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1.
Cell ; 133(5): 864-77, 2008 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-18510930

RESUMO

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore gamma-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after gamma-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy.


Assuntos
Apoptose , Caspase 2/metabolismo , Dano ao DNA , Proteínas Quinases/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Caspase 3/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Raios gama , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Blood ; 126(26): 2811-20, 2015 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-26385351

RESUMO

Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)green fluorescent protein (GFP)(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-)GFP(+/+) HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Hemangioblastos/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula/fisiologia , Eletroporação , Embrião de Mamíferos , Embrião não Mamífero , Citometria de Fluxo , Imuno-Histoquímica , Mesonefro/embriologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Peixe-Zebra
3.
PLoS Genet ; 7(3): e1001357, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21483806

RESUMO

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.


Assuntos
Proteína BRCA2/fisiologia , Instabilidade Genômica , Neoplasias de Tecido Gonadal/genética , Oócitos/fisiologia , Oogênese , Espermatogênese , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/genética , Proteína BRCA2/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Anemia de Fanconi/genética , Feminino , Genes p53/genética , Genes p53/fisiologia , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Insercional/genética , Oócitos/citologia , Fenótipo , Espermatócitos/citologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
4.
Blood ; 118(4): 903-15, 2011 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-21653321

RESUMO

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18(hi1727/+) embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.


Assuntos
Ciclo Celular/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Alelos , Animais , Western Blotting , Separação Celular , Embrião não Mamífero , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Humanos , Hibridização In Situ , Mutagênese Sítio-Dirigida , Mutação , Células Mieloides/citologia , Células Mieloides/metabolismo , Reação em Cadeia da Polimerase , Proteínas de Peixe-Zebra/genética
5.
Blood ; 117(15): 3996-4007, 2011 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-21330472

RESUMO

A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3' untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP(+) cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Mutagênese/fisiologia , Fenótipo , Peixe-Zebra
6.
Hum Mol Genet ; 19(23): 4643-53, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20858602

RESUMO

Neurofibromatosis type 1 is the most commonly inherited human cancer predisposition syndrome. Neurofibromin (NF1) gene mutations lead to increased risk of neurofibromas, schwannomas, low grade, pilocytic optic pathway gliomas, as well as malignant peripheral nerve sheath tumors and glioblastomas. Despite the evidence for NF1 tumor suppressor function in glial cell tumors, the mechanisms underlying transformation remain poorly understood. In this report, we used morpholinos to knockdown the two nf1 orthologs in zebrafish and show that oligodendrocyte progenitor cell (OPC) numbers are increased in the developing spinal cord, whereas neurons are unaffected. The increased OPC numbers in nf1 morphants resulted from increased proliferation, as detected by increased BrdU labeling, whereas TUNEL staining for apoptotic cells was unaffected. This phenotype could be rescued by the forced expression of the GTPase-activating protein (GAP)-related domain of human NF1. In addition, the in vivo analysis of OPC migration following nf1 loss using time-lapse microscopy demonstrated that olig2-EGFP(+) OPCs exhibit enhanced cell migration within the developing spinal cord. OPCs pause intermittently as they migrate, and in nf1 knockdown animals, they covered greater distances due to a decrease in average pause duration, rather than an increase in velocity while in motion. Interestingly, nf1 knockdown also leads to an increase in ERK signaling, principally in the neurons of the spinal cord. Together, these results show that negative regulation of the Ras pathway through the GAP activity of NF1 limits OPC proliferation and motility during development, providing insight into the oncogenic mechanisms through which NF1 loss contributes to human glial tumors.


Assuntos
Genes da Neurofibromatose 1 , Células-Tronco Mesenquimais/metabolismo , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Medula Espinal/citologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Contagem de Células , Movimento Celular , Modelos Animais de Doenças , Imunofluorescência , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Hibridização In Situ , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurofibromatose 1 , Neurônios/metabolismo , Oligodesoxirribonucleotídeos Antissenso , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Peixe-Zebra/metabolismo
7.
Blood ; 115(16): 3329-40, 2010 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-20197555

RESUMO

Mutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1(+) primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53(m/m) embryos expressing NPMc+, where mpx(+) and csf1r(+) cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41(+) cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell.


