RESUMO
Isocitrate dehydrogenase 1 mutation (IDH1-R132H) was recently identified in acute myeloid leukemia with normal cytogenetics. The mutant enzyme is thought to convert α-ketoglutarate to the pathogenic 2-hydroxyglutarate (2-HG) that affects DNA methylation via inhibition of ten-eleven translocation 2. However, the role of wild-type IDH1 in normal hematopoiesis and its relevance to acute myeloid leukemia is unknown. Here we showed that zebrafish idh1 (zidh1) knockdown by morpholino and targeted mutagenesis by transcription activator-like effector nuclease might induce blockade in myeloid differentiation, as evident by an increase in pu.1 and decrease in mpo, l-plastin, and mpeg1 expression, and significantly reduce definitive hematopoiesis. Morpholino knockdown of zidh2 also induced a blockade in myeloid differentiation but definitive hematopoiesis was not affected. The hematopoietic phenotype of zidh1 knockdown was not rescuable by zidh2 messenger RNA, suggesting nonredundant functions. Overexpression of human IDH1-R132H or its zebrafish ortholog resulted in 2-HG elevation and expansion of myelopoiesis in zebrafish embryos. A human IDH1-R132H-specific inhibitor (AGI-5198) significantly ameliorated both hematopoietic and 2-HG responses in human but not zebrafish IDH1 mutant expression. The results provided important insights to the role of zidh1 in myelopoiesis and definitive hematopoiesis and of IDH1-R132H in leukemogenesis.
Assuntos
Embrião não Mamífero/metabolismo , Hematopoese/fisiologia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação/genética , Mielopoese/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Benzenoacetamidas/farmacologia , Western Blotting , Células Cultivadas , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Cromatografia Gasosa-Espectrometria de Massas , Glutaratos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imidazóis/farmacologia , Técnicas Imunoenzimáticas , Isocitrato Desidrogenase/antagonistas & inibidores , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
FMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD(+)) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD(+)) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD(+) and FLT3-TKD(+) AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb (definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1(+), mpo(+), and cebpα(+)) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD(+) and FLT3-TKD(+) AML that may facilitate high-throughput screening of novel and personalized agents.
Assuntos
Hematopoese/genética , Leucemia Mieloide Aguda/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência Conservada , Embrião não Mamífero , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Sequências de Repetição em Tandem , Transcriptoma , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/química , Tirosina Quinase 3 Semelhante a fms/químicaRESUMO
Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% of acute myeloid leukemias (AMLs) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD(+) AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow myeloblasts after 27 (range 21-84) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54-287) days but the FLT3 and downstream effectors remained suppressed. Gene expression profiling showed that leukemia cells that have become sorafenib resistant expressed several genes including ALDH1A1, JAK3, and MMP15, whose functions were unknown in AML. Nonobese diabetic/severe combined immunodeficiency mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain mutations at D835 were identified in leukemia initiating cells (LICs) from samples before sorafenib treatment. LICs bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggest that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations, and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.
Assuntos
Antineoplásicos/administração & dosagem , Benzenossulfonatos/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Piridinas/administração & dosagem , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Animais , Antineoplásicos/efeitos adversos , Benzenossulfonatos/efeitos adversos , Medula Óssea/enzimologia , Medula Óssea/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Janus Quinase 3/biossíntese , Janus Quinase 3/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Masculino , Metaloproteinase 15 da Matriz/biossíntese , Metaloproteinase 15 da Matriz/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Niacinamida/análogos & derivados , Compostos de Fenilureia , Estrutura Terciária de Proteína , Piridinas/efeitos adversos , Retinal Desidrogenase , Sorafenibe , Fatores de Tempo , Transplante Heterólogo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
In a chemical screening, we tested the antiangiogenic effects of fumagillin derivatives and identified fumagillin as an inhibitor of definitive hematopoiesis in zebrafish embryos. Fumagillin is known to target methionine aminopeptidase II (MetAP2), an enzyme whose function in hematopoiesis is unknown. We investigated the role of MetAP2 in hematopoiesis by using zebrafish embryo and human umbilical cord blood models. Zebrafish metap2 was expressed ubiquitously during early embryogenesis and later in the somitic region, the caudal hematopoietic tissue, and pronephric duct. metap2 was inhibited by morpholino and fumagillin treatment, resulting in increased mpo expression at 18 hours postfertilization and reduced c-myb expression along the ventral wall of dorsal aorta at 36 hours postfertilization. It also disrupted intersegmental vessels in Tg(fli1:gfp) embryos without affecting development of major axial vasculatures. Inhibition of MetAP2 in CB CD34(+) cells by fumagillin had no effect on overall clonogenic activity but significantly reduced their engraftment into immunodeficient nonobese diabetes/severe combined immunodeficiency mice. metap2 knock-down in zebrafish and inhibition by fumagillin in zebrafish and human CB CD34(+) cells inhibited Calmodulin Kinase II activity and induced ERK phosphorylation. This study demonstrated a hitherto-undescribed role of MetAP2 in definitive hematopoiesis and a possible link to noncanonical Wnt and ERK signaling.
