RESUMEN
Through long-term laboratory selection (over 200 generations), we have generated Drosophila melanogaster populations that tolerate severe, normally lethal, levels of hypoxia. Because of initial experiments suspecting genetic mechanisms underlying this adaptation, we compared the genomes of the hypoxia-selected flies with those of controls using deep resequencing. By applying unique computing and analytical methods we identified a number of DNA regions under selection, mostly on the X chromosome. Several of the hypoxia-selected regions contained genes encoding or regulating the Notch pathway. In addition, previous expression profiling revealed an activation of the Notch pathway in the hypoxia-selected flies. We confirmed the contribution of Notch activation to hypoxia tolerance using a specific γ-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), which significantly reduced adult survival and life span in the hypoxia-selected flies. We also demonstrated that flies with loss-of-function Notch mutations or RNAi-mediated Notch knockdown had a significant reduction in hypoxia tolerance, but those with a gain-of-function had a dramatic opposite effect. Using the UAS-Gal4 system, we also showed that specific overexpression of the Notch intracellular domain in glial cells was critical for conferring hypoxia tolerance. Unique analytical tools and genetic and bioinformatic strategies allowed us to discover that Notch activation plays a major role in this hypoxia tolerance in Drosophila melanogaster.
Asunto(s)
Adaptación Fisiológica/genética , Cromosomas de Insectos/genética , Proteínas de Drosophila/genética , Hipoxia/genética , Receptores Notch/genética , Selección Genética , Cromosoma X/genética , Adaptación Fisiológica/efectos de los fármacos , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Cromosomas de Insectos/metabolismo , ADN/genética , ADN/metabolismo , Dipéptidos/farmacología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Hipoxia/metabolismo , Mutación , Estructura Terciaria de Proteína , Receptores Notch/metabolismo , Cromosoma X/metabolismoRESUMEN
We have previously discovered that the adult Drosophila melanogaster is tolerant to a low O2 environment, withstanding hours of total O2 deprivation without showing any evidence of cell injury. Subsequently, our laboratory embarked on the study of hypoxia tolerance using a mutagenesis and overexpression screens to begin to investigate loss-of-function or gain-of-function phenotypes. Both have given us promising results and, in this article, we detail some of the interesting results. Furthermore, several years ago, we have also started an experimental "Darwinian" selection to generate a fly strain that can perpetuate through all of its life cycle stages in hypoxic environments. Through microarrays and bioinformatic analyses, we have obtained genes (e.g. Notch pathway genes) that play an important role in hypoxia resistance. In addition, we also detail a proof of principle that Drosophila genes that are beneficial in fly resistance to hypoxia can also be as well in mammalian cells. We believe that the mechanisms that we are uncovering in Drosophila will allow us to gain insight regarding susceptibility and tolerance to low O2 and will therefore pave the way to develop better therapies for ailments that afflict humans as a consequence of low O2 delivery or low blood O2 levels.