Identification of Genes Underlying Hypoxia Tolerance inDrosophilaby a P-element Screen

Abstract

Hypoxia occurs in physiologic conditions (e.g. high altitude) or during pathologic states (e.g. ischemia). Our research is focused on understanding the molecular mechanisms that lead to adaptation and survival or injury to hypoxic stress using Drosophila as a model system. To identify genes involved in hypoxia tolerance, we screened the P-SUP P-element insertion lines available for all the chromosomes of Drosophila. We screened for the eclosion rates of embryos developing under 5% O2 condition and the number of adult flies surviving one week after eclosion in the same hypoxic environment. Out of 2187 lines (covering ∼1870 genes) screened, 44 P-element lines representing 44 individual genes had significantly higher eclosion rates (i.e. >70%) than those of the controls (i.e. ∼7–8%) under hypoxia. The molecular function of these candidate genes ranged from cell cycle regulation, DNA or protein binding, GTP binding activity, and transcriptional regulators. In addition, based on pathway analysis, we found these genes are involved in multiple pathways, such as Notch, Wnt, Jnk, and Hedgehog. Particularly, we found that 20 out of the 44 candidate genes are linked to Notch signaling pathway, strongly suggesting that this pathway is essential for hypoxia tolerance in flies. By employing the UAS/RNAi-Gal4 system, we discovered that genes such as osa (linked to Wnt and Notch pathways) and lqf (Notch regulator) play an important role in survival and development under hypoxia in Drosophila. Based on these results and our previous studies, we conclude that hypoxia tolerance is a polygenic trait including the Notch pathway.

Multiscale modeling of the causal functional roles of nsSNPs in a genome-wide association study: application to hypoxia

“…Using integrated structural and network analysis, we hypothesize that nsSNPs in H, Rad51D, Ulp1, Sol, Wnt5, CG33714, GalNAc-T2, Dys, and HDAC4, may all lead to the functional modification of these genes via allosteric regulation and protein-protein/DNA/RNA interactions and hence are driver mutations defining the hypoxia tolerance phenotype. Our predictions are supported by experimental evidence [23,26]. Moreover, multiscale modeling may identify potential epistasis using a very small sample size.…”

“…The reduced activation of Notch signaling by a specific γ-secretase inhibitor significantly reduces the survival and life-span of hypoxia tolerant D. melanogaster strains [23]. The critical role of Notch signaling in hypoxia tolerance is further supported by UAS-Gal4 over-expression and RNAi knockdown of genes involved in Notch signaling [26]. Other experimental evidence from the literatures, as detailed below, also support our predictions.…”

“…This pathway analysis of the MSSN identifies four core pathways: up-regulated Notch and Gurken/Epidermal Growth Factor Receptor (EGFR), and down-regulated Toll and Torso/Receptor Tyrosine Kinase (RTK) pathways. Using Notch signaling inhibition and a P-element screen, we have experimentally validated that the up-regulation of Notch signaling is critical to the survival of hypoxia tolerant Drosophila strains [23,26]. Thus the up-regulation of Notch signaling is confirmed as a driver for hypoxia tolerance in D. melanogaster .…”

Population genetic evidence for cold adaptation in European Drosophila melanogaster populations

“…This is interesting in the context of its possible role in hypoxia tolerance (Azad et al . ), which it shares with dpr8 (Weber et al . ), and its temperature‐sensitive effects on developmental time (Mensch et al .…”

Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster

“…These stocks can be screened for specific phenotypes. Numerous screens have used this strategy for bristle number variation (Norga et al, 2003), synaptic transmission (Liebl et al, 2006), hypoxia tolerance (Azad et al, 2012), and olfactory behavior (Tunstall et al, 2012), among others. Once interesting phenotypes have been identified, one needs to ascertain that the TE insertion causes the phenotype, preferably by doing three experiments: precise excision of the TE and reversion of the phenotype (Hummel and Klambt, 2008), failure to complement the phenotype with a deficiency (Cook et al, 2010a), and rescue with a P[acman] transgene encompassing the genomic fragment (Venken et al, 2006; Venken et al, 2009; Venken et al, 2010; Szabad et al, 2012).…”