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1.
J Environ Sci (China) ; 107: 160-170, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34412779

RESUMO

Elevated temperature could influence the sex differentiation by altering the expression of sex-related genes in fish. However, the underlying mechanisms by which the gene expression is altered remain poorly understood. Here, we aimed to explore the role of DNA methylation in sex differentiation of zebrafish (Danio rerio) in response to elevated temperature. The results showed that high temperature (33°C) exposure of fish from 20 to 30 days post fertilization (dpf), compared to normal temperature (28°C), resulted in male-biased sex ratio and decreased expression of female-related genes including cyp19a1a, sox9b and esr1. Meanwhile, the expressions of DNA methyltransferases dnmt3a1 and dnmt3a2, and the DNA methylation levels in sox9b and esr1 promoter were significantly increased by high temperature, strongly implying that DNA methylation is involved in high temperature-induced masculinization of zebrafish. Co-treatment with 5-aza-2'-deoxycytidine (a DNA methylation inhibitor) attenuated the high temperature-induced masculinizing effect, recovered the expression of esr1 and sox9b, suppressed the transcription of dnmt3a1 and dnmt3a2, and decreased the methylation of esr1 and sox9b promoter, further confirming that DNA methylation plays an important role in high temperature-induced masculinization of zebrafish. Furthermore, the methylation of sox9b promoter decreased the enrichment of transcription factor CREB (cAMP-responsive element binding proteins). Overall, these findings suggest that high temperature induce masculinization of zebrafish by down-regulation of female-related genes via DNA methylation, providing a new insight in understanding the epigenetic mechanism of thermal-mediated sex differentiation in fish.


Assuntos
Metilação de DNA , Receptor alfa de Estrogênio/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Regulação para Baixo , Epigênese Genética , Feminino , Masculino , Fatores de Transcrição SOX9/genética , Temperatura , Peixe-Zebra/genética
2.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360576

RESUMO

Noncoding RNAs have been known to contribute to a variety of fundamental life processes, such as development, metabolism, and circadian rhythms. However, much remains unrevealed in the huge noncoding RNA datasets, which require further bioinformatic analysis and experimental investigation-and in particular, the coding potential of lncRNAs and the functions of lncRNA-encoded peptides have not been comprehensively studied to date. Through integrating the time-course experimentation with state-of-the-art computational techniques, we studied tens of thousands of zebrafish lncRNAs from our own experiments and from a published study including time-series transcriptome analyses of the testis and the pineal gland. Rhythmicity analysis of these data revealed approximately 700 rhythmically expressed lncRNAs from the pineal gland and the testis, and their GO, COG, and KEGG pathway functions were analyzed. Comparative and conservative analyses determined 14 rhythmically expressed lncRNAs shared between both the pineal gland and the testis, and 15 pineal gland lncRNAs as well as 3 testis lncRNAs conserved among zebrafish, mice, and humans. Further, we computationally analyzed the conserved lncRNA-encoded peptides, and revealed three pineal gland and one testis lncRNA-encoded peptides conserved among these three species, which were further investigated for their three-dimensional (3D) structures and potential functions. Our computational findings provided novel annotations and regulatory mechanisms for hundreds of rhythmically expressed pineal gland and testis lncRNAs in zebrafish, and set the stage for their experimental studies in the near future.


Assuntos
Ritmo Circadiano , Glândula Pineal/metabolismo , RNA Longo não Codificante/genética , Testículo/metabolismo , Transcriptoma , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Biologia Computacional , Perfilação da Expressão Gênica , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
3.
Nucleic Acids Res ; 49(15): 8961-8973, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365506

