tumor cells. We show that deletion of a single HDAC is not sufficient to induce cell death, but that HDAC1 and 2 play redundant and essential roles in tumor cell survival. Their deletion leads to nuclear bridging, nuclear fragmentation, and mitotic catastrophe, mirroring the effects of HDACi on cancer cells. These findings suggest that pharmacological inhibition of HDAC1 and 2 may be sufficient for anticancer activity, providing an experimental framework for the development of isoform-specific HDAC inhibitors.
Assuntos
Histona Desacetilases/genética , Histona Desacetilases/fisiologia , Animais , Antineoplásicos/farmacologia , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases , Humanos , Camundongos , Camundongos Nus , Modelos Genéticos , Transplante de Neoplasias , Neoplasias/metabolismo , Isoformas de ProteínasRESUMO
Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.
Assuntos
Traumatismos da Medula Espinal , Peixe-Zebra , Adulto , Humanos , Animais , Recém-Nascido , Miostatina , Neurogênese , Traumatismos da Medula Espinal/genética , Recuperação de Função Fisiológica , Fator 1 de Crescimento de Fibroblastos , MamíferosRESUMO
Adipocyte differentiation is a well defined process that is under the control of transcriptional activators and repressors. We show that histone deacetylase (HDAC) inhibitors efficiently block adipocyte differentiation in vitro. This effect is specific to adipogenesis, as another mesenchymal differentiation process, osteoblastogenesis, is enhanced upon HDAC inhibition. Through the systematic genetic deletion of HDAC genes in cultured mesenchymal precursor cells, we show that deletion of HDAC1 and HDAC2 leads to reduced lipid accumulation, revealing redundant and requisite roles of these class I HDACs in adipogenesis. These findings unveil a previously unrecognized role for HDACs in the control of adipogenesis.
Assuntos
Adipogenia/fisiologia , Embrião de Mamíferos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Western Blotting , Butiratos/farmacologia , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/genética , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Osteogênese , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.
Assuntos
Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Animais , Axônios/fisiologia , Regeneração Nervosa/fisiologia , Medula Espinal , Regeneração da Medula Espinal/fisiologia , Peixe-Zebra , Proteínas de Peixe-ZebraRESUMO
Adult zebrafish are widely used to interrogate mechanisms of disease development and tissue regeneration. Yet, the prospect of large-scale genetics in adult zebrafish has traditionally faced a host of biological and technical challenges, including inaccessibility of adult tissues to high-throughput phenotyping and the spatial and technical demands of adult husbandry. Here, we describe an experimental pipeline that combines high-efficiency CRISPR/Cas9 mutagenesis with functional phenotypic screening to identify genes required for spinal cord repair in adult zebrafish. Using CRISPR/Cas9 dual-guide ribonucleic proteins, we show selective and combinatorial mutagenesis of 17 genes at 28 target sites with efficiencies exceeding 85% in adult F0 "crispants". We find that capillary electrophoresis is a reliable method to measure indel frequencies. Using a quantifiable behavioral assay, we identify seven single- or duplicate-gene crispants with reduced functional recovery after spinal cord injury. To rule out off-target effects, we generate germline mutations that recapitulate the crispant regeneration phenotypes. This study provides a platform that combines high-efficiency somatic mutagenesis with a functional phenotypic readout to perform medium- to large-scale genetic studies in adult zebrafish.
Assuntos
Sistemas CRISPR-Cas , Peixe-Zebra , Animais , Mutação INDEL , Mutagênese , Fenótipo , Peixe-Zebra/genéticaRESUMO
Anti-regenerative scarring obstructs spinal cord repair in mammals and presents a major hurdle for regenerative medicine. In contrast, adult zebrafish possess specialized glial cells that spontaneously repair spinal cord injuries by forming a pro-regenerative bridge across the severed tissue. To identify the mechanisms that regulate differential regenerative capacity between mammals and zebrafish, we first defined the molecular identity of zebrafish bridging glia and then performed cross-species comparisons with mammalian glia. Our transcriptomics show that pro-regenerative zebrafish glia activate an epithelial-to-mesenchymal transition (EMT) gene program and that EMT gene expression is a major factor distinguishing mammalian and zebrafish glia. Functionally, we found that localized niches of glial progenitors undergo EMT after spinal cord injury in zebrafish and, using large-scale CRISPR-Cas9 mutagenesis, we identified the gene regulatory network that activates EMT and drives functional regeneration. Thus, non-regenerative mammalian glia lack an essential EMT-driving gene regulatory network that reprograms pro-regenerative zebrafish glia after injury.