Assuntos
Hematopoese/genética , Leucemia Mieloide Aguda/genética , Células Mieloides/fisiologia , Proteínas Nucleares/genética , Animais , Apoptose/genética , Sequência de Bases , Western Blotting , Separação Celular , Citoplasma/metabolismo , Embrião não Mamífero , Citometria de Fluxo , Imunofluorescência , Células-Tronco Hematopoéticas/fisiologia , Humanos , Imunoprecipitação , Leucemia Mieloide Aguda/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Nucleofosmina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Peixe-Zebra
8.
Proc Natl Acad Sci U S A ; 106(52): 22305-10, 2009 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-19966217

RESUMO

Von Recklinghausen neurofibromatosis is a common autosomal dominant genetic disorder characterized by benign and malignant tumors of neural crest origin. Significant progress in understanding the pathophysiology of this disease has occurred in recent years, largely aided by the development of relevant animal models. Von Recklinghausen neurofibromatosis is caused by mutations in the NF1 gene, which encodes neurofibromin, a large protein that modulates the activity of Ras. Here, we describe the identification and characterization of zebrafish nf1a and nf1b, orthologues of NF1, and show neural crest and cardiovascular defects resulting from morpholino knockdown, including vascular and cardiac valvular abnormalities. Development of a zebrafish model of von Recklinghausen neurofibromatosis will allow for structure-function analysis and genetic screens in this tractable vertebrate system.


Assuntos
Fenômenos Fisiológicos Cardiovasculares/genética , Genes da Neurofibromatose 1 , Neurofibromatose 1/genética , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Sequência de Bases , Anormalidades Cardiovasculares/embriologia , Anormalidades Cardiovasculares/genética , Modelos Animais de Doenças , Humanos , Hibridização In Situ , Mutação , Neurofibromatose 1/patologia , Neurofibromatose 1/fisiopatologia , Oligodesoxirribonucleotídeos Antissenso/genética , Filogenia , Especificidade da Espécie , Peixe-Zebra/embriologia
9.
Br J Haematol ; 152(3): 307-21, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21155757

RESUMO

Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.


Assuntos
Sistema Hematopoético/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Peixe-Zebra/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biologia Computacional , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Humanos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Fases de Leitura Aberta , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade da Espécie , Peixe-Zebra/genética
10.
Blood ; 114(21): 4654-63, 2009 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19729519

RESUMO

The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.


Assuntos
Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Hematopoese/fisiologia , Hibridização In Situ , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Elementos Reguladores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
11.
Dev Cell ; 8(1): 97-108, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15621533

RESUMO

The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis, allowing for the critical in vivo analysis of cell lineage determination. In this study, we identify zebrafish myeloerythroid progenitor cells (MPCs) that are likely to represent the functional equivalent of mammalian common myeloid progenitors. Utilizing transgenic pu.1-GFP fish, real-time MPC differentiation was correlated with dynamic changes in cell motility, morphology, and gene expression. Unlike mammalian hematopoiesis, embryonic zebrafish myelopoiesis and erythropoiesis occur in anatomically separate locations. Gene knockdown experiments and transplantation assays demonstrated the reciprocal negative regulation of pu.1 and gata1 and their non-cell-autonomous regulation that determines myeloid versus erythroid MPC fate in the distinct blood-forming regions. Furthermore, forced expression of pu.1 in the bloodless mutant cloche resulted in myelopoietic rescue, providing intriguing evidence that this gene can function in the absence of some stem cell genes, such as scl, in governing myelopoiesis.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Células Precursoras Eritroides/fisiologia , Células Progenitoras Mieloides/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Ligação a DNA/genética , Indução Embrionária , Fatores de Ligação de DNA Eritroide Específicos , Citometria de Fluxo/métodos , Fator de Transcrição GATA1 , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hematopoese/fisiologia , Hibridização In Situ/métodos , Microinjeções/métodos , Modelos Biológicos , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transativadores/genética , Fatores de Transcrição/genética , Transplante/métodos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Dev Cell ; 5(6): 903-14, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14667412