RESUMO

Histone recognition constitutes a key epigenetic mechanism in gene regulation and cell fate decision. PHF14 is a conserved multi-PHD finger protein that has been implicated in organ development, tissue homeostasis, and tumorigenesis. Here we show that PHF14 reads unmodified histone H3(1-34) through an integrated PHD1-ZnK-PHD2 cassette (PHF14PZP). Our binding, structural and HDX-MS analyses revealed a feature of bipartite recognition, in which PHF14PZP utilizes two distinct surfaces for concurrent yet separable engagement of segments H3-Nter (e.g. 1-15) and H3-middle (e.g. 14-34) of H3(1-34). Structural studies revealed a novel histone H3 binding mode by PHD1 of PHF14PZP, in which a PHF14-unique insertion loop but not the core ß-strands of a PHD finger dominates H3K4 readout. Binding studies showed that H3-PHF14PZP engagement is sensitive to modifications occurring to H3 R2, T3, K4, R8 and K23 but not K9 and K27, suggesting multiple layers of modification switch. Collectively, our work calls attention to PHF14 as a 'ground' state (unmodified) H3(1-34) reader that can be negatively regulated by active marks, thus providing molecular insights into a repressive function of PHF14 and its derepression.


Assuntos
Histonas/química , Histonas/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Regulação Alostérica , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese , Proteínas Nucleares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Proteínas de Peixe-Zebra/genética
4.
Ecotoxicol Environ Saf ; 222: 112514, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34280841

RESUMO

Pendimethalin (PND) is one of the best sellers of selective herbicide in the world and has been frequently detected in the water. However, little is known about its effects on cardiac development. In this study, we used zebrafish to investigate the developmental and cardiac toxicity of PND. We exposed the zebrafish embryos with a serial of concentrations at 3, 4, and 5 mg/L at 5.5-72 h post-fertilization (hpf). We found that PND exposure can reduce the heart rate, survival rate, and body length of zebrafish embryos. Furthermore, we identified many malformations including pericardial and yolk sac edema, spinal deformity, and cardiac looping abnormality. In addition, PND increased the expression of reactive oxygen species and malondialdehyde and reduced the activity of superoxide dismutase (Antioxidant enzymes); We examined the expression of cardiac development-related genes and the apoptosis markers, and found changes of the following marker: vmhc, nppa, tbx5a, nkx2.5, gata4, tbx2b and FoxO1, bax, bcl-2, p53, casp-9, casp-3. Our data showed that activation of Wnt pathway can rescue the cardiac abnormalities caused by PND. Our results provided new evidence for the toxicity of PND and suggested that the PND residual should be treated as a hazard in the environment.


Assuntos
Embrião não Mamífero , Peixe-Zebra , Compostos de Anilina , Animais , Apoptose , Cardiotoxicidade/metabolismo , Embrião não Mamífero/metabolismo , Estresse Oxidativo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Nat Commun ; 12(1): 4484, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301940

RESUMO

Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that Cx41.8 is important to lower ROS levels in HSPCs. We also demonstrate a new role for ifi30, known to be involved in the immune response. In the hematopoietic niche, Ifi30 can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver.


Assuntos
Conexinas/genética , Células-Tronco Hematopoéticas/metabolismo , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Transdução de Sinais/genética , Nicho de Células-Tronco , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Conexinas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Humanos , Microscopia Confocal , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Imagem com Lapso de Tempo/métodos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
Nat Commun ; 12(1): 4488, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301944

RESUMO

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Optogenética/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Opsinas/genética , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapses/genética , Sinapses/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
7.
Gene ; 801: 145848, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34293450

RESUMO

The homeodomain-containing transcription factors dlx1a, dlx2a, dlx5a and dlx6a are expressed in the zebrafish brain in overlapping patterns and are important in vertebrate development. Previous work in mice have suggested the overlapping expression pattern is in part due to cross-regulatory interactions among the aforementioned dlx genes. However, the extent of these interactions and whether they are conserved among vertebrates remains to be determined. Through whole-mount in situ hybridization in zebrafish dlx mutants produced by CRISPR-Cas9 mutagenesis, cross-regulatory interactions between dlx1a, dlx2a, dlx5a and dlx6a were examined from 24 to 72 h post fertilization (hpf). Notably, and different from previous work done in mouse, zebrafish dlx2a-/- mutants continue to express dlx5a until 72hpf, whereas deletion of both enhancers within the dlx5a/dlx6a locus resulted in delayed dlx5a/dlx6a expression and relative increased dlx2a expression. These results suggest alternative regulatory elements and pathways exist to mediate dlx expression in zebrafish and may highlight evolutionary differences in gene interactions between vertebrates.


