Hypoxia-selected Drosophila melanogaster
Experimental selection of hypoxia-adapted Drosophila melanogaster
Genomic DNA was isolated from a pool of 100 male and 100 female adult flies collected from hypoxia-selected populations or generation matched control populations by standard phenol:chloroform extraction followed by treatment with DNase-free RNase. DNA quality was assessed by spectrophotometry (260/280 and 260/230) and gel electrophoresis. A total of 3µg was sheared DNA (Covaris, Woburn, MA) and used to construct library for paired-end sequencing. The DNA fragments were subject to end repair using End-IT DNA End-Repair Kit (Epicentre, Madison, WI) and then ligated to Illumina PE adapters. The adapter-ligated products were purified on Qiaquick spin columns (Qiagen Inc., Valencia, CA) and PCR-amplified with high fidelity DNA Polymerase in 12 cycles using Illumina’s PE primer set (Illumina, San Diego, CA). Cluster generation was performed using the Illumina cluster station and cluster generation kit v2. The 54+54 paired-end sequencing was performed using genome analyzer II (Illumina, San Diego, CA) and sequencing kit v3. The fluorescent images were processed to sequences using the Illumina base-calling pipeline (GA Pipeline-1.4.0).
Through long-term laboratory selection (over 200 generations), we have generated Drosophila melanogaster populations that tolerate severe, normally lethal, level 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 re-sequencing.  By applying novel 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 ?-secretase inhibitor, DAPT, which significantly reduced adult survival and lifespan 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 over-expression of the Notch intracellular domain in glial cells was critical for conferring hypoxia tolerance. Novel analytical tools, genetic and bioinformatic strategies allowed us to discover that Notch activation plays a major role in this hypoxia tolerance in Drosophila melanogaster.