RESUMO

Mechanisms underlying the multiple developmental defects observed in Fanconi anemia (FA) patients are not well defined. We have identified the zebrafish homolog of human FANCD2, which encodes a nuclear effector protein that is monoubiquitinated in response to DNA damage, targeting it to nuclear foci where it preserves chromosomal integrity. Fancd2-deficient zebrafish embryos develop defects similar to those found in children with FA, including shortened body length, microcephaly, and microophthalmia, which are due to extensive cellular apoptosis. Developmental defects and increased apoptosis in Fancd2-deficient zebrafish were corrected by injection of human FANCD2 or zebrafish bcl2 mRNA, or by knockdown of p53, indicating that in the absence of Fancd2, developing tissues spontaneously undergo p53-dependent apoptosis. Thus, Fancd2 is essential during embryogenesis to prevent inappropriate apoptosis in neural cells and other tissues undergoing high levels of proliferative expansion, implicating this mechanism in the congenital abnormalities observed in human infants with FA.


Assuntos
Anormalidades Múltiplas/genética , Apoptose/fisiologia , Anemia de Fanconi/genética , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Animais , Reagentes de Ligações Cruzadas/farmacologia , Compostos de Epóxi/farmacologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/farmacologia , Regulação para Cima/genética , Peixe-Zebra
14.
Exp Hematol ; 35(2): 230-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17258072

RESUMO

OBJECTIVE: We investigated the role of CCAAT enhancer-binding protein-alpha (C/EBPalpha) during zebrafish embryonic blood development. METHODS: Whole-mount mRNA in situ hybridization was performed to determine the spatio-temporal expression pattern of zebrafish cebpa in developing hematopoietic progenitors. A deletion mutation of cebpa (zD420), which mimics the human dominant-negative mutations of C/EBPalpha, was transfected into CV1 cell line to evaluate its transcriptional activity in vitro and injected into zebrafish embryos at the one- to two-cell stage to examine its effects on primitive hematopoiesis during early zebrafish development. RESULTS: Zebrafish cebpa is expressed in the anterior and posterior lateral plate mesoderm at 12 hours postfertilization, along with scl, pu.1, and gata1 in developing hematopoietic progenitors. In vitro, the deletion mutation of cebpa (zD420) prevents expression of the full-length protein, allowing the expression of truncated isoforms from internal translational initiation sites. As in the human, the truncated zebrafish C/EBPalpha proteins did not activate the expression of known target granulocytic genes, and in fact suppressed transactivation that was induced in vitro by the full-length protein. Forced expression of the zD420 mRNA in zebrafish embryos led to an expansion of primitive erythropoiesis, without a discernible effect on granulopoiesis. CONCLUSION: Expression of the truncated isoforms of cebpa alters the developmental pattern of hematopoietic progenitor cells during embryogenesis.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Peixe-Zebra/genética , Animais , Sequência de Bases , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , DNA Complementar/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA , Translocação Genética/genética , Translocação Genética/fisiologia , Transplante Heterólogo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
15.
Cancer Res ; 64(20): 7256-62, 2004 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15492244

RESUMO

The zebrafish model organism has been used extensively for studies of genetic pathways in development, indicating its potential applicability to cancer. Here we show that targeted expression of MYCN in cells of the pancreatic islet induces neuroendocrine carcinoma. Four transgenic fish developed abdominal tumors between 4 and 6 months of age, and histologic analysis revealed lobulated arrangements of neoplastic cells with expression of the MYCN transgene. The tumors also expressed insulin mRNA, and pancreatic exocrine cells and ducts were identified within the neoplasms, indicating a pancreatic origin for the tumor. Transmission electron microscopy revealed cytoplasmic, endocrine-dense core granules, analogous to those found in human neuroendocrine tumors. Our studies establish a zebrafish transgenic model of pancreatic neuroendocrine carcinoma, setting the stage to evaluate molecular pathways downstream of MYCN in this vertebrate forward genetic model system.