Assuntos
Aminopeptidases/fisiologia , Glicoproteínas/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Metaloendopeptidases/fisiologia , Aminopeptidases/genética , Aminopeptidases/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Animais Geneticamente Modificados , Divisão Celular/fisiologia , Cicloexanos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácidos Graxos Insaturados/farmacologia , Sangue Fetal/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glicoproteínas/genética , Hematopoese/fisiologia , Humanos , Leucócitos Mononucleares/citologia , Metaloendopeptidases/genética , Metionil Aminopeptidases , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mielopoese/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Sesquiterpenos/farmacologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Peixe-ZebraRESUMO
Metformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.
RESUMO
ADP-ribosylation factor-like 4aa (Arl4aa) is a member of the ADP-ribosylation factor family. It is expressed in hematopoietic tissue during embryonic development, but its function was unknown. Zebrafish arl4aa is preferentially expressed in the ventral wall of the dorsal aorta (VDA) at 24 and 36 hpf and in caudal hematopoietic tissue at 48 hpf. Morpholino knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aa significantly reduced expression of genes associated with definitive hematopoietic stem cells (HSCs). Golgi complex integrity in VDA was disrupted as shown by transmission electron microscopy and immunostaining of Golgi membrane Giantin. Mechanistically, arl4aa knockdown reduced Notch signaling in the VDA and its target gene expression. Protein expression of NICD was also reduced. Effects of arl4aa knockdown on definitive hematopoiesis could be restored by NICD expression. This study identified arl4aa as a factor regulating initiation of definitive HSCs by maintaining the integrity of Golgi complex and, secondarily, maturation of the Notch receptor.
Assuntos
Complexo de Golgi/metabolismo , Hemangioblastos/metabolismo , Hematopoese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Cruzamentos Genéticos , Regulação para Baixo , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Modelos Biológicos , Mutação/genética , Receptores Notch/metabolismo , Transdução de Sinais , Nucleases dos Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
OBJECTIVES: Phospholipase C (PLC) gamma 1 has been shown to mediate signal transduction of tyrosine kinases and affect function of hematopoietic cells. However, its role in hematopoiesis during embryonic development is currently unclear. In this study, we examined this issue using morpholino (MO) gene knockdown in zebrafish embryos METHODS: MO targeting at the exon-1-intron-1 junction of zebrafish PLC-gamma1 was injected into embryos at the one- to four-cell stage (referred herein zPLC-gamma1(MO) embryos). Primitive hematopoiesis was examined quantitatively by flow cytometry in Tg(gata1:GFP) embryos and by real-time quantitative polymerase chain reaction at 18 hours-post-fertilization (hpf), before the onset of circulation. The embryos were also treated with receptor inhibitors of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor at 25, 1, and 30 micromol/L, respectively, from one cell until 48 hpf. RESULTS: Erythropoiesis was reduced in zPLC-gamma1(MO) embryos, as shown by the reduction in gata1(+) cells (wild-type: 4.32% +/- 0.10% vs zPLC-gamma1(MO): 2.38% +/- 0.11%, p = 0.021) and gata1 and alpha-embryonic hemoglobin expression [0.47 +/- 0.06-fold (p = 0.013) and 0.46 +/- 0.04-fold (p = 0.013)]. Expression of scl, lmo-2 (early hematopoietic progenitors), pu.1, and l-plastin (myelomonocytic lineages) as well as fli1 (vascular progenitors) were unaffected. Fli1(+) cells in Tg(fli1:GFP) embryos were also unaffected by zPLC-gamma1(MO). When embryos were incubated with receptor inhibitors of vascular endothelial growth factor (VEGFRTKI), fibroblast growth factor (SU5402), or platelet-derived growth factor (AG1296), only VEGFRTKI reduced erythropoiesis [VEGFRTKI: 2.10% +/- 0.07% (p = 0.021) vs SU5402: 4.08% +/- 0.12% (p = 0.248) vs AG1296: 4.12% +/- 0.14% (p = 0.149)]. CONCLUSION: PLC-gamma1 is involved in the regulation of primitive hematopoiesis in zebrafish embryos, which is distinct from its later effect on vascular formation.
