RESUMEN
The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Mielopoyesis/genética , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Pez Cebra/embriología , Pez Cebra/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/análisis , Proteínas Potenciadoras de Unión a CCAAT/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/genética , Técnicas Genéticas , Granulocitos/fisiología , Hibridación Fluorescente in Situ , Macrófagos/fisiología , Metaloendopeptidasas/análisis , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/genética , Microinyecciones , Modelos Animales , Mutación/genética , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN/análisis , ARN/metabolismo , Transactivadores/análisis , Transactivadores/biosíntesis , Transactivadores/genéticaRESUMEN
The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd(mi149)), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.
Asunto(s)
Hemorragia Cerebral/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Pez Cebra/genética , Empalme Alternativo , Animales , Hemorragia Cerebral/embriología , Circulación Cerebrovascular/genética , Mapeo Cromosómico , Embrión no Mamífero , Genes Recesivos , Variación Genética , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Serina-Treonina Quinasas/deficiencia , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Proteínas de Pez Cebra/genética , Quinasas p21 ActivadasRESUMEN
Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.