Assuntos
Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , DNA Intergênico , Regulação da Expressão Gênica no Desenvolvimento , Larva , Mutação
8.
Cell Prolif ; 54(8): e13096, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34240779

RESUMO

OBJECTIVES: PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate-limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. MATERIALS AND METHODS: In this study, we used CRISPR-based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA-seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. RESULTS: The splicing sites of PKM, a 5' donor site of GT and a 3' acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax-NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5' donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non-canonical PKM isoforms and produced some novel splicing isoforms. CONCLUSIONS: This work proved that CRISPR-based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.


Assuntos
Edição de Genes , Piruvato Quinase/metabolismo , Processamento Alternativo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação para Baixo , Éxons , Feminino , Células HCT116 , Humanos , Mutagênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinase/genética , Regulação para Cima , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298970

RESUMO

Dominant spinocerebellar ataxias (SCAs) are progredient neurodegenerative diseases commonly affecting the survival of Purkinje cells (PCs) in the human cerebellum. Spinocerebellar ataxia type 1 (SCA1) is caused by the mutated ataxin1 (Atx1) gene product, in which a polyglutamine stretch encoded by CAG repeats is extended in affected SCA1 patients. As a monogenetic disease with the Atx1-polyQ protein exerting a gain of function, SCA1 can be genetically modelled in animals by cell type-specific overexpression. We have established a transgenic PC-specific SCA1 model in zebrafish coexpressing the fluorescent reporter protein mScarlet together with either human wild type Atx1[30Q] as control or SCA1 patient-derived Atx1[82Q]. SCA1 zebrafish display an age-dependent PC degeneration starting at larval stages around six weeks postfertilization, which continuously progresses during further juvenile and young adult stages. Interestingly, PC degeneration is observed more severely in rostral than in caudal regions of the PC population. Although such a neuropathology resulted in no gross locomotor control deficits, SCA1-fish with advanced PC loss display a reduced exploratory behaviour. In vivo imaging in this SCA1 model may help to better understand such patterned PC death known from PC neurodegeneration diseases, to elucidate disease mechanisms and to provide access to neuroprotective compound characterization in vivo.


Assuntos
Ataxina-1/genética , Modelos Animais de Doenças , Ataxias Espinocerebelares/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Ataxina-1/fisiologia , Morte Celular , Progressão da Doença , Comportamento Exploratório , Genes Reporter , Humanos , Larva , Proteínas Luminescentes/genética , Células de Purkinje/patologia , Transgenes , Expansão das Repetições de Trinucleotídeos , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/fisiologia
10.
Arterioscler Thromb Vasc Biol ; 41(9): 2454-2468, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34261327

RESUMO

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.


Assuntos
Sistema Cardiovascular/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistema Cardiovascular/embriologia , Separação Celular , Microscopia Crioeletrônica , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
11.
Biomed Pharmacother ; 138: 111521, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34311525