Assuntos
Tumores Neuroectodérmicos/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Neoplasias Pancreáticas/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Carcinoma de Células das Ilhotas Pancreáticas/genética , Carcinoma de Células das Ilhotas Pancreáticas/metabolismo , Humanos , Insulina/biossíntese , Insulina/genética , Ilhotas Pancreáticas/patologia , Proteína Proto-Oncogênica N-Myc , Tumores Neuroectodérmicos/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Oncogênicas/biossíntese , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transgenes , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
16.
Exp Hematol ; 33(9): 997-1006, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16140147

RESUMO

Zebrafish produce nearly identical hematopoeitic cell lineages to those found in mammals and other higher vertebrates. As in mammals, blood cell development proceeds in distinct waves, constituting embryonic (primitive) and adult (definitive) hematopoiesis. The conservation of genes such as scl, pu.1, c/ebpalpha, mpo, l-plastin, and lysozyme C in myelopoiesis and the corresponding expression patterns in zebrafish suggests that shared genetic pathways regulate this complex developmental process. In the zebrafish model system, experimental approaches have been applied, including RNA in situ hybridization, morpholino injections, and the analysis of mutant and transgenic fish lines, leading to improved understanding of the regulation in vivo of key molecular pathways with conserved roles in vertebrate myelopoiesis.


Assuntos
Desenvolvimento Embrionário/genética , Mielopoese/fisiologia , Animais , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Hematopoese/fisiologia , Modelos Animais , Mielopoese/genética , Peixe-Zebra
17.
Biotechniques ; 60(1): 13-20, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26757807

RESUMO

The zebrafish represents a revolutionary tool in large-scale genetic and small-molecule screens for gene and drug discovery. Transgenic zebrafish are often utilized in these screens. Many transgenic fish lines are maintained in the heterozygous state due to the lethality associated with homozygosity; thus, their progeny must be sorted to ensure a population expressing the transgene of interest for use in screens. Sorting transgenic embryos under a fluorescence microscope is very labor-intensive and demands fine-tuned motor skills. Here we report an efficient transgenic method of utilizing pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. This was accomplished by co-injecting two constructs with the I-SceI meganuclease enzyme into pigmentless nacre embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation and I-SceI-zpromoter:gene-of-interest-I-SceI to express the gene of interest under a zebrafish promoter (zpromoter). Pigmentation rescue reliably predicted transgene integration. Compared with other transgenic techniques, our approach significantly increases the overall percentage of founders and facilitates accurate naked-eye identification of stable transgenic fish, greatly reducing laborious fluorescence microscope sorting and PCR genotyping. Thus, this approach is ideal for generating transgenic fish for large-scale screens.


Assuntos
Técnicas de Transferência de Genes , Fator de Transcrição Associado à Microftalmia/genética , Pigmentação/genética , Regiões Promotoras Genéticas , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Genótipo , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Peixe-Zebra/genética , Peixe-Zebra/fisiologia
18.
Biotechniques ; 39(2): 227-37, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16116796

RESUMO

The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200-300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.


Assuntos
Modelos Animais de Doenças , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Predisposição Genética para Doença/embriologia , Predisposição Genética para Doença/genética , Neoplasias/embriologia , Peixe-Zebra/embriologia
19.
Biotechniques ; 35(5): 956-8, 960, 962 passim, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14628669

RESUMO

High fecundity, rapid generation time, and external development of optically clear embryos make the zebrafish (Danio rerio) a convenient vertebrate model for genetic, developmental, and disease studies. Efficient sperm cryopreservation enhances the zebrafish model system by optimizing productive use of facility space, extending the reproductive lifetime of males, providing an alternative to live stocks for strain recovery, and ensuring the survival of valuable mutant lines. Here we identify a cryoprotective medium, 10% N,N-dimethylacetamide (DMA) (v/v) diluted in buffered sperm motility-inhibiting solution (BSMIS), as well as parameters for zebrafish sperm cryopreservation that enhance cryopreservation efficiency and significantly increase the yield of live embryos from archived stocks. Our experiments emphasize the effect of the ratio of sperm and medium volume and the use of large egg clutches to maximize the recovery of viable embryos.


Assuntos
Acetamidas/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Masculino , Peixe-Zebra
20.
Cancer Cell ; 21(3): 362-73, 2012 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-22439933

RESUMO

Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analog following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma.


Assuntos
Neuroblastoma/genética , Proteínas Nucleares/fisiologia , Proteínas Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Peixe-Zebra/genética , Quinase do Linfoma Anaplásico , Animais , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Organismos Geneticamente Modificados , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo
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