Assuntos
Hematopoese/fisiologia , Fosfolipase C gama/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Eritropoese/efeitos dos fármacos , Eritropoese/fisiologia , Fator 1 de Crescimento de Fibroblastos/administração & dosagem , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes/métodos , Proteínas de Fluorescência Verde/genética , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Oligonucleotídeos Antissenso/administração & dosagem , Fosfolipase C gama/efeitos dos fármacos , Fosfolipase C gama/genética , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Peixe-Zebra/genéticaRESUMO
RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.
Assuntos
Síndrome de Costello/genética , Displasia Ectodérmica/genética , Insuficiência de Crescimento/genética , Cardiopatias Congênitas/genética , Neurofibromatose 1/genética , Síndrome de Noonan/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas ras/genética , Adolescente , Animais , Bioensaio , Criança , Pré-Escolar , Biologia Computacional , Síndrome de Costello/patologia , Displasia Ectodérmica/patologia , Fácies , Insuficiência de Crescimento/patologia , Feminino , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa , Cardiopatias Congênitas/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , MAP Quinase Quinase 1/genética , Masculino , Mutação de Sentido Incorreto , Neurofibromatose 1/patologia , Síndrome de Noonan/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/genética , Peixe-Zebra , alfa-Macroglobulinas/genéticaRESUMO
The recent advent of next-generation sequencing (NGS) has greatly accelerated identification of gene mutations in myeloid malignancies at unprecedented speed that will soon outpace their functional validation by conventional laboratory techniques and animal models. A high-throughput whole-organism model is useful for the functional validation of new mutations. We recently reported the use of zebrafish to evaluate the hematopoietic function of isocitrate dehydrogenase 1 (IDH1) and the effects of expressing human IDH1-R132H that is frequently identified in human acute myeloid leukemia (AML), in myelopoiesis, with a view to develop zebrafish as a model of AML. Here, we use IDH1 as an example to describe a comprehensive approach to evaluate hematopoietic gene function and the effects of mutations using zebrafish as a model.
Assuntos
Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Mutação , Animais , Embrião não Mamífero/citologia , Hematopoese , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/metabolismo , Mutagênese Sítio-Dirigida , Fenótipo , Peixe-ZebraRESUMO
Transcription activator-like effectors (TALEs) are important genomic tools with customizable DNA-binding motifs for locus-specific modifications. In particular, TALE nucleases or TALENs have been successfully used in the zebrafish model system to introduce targeted mutations via repair of double-stranded breaks (DSBs) either through nonhomologous end joining (NHEJ) or by homology-directed repair (HDR) and homology-independent repair in the presence of a donor template. Compared with other customizable nucleases, TALENs offer high binding specificity and fewer sequence constraints in targeting the genome, with comparable mutagenic activity. Here, we describe a detailed in silico design tool for zebrafish genome editing for TALENs and CRISPR/Cas9 custom restriction enzymes using Mojo Hand 2.0 software.
Assuntos
Edição de Genes/métodos , Mutagênese/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Peixe-Zebra/genéticaRESUMO
Recent advent in next generation sequencing (NGS) and bioinformatics has generated an unprecedented amount of genetic information in myeloidmalignancies. This information may shed lights to the pathogenesis, diagnosis and prognostication of these diseases and provide potential targets for therapeutic intervention. However, the rapid emergence of genetic information will quickly outpace their functional validation by conventional laboratory platforms. Foundational knowledge about zebrafish hematopoiesis accumulated over the past two decades and novel genomeediting technologies and research strategies in thismodel organismhavemade it a unique and timely research tool for the study of human blood diseases. Recent studies modeling human myeloid malignancies in zebrafish have also highlighted the technical feasibility and clinical relevance of thesemodels. Careful validation of experimental protocols and standardization among laboratorieswill further enhance the application of zebrafish in the scientific communities and provide important insights to the personalized treatment ofmyeloid malignancies.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Transtornos Mieloproliferativos/genética , Animais , Modelos Animais de Doenças , Descoberta de Drogas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Fenótipo , Medicina de Precisão , Peixe-ZebraRESUMO
OBJECTIVE: The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV. MATERIALS AND METHODS: Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites. RESULTS: Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019). CONCLUSION: The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.
Assuntos
Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Mutação , Policitemia Vera/patologia , Proteínas Tirosina Quinases/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Modelos Animais de Doenças , Embrião não Mamífero/enzimologia , Embrião não Mamífero/metabolismo , Eritropoese/genética , Eritropoetina/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Janus Quinase 2/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Policitemia Vera/enzimologia , Policitemia Vera/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Homologia de Sequência de Aminoácidos , Sulfonamidas/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismoRESUMO
Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2(617V>F)) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5' untranslated region (UTR) (jak2a(UTR)-MO) and splice-site junction (jak2a(SS)-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2a(ca)). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1(+) (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, alphahe1 and betahe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2a(ca) significantly increased erythropoiesis and expression of gata1, alphahe1 and betahe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.