RESUMO

Euphorbiae pekinensis Radix (EP) is effective in treating various diseases, but it's toxicity is a major obstacle in use in clinical. Although EP was processed with vinegar to reduce it's toxicity, the detailed mechanism of toxicity in EP have not been clearly delineated. This study investigate the toxicity attenuation-mechanism of Euphorbiae pekinensis after being processed with vinegar (VEP) and the toxic mechanism of four compounds from EP on zebrafish embryos. The contents of four compounds decreased obviously in VEP. Correspondingly, slower development on embryos can be seen as some symptoms like reduction of heart rate, liver area and gastrointestinal peristalsis after exposed to the compounds. Some obvious pathological signals such as pericardial edema and yolk sac edema were observed. Furthermore, the compounds could increase the contents of MDA and GSH-PX and induce oxidative damage by inhibiting the activity of SOD. Also, four compounds could provoke apoptosis by up-regulating the expression level of p53, MDM2, Bax, Bcl-2 and activating the activity of caspase-3, caspase-9. In conclusion, the four compounds play an important role in the toxicity attenuation effects of VEP, which may be related to the apoptosis induction and oxidative damage. This would contribute to the clinical application and further toxicity-reduction mechanism research.


Assuntos
Euphorbia/toxicidade , Trato Gastrointestinal/efeitos dos fármacos , Coração/efeitos dos fármacos , Fígado/efeitos dos fármacos , Compostos Fitoquímicos/toxicidade , Extratos Vegetais/toxicidade , Peixe-Zebra/embriologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cardiotoxicidade , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Euphorbia/química , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Coração/embriologia , Fígado/embriologia , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Compostos Fitoquímicos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
J Immunol ; 207(3): 784-798, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290106

RESUMO

In mammals, cyclic GMP-AMP synthase (cGAS) recognizes cytosolic dsDNA to induce the type I IFN response. However, the functional role of cGAS in the IFN response of fish remains unclear or controversial. In this study, we report that cGAS orthologs from crucian carp Carassius auratus (CacGAS) and grass carp Ctenopharyngodon idellus (CicGAS) target the dsRNA sensor retinoic acid-inducible gene I (RIG-I) for negative regulation of the IFN response. First, poly(deoxyadenylic-deoxythymidylic) acid-, polyinosinic-polycytidylic acid-, and spring viremia of carp virus-induced IFN responses were impaired by overexpression of CacGAS and CicGAS. Then, CacGAS and CicGAS interacted with CiRIG-I and CiMAVS and inhibited CiRIG-I- and CiMAVS-mediated IFN induction. Moreover, the K63-linked ubiquitination of CiRIG-I and the interaction between CiRIG-I and CiMAVS were attenuated by CacGAS and CicGAS. Finally, CacGAS and CicGAS decreased CiRIG-I-mediated the cellular antiviral response and facilitated viral replication. Taken together, data in this study identify CacGAS and CicGAS as negative regulators in RIG-I-like receptor signaling, which extends the current knowledge regarding the role of fish cGAS in the innate antiviral response.


Assuntos
Proteínas de Peixes/genética , Interferon Tipo I/metabolismo , Nucleotidiltransferases/genética , Infecções por Rhabdoviridae/imunologia , Rhabdoviridae/fisiologia , Animais , Carpas , Cyprinidae , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Carpa Dourada , Células HEK293 , Humanos , Imunidade Inata/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Ubiquitinação , Proteínas de Peixe-Zebra/genética
13.
J Biol Chem ; 297(1): 100853, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34090874

RESUMO

The highly conserved dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) plays crucial roles during central nervous system development and homeostasis. Furthermore, its hyperactivity is considered responsible for some neurological defects in individuals with Down syndrome. We set out to establish a zebrafish model expressing human Dyrk1A that could be further used to characterize the interaction between Dyrk1A and neurological phenotypes. First, we revealed the prominent expression of dyrk1a homologs in cerebellar neurons in the zebrafish larval and adult brains. Overexpression of human dyrk1a in postmitotic cerebellar Purkinje neurons resulted in a structural misorganization of the Purkinje cells in cerebellar hemispheres and a compaction of this cell population. This impaired Purkinje cell organization was progressive, leading to an age-dependent dispersal of Purkinje neurons throughout the cerebellar molecular layer with larval swim deficits resulting in miscoordination of swimming and reduced exploratory behavior in aged adults. We also found that the structural misorganization of the larval Purkinje cell layer could be rescued by pharmacological treatment with Dyrk1A inhibitors. We further reveal the in vivo efficiency of a novel selective Dyrk1A inhibitor, KuFal194. These findings demonstrate that the zebrafish is a well-suited vertebrate organism to genetically model severe neurological diseases with single cell type specificity. Such models can be used to relate molecular malfunction to cellular deficits, impaired tissue formation, and organismal behavior and can also be used for pharmacological compound testing and validation.


