RESUMO
Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.
Assuntos
Proteínas de Transporte/genética , Morfogênese/genética , Deficiência de Vitamina B 12/genética , Vitamina B 12/genética , Proteínas de Peixe-Zebra/genética , Animais , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Camundongos , Mutação/genética , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/patologia , Oxirredutases/genética , Retina/crescimento & desenvolvimento , Retina/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
BACKGROUND: Vertebrate hematopoiesis is a complex developmental process that is controlled by genes in diverse pathways. To identify novel genes involved in early hematopoiesis, we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in zebrafish. The mummy (mmy) line was investigated because of its multiple hematopoietic defects. RESULTS: Homozygous mmy embryos lacked circulating blood cell types and were dead by 30 hr post-fertilization (hpf). The mmy mutants did not express myeloid markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation in dhx8 in the mmy fish. dhx8 is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. mmy mutants had splicing defects in many genes, including several hematopoietic genes. mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. CONCLUSIONS: Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos.
Assuntos
Divisão Celular/genética , RNA Helicases DEAD-box/genética , Hematopoese/genética , Splicing de RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/metabolismo , Embrião não Mamífero/metabolismo , Sistema Hematopoético/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first committed step in the synthesis of phosphoinositides, important lipid regulators of signaling and trafficking pathways. Here we cloned Pik4a, one of the zebrafish PI4K enzymes, and studied its role(s) in vertebrate development using morpholino oligonucleotide-based gene silencing in zebrafish. Downregulation of Pik4a led to multiple developmental abnormalities, affecting the brain, heart, trunk and most prominently causing loss of pectoral fins. Strikingly similar defects were caused by treatment of the developing embryos with the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. To investigate the cause of the pectoral fin developmental defect, we focused on fibroblast growth factor (FGF) signaling pathways because vertebrate limb development requires the concerted action of a series of FGF ligands. Using in situ hybridization, the pectoral fin defect was traced to disruption of the early FGF signaling loops that are crucial for the establishment of the sharp signaling center formed by the apical ectodermal ridge and the underlying mesenchyme. This, in turn caused a prominent loss of the induction of one of the mitogen-activated protein kinase (MAPK) phosphatases, Mkp3, an essential intermediate in vertebrate limb development. These changes were associated with impaired proliferation in the developing fin bud due to a loss of balance between the MAPK and PI3K branch of FGF-initiated signals. Our results identify Pik4a as an upstream partner of PI3Ks in the signaling cascade orchestrated by FGF receptors with a prominent role in forelimb development.
Assuntos
1-Fosfatidilinositol 4-Quinase/fisiologia , Extremidades/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas de Peixe-Zebra/fisiologia , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Cromonas/farmacologia , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização in Situ Fluorescente , Marcação In Situ das Extremidades Cortadas , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Testicular germ cell tumors (TGCTs) are the most common malignancy in young men. However, there are few in vivo animal models that have been developed to study this disease. We have used the pufferfish (fugu) lymphocyte-specific protein tyrosine kinase (flck) promoter, which has been shown to enforce high-level expression in the testes of transgenic mice, to express Simian virus 40 large T-antigen in zebrafish testes. Zebrafish that express T-antigen develop TGCTs after a long latency of >1 year. Although overt TGCTs are only evident in 20% of the fish, occult TGCTs can be detected in 90% of the transgenic fish by 36 month of age. The TGCTs resemble the human disease in terms of morphology and gene expression pattern, and can be transplanted to healthy wild-type recipient fish. In addition, enforced expression of the zebrafish stem cell leukemia (scl) gene in the zebrafish testes also generated TGCTs in transgenic fish. These results demonstrate the feasibility of studying TGCTs in a model organism.