RESUMEN
BACKGROUND: Normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. The molecular mechanisms that regulate these processes remain poorly understood, and thus further in vivo study is required. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that uniquely also promotes mitosis and cell proliferation. Postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. Prenatally, survivin is also highly expressed in several tissues. Fully delineating the properties of survivin in vivo in mice has been confounded by early lethal phenotypes following survivin gene inactivation. RESULTS: To gain further insights into the properties of survivin, we used the zebrafish model. There are 2 zebrafish survivin genes (Birc5a and Birc5b) with overlapping expression patterns during early development, prominently in neural and vascular structures. Morpholino-induced depletion of Birc5a causes profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. Similar abnormalities, all less severe except for hematopoiesis, were evident with suppression of Birc5b. The phenotypes induced by morpholino knockdown of one survivin gene, were rescued by overexpression of the other, indicating that the Birc5 paralogs may compensate for each. The potent vascular endothelial growth factor (VEGF) also entirely rescues the phenotypes induced by depletion of either Birc5a and Birc5b, highlighting its multi-functional properties, as well as the power of the model in characterizing the activities of growth factors. CONCLUSION: Overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. These properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this IAP in the pathogenesis of human disease, and for exploring its potential as a therapeutic target.
Asunto(s)
Embrión no Mamífero/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Apoptosis/fisiología , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Corazón/embriología , Hematopoyesis/genética , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Microinyecciones , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/fisiología , Datos de Secuencia Molecular , Miocardio/metabolismo , Neurogénesis/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Survivin , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiologíaRESUMEN
We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivin(lox/lox) mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.
Asunto(s)
Células Endoteliales/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Miocardio/metabolismo , Neovascularización Patológica , Cresta Neural/citología , Defectos del Tubo Neural/genética , Animales , Apoptosis , Cruzamientos Genéticos , Medios de Cultivo Condicionados/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/citología , Genotipo , Proteínas Inhibidoras de la Apoptosis , Ratones , Ratones Transgénicos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Proteínas Represoras , SurvivinRESUMEN
Approaches to regulating angiogenesis in the brain, which may diminish parenchymal damage after stroke, are lacking. Survivin, the inhibitor of apoptosis protein, is up-regulated in vitro in vascular endothelial cells by angiogenic factors, including vascular endothelial cell growth factor (VEGF). To evaluate the in vivo role of survivin in the brain in response to hypoxia/ischemia, we used a mouse model of stroke and show that 2 days after permanent middle cerebral artery occlusion, survivin is uniquely expressed by microvessels that form in the peri-infarct and infarct regions. The extent of vascularization of the infarct is dependent on expression of survivin, since vessel density is significantly reduced in mice with heterozygous deficiency of the survivin gene (survivin+/- mice), even though infarct sizes were not different. Hypoxia alone induces survivin expression in the brain, by cultured endothelial cells and by embryonic stem cells, but this response is at least partially independent of VEGF, hypoxia inducible factor 1alpha, or placental growth factor. Delineating the spatiotemporal pattern of expression of survivin after stroke, and the molecular mechanisms by which this is regulated, may provide novel approaches to therapeutically optimize angiogenesis in a variety of ischemic disorders.