Assuntos
Cerebelo/metabolismo , Síndrome de Down/genética , Neurônios/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas de Peixe-Zebra/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cerebelo/patologia , Modelos Animais de Doenças , Síndrome de Down/patologia , Humanos , Neurônios/patologia , Fosforilação/genética , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Peixe-Zebra/genética
14.
Commun Biol ; 4(1): 695, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099848

RESUMO

The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.


Assuntos
Melanoma/genética , Crista Neural/embriologia , Fatores de Transcrição SOXE/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Crista Neural/metabolismo
15.
Commun Biol ; 4(1): 676, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083742

RESUMO

Myopia is the most common developmental disorder of juvenile eyes, and it has become an increasing cause of severe visual impairment. The GJD2 locus has been consistently associated with myopia in multiple independent genome-wide association studies. However, despite the strong genetic evidence, little is known about the functional role of GJD2 in refractive error development. Here, we find that depletion of gjd2a (Cx35.5) or gjd2b (Cx35.1) orthologs in zebrafish, cause changes in the biometry and refractive status of the eye. Our immunohistological and scRNA sequencing studies show that Cx35.5 (gjd2a) is a retinal connexin and its depletion leads to hyperopia and electrophysiological changes in the retina. These findings support a role for Cx35.5 (gjd2a) in the regulation of ocular biometry. Cx35.1 (gjd2b) has previously been identified in the retina, however, we found an additional lenticular role. Lack of Cx35.1 (gjd2b) led to a nuclear cataract that triggered axial elongation. Our results provide functional evidence of a link between gjd2 and refractive error.


Assuntos
Conexinas/genética , Modelos Animais de Doenças , Proteínas do Olho/genética , Mutação , Erros de Refração/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Catarata/genética , Conexinas/metabolismo , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Miopia/genética , RNA-Seq/métodos , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Análise de Célula Única/métodos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
16.
Commun Biol ; 4(1): 680, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083748

RESUMO

Genetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Local field potential recordings (LFP) were used to screen ∼3300 larvae. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.


Assuntos
Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Epilepsia/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Criança , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Epilepsia/patologia , Epilepsia/fisiopatologia , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva/genética , Mutação , Fenótipo , Análise de Sobrevida , Sequenciamento Completo do Exoma/métodos , Peixe-Zebra/embriologia
17.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34155518

RESUMO

Mutations in CEP290 (also known as NPHP6), a large multidomain coiled coil protein, are associated with multiple cilia-associated syndromes. Over 130 CEP290 mutations have been linked to a wide spectrum of human ciliopathies, raising the question of how mutations in a single gene cause different disease syndromes. In zebrafish, the expressivity of cep290 deficiencies were linked to the type of genetic ablation: acute cep290 morpholino knockdown caused severe cilia-related phenotypes, whereas deficiencies in a CRISPR/Cas9 genetic mutant were restricted to photoreceptor defects. Here, we show that milder phenotypes in genetic mutants were associated with the upregulation of genes encoding the cilia-associated small GTPases arl3, arl13b and unc119b. Upregulation of UNC119b was also observed in urine-derived renal epithelial cells from human Joubert syndrome CEP290 patients. Ectopic expression of arl3, arl13b and unc119b in cep290 morphant zebrafish embryos rescued Kupffer's vesicle cilia and partially rescued photoreceptor outer segment defects. The results suggest that genetic compensation by upregulation of genes involved in a common subcellular process, lipidated protein trafficking to cilia, may be a conserved mechanism contributing to genotype-phenotype variations observed in CEP290 deficiencies. This article has an associated First Person interview with the first author of the paper.


