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1.
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
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.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361083

RESUMO

The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target structures in the outward open and inward open conformations. The resulting models are then embedded in a model membrane and investigated via a rigorous molecular dynamics simulation protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration phase and to assess a structural comparison between proteins in different states, and between different proteins in the same state. Our study clearly identifies a network of key residues at the interface between the gate and the core domains of prestin that might be responsible for the conformational change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl- ions in the presence of an applied electric field towards their putative binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.


Assuntos
Proteínas de Transporte de Ânions/química , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular , Transportadores de Sulfato/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions/metabolismo , Sítios de Ligação , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência , Transportadores de Sulfato/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
4.
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
5.
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
6.
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
7.
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
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.
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
10.
Int J Mol Sci ; 22(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34281244

RESUMO

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.


Assuntos
Channelrhodopsins/metabolismo , Percepção Olfatória/genética , Olfato/genética , Animais , Animais Geneticamente Modificados/genética , Channelrhodopsins/genética , Sinais (Psicologia) , Larva/fisiologia , Luz , Rede Nervosa/metabolismo , Neurônios/metabolismo , Odorantes , Optogenética/métodos , Estimulação Luminosa , Regiões Promotoras Genéticas/genética , Natação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
11.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206401

RESUMO

Wingless-type MMTV integration site family, member 16 (wnt16), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16-/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16, which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16-/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16-/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein-protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal development.


Assuntos
Osso e Ossos/anormalidades , Anormalidades Musculoesqueléticas/genética , Anormalidades Musculoesqueléticas/metabolismo , Osteogênese/genética , Proteínas Wnt/deficiência , Proteínas de Peixe-Zebra/deficiência , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Ontologia Genética , Anotação de Sequência Molecular , Anormalidades Musculoesqueléticas/diagnóstico , Fenótipo , Transcriptoma , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
12.
Cancer Sci ; 112(9): 3884-3894, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34077586

RESUMO

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15-/- zebrafish using transcription activator-like effector nuclease technology. Whole-mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15-/- zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15-/- ; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression.


Assuntos
RNA Helicases DEAD-box/metabolismo , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Resposta a Proteínas não Dobradas/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Apoptose/genética , Proliferação de Células/genética , RNA Helicases DEAD-box/genética , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hibridização In Situ , Leucemia Mieloide Aguda/metabolismo , Mutação , RNA Helicases/genética , RNA Helicases/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
13.
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
14.
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
15.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131730

RESUMO

Noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling has been implicated in endoderm morphogenesis. However, the underlying cellular and molecular mechanisms of this process are unclear. We found that, during convergence and extension (C&E) in zebrafish, gut endodermal cells are polarized mediolaterally, with GFP-Vangl2 enriched at the anterior edges. Endoderm cell polarity is lost and intercalation is impaired in the absence of glypican 4 (gpc4), a heparan-sulfate proteoglycan that promotes Wnt/PCP signaling, suggesting that this signaling is required for endodermal cell polarity. Live imaging revealed that endoderm C&E is accomplished by polarized cell protrusions and junction remodeling, which are impaired in gpc4-deficient endodermal cells. Furthermore, in the absence of gpc4, Cadherin 2 expression on the endodermal cell surface is increased as a result of impaired Rab5c-mediated endocytosis, which partially accounts for the endodermal defects in these mutants. These findings indicate that Gpc4 regulates endodermal planar cell polarity during endoderm C&E by influencing the localization of Cadherin 2. Thus, our study uncovers a new mechanism by which Gpc4 regulates planar cell polarity and reveals the role of Wnt/PCP signaling in endoderm morphogenesis.


Assuntos
Caderinas/metabolismo , Polaridade Celular/fisiologia , Endoderma/metabolismo , Glipicanas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas de Heparan Sulfato/metabolismo , Morfogênese , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/metabolismo , Proteínas rab5 de Ligação ao GTP
16.
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
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.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34061172

RESUMO

Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.


Assuntos
Nadadeiras de Animais/fisiologia , Expressão Ectópica do Gene/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/anatomia & histologia , Animais , Sistemas CRISPR-Cas , Calcineurina/metabolismo , Proliferação de Células , Expressão Ectópica do Gene/genética , Éter , Canais de Potássio Éter-A-Go-Go/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Tamanho do Órgão , Regeneração/fisiologia , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
19.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34061173

RESUMO

During early embryogenesis, the vertebrate embryo extends from anterior to posterior because of the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for NMps to retain mesodermal potential and, hence, normal axis development. We recently showed that Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor-binding sites, we identified more than 500 potential Hox13 target genes in zebrafish. Importantly, we found two highly conserved Hox13-binding elements far from the tbxta transcription start site that also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis-stage expression to a tbxta promoter that, on its own, only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.


Assuntos
Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Sítios de Ligação , Padronização Corporal , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Somitos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/embriologia
20.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074030

RESUMO

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca2+ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca2+ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl3 and capsazepine and treatment with the inhibitors for CaMKII (Ca²2/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca2+-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Hibridização In Situ , Lantânio/farmacologia , Microscopia de Fluorescência , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
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