Assuntos
Antígenos de Neoplasias , Proteínas de Ciclo Celular , Cílios , Proteínas do Citoesqueleto , Proteínas Monoméricas de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Cílios/genética , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Humanos , Proteínas Associadas aos Microtúbulos , Mutação/genética , Regulação para Cima/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
18.
Genes (Basel) ; 12(5)2021 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066767

RESUMO

The molecular mechanisms underlying fish tolerance to soybean meal (SBM) remain unclear. Identifying these mechanisms would be beneficial, as this trait favors growth. Two fish replicates from 19 experimental families were fed fishmeal-(100FM) or SBM-based diets supplemented with saponin (50SBM + 2SPN) from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (HG-50SBM + 2SPN, 170 ± 18 mg) or lower (LG-50SBM + 2SPN, 76 ± 10 mg) weight gain on 50SBM + 2SPN for intestinal transcriptomic analysis. A histological evaluation confirmed middle intestinal inflammation in the LG- vs. HG-50SBM + 2SPN group. Enrichment analysis of 665 differentially expressed genes (DEGs) identified pathways associated with immunity and lipid metabolism. Genes linked to intestinal immunity were downregulated in HG fish (mpx, cxcr3.2, cftr, irg1l, itln2, sgk1, nup61l, il22), likely dampening inflammatory responses. Conversely, genes involved in retinol signaling were upregulated (rbp4, stra6, nr2f5), potentially favoring growth by suppressing insulin responses. Genes associated with lipid metabolism were upregulated, including key components of the SREBP (mbtps1, elov5l, elov6l) and cholesterol catabolism (cyp46a1), as well as the downregulation of cyp7a1. These results strongly suggest that transcriptomic changes in lipid metabolism mediate SBM tolerance. Genotypic variations in DEGs may become biomarkers for improving early selection of fish tolerant to SMB or others plant-based diets.


Assuntos
Imunidade Inata , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos , Proteínas de Soja/imunologia , Transcriptoma , Proteínas de Peixe-Zebra/genética , Animais , Mucosa Intestinal/imunologia , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
19.
Development ; 148(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34104942

RESUMO

Epithelial cilia, whether motile or primary, often display an off-center planar localization within the apical cell surface. This form of planar cell polarity (PCP) involves the asymmetric positioning of the ciliary basal body (BB). Using the monociliated epithelium of the embryonic zebrafish floor-plate, we investigated the dynamics and mechanisms of BB polarization by live imaging. BBs were highly motile, making back-and-forth movements along the antero-posterior (AP) axis and contacting both the anterior and posterior membranes. Contacts exclusively occurred at junctional Par3 patches and were often preceded by membrane digitations extending towards the BB, suggesting focused cortical pulling forces. Accordingly, BBs and Par3 patches were linked by dynamic microtubules. Later, BBs became less motile and eventually settled at posterior apical junctions enriched in Par3. BB posterior positioning followed Par3 posterior enrichment and was impaired upon Par3 depletion or disorganization of Par3 patches. In the PCP mutant vangl2, BBs were still motile but displayed poorly oriented membrane contacts that correlated with Par3 patch fragmentation and lateral spreading. Thus, we propose an unexpected function for posterior Par3 enrichment in controlling BB positioning downstream of the PCP pathway.


Assuntos
Corpos Basais/metabolismo , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas de Transporte/genética , Polaridade Celular , Feminino , Masculino , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Transcriptoma , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
20.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071043

RESUMO

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR-TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Cardiomiopatia Dilatada/genética , Mutação com Ganho de Função , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Mutação Puntual , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Substituição de Aminoácidos , Animais , Autofagia , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cardiomiopatia Dilatada/terapia , Células Cultivadas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Ventrículos do Coração/citologia , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos Wistar , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
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