Mechanisms Underlying Hypoxia Tolerance in Drosophila melanogaster : hairy as a Metabolic Switch Dan Zhou1,2*, Jin Xue1,2, James C. K. Lai3, Nicholas J. Schork4, Kevin P. White5,6, Gabriel G. Haddad1,2* 1 Departments of Pediatrics (Section of Respiratory Medicine) and Neuroscience, University of California San Diego, La Jolla, California, United States of America, 2 Rady Children’s Hospital – San Diego, San Diego, California, United States of America, 3 College of Pharmacy, Idaho State University, Pocatello, Idaho, United States of America, 4 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America, 5 Institute for Genomics and Systems Biology, The University of Chicago, Chicago, Illinois, United States of America, 6 Departments of Human Genetics and Ecology and Evolution, The University of Chicago, Chicago, Illinois, United States of America Abstract Hypoxia-induced cell injury has been related to multiple pathological conditions. In order to render hypoxia-sensitive cells and tissues resistant to low O2 environment, in this current study, we used Drosophila melanogaster as a model to dissect the mechanisms underlying hypoxia-tolerance. A D. melanogaster strain that lives perpetually in an extremely low-oxygen environment (4% O2, an oxygen level that is equivalent to that over about 4,000 m above Mt. Everest) was generated through laboratory selection pressure using a continuing reduction of O2 over many generations. This phenotype is genetically stable since selected flies, after several generations in room air, survive at this low O2 level. Gene expression profiling showed striking differences between tolerant and na¨ıve flies, in larvae and adults, both quantitatively and qualitatively. Up-regulated genes in the tolerant flies included signal transduction pathways (e.g., Notch and Toll/Imd pathways), but metabolic genes were remarkably down-regulated in the larvae. Furthermore, a different allelic frequency and enzymatic activity of the triose phosphate isomerase (TPI) was present in the tolerant versus na¨ıve flies. The transcriptional suppressor, hairy, was up-regulated in the microarrays and its binding elements were present in the regulatory region of the specifically down-regulated metabolic genes but not others, and mutations in hairy significantly reduced hypoxia tolerance. We conclude that, the hypoxia-selected flies: (a) altered their gene expression and genetic code, and (b) coordinated their metabolic suppression, especially during development, with hairy acting as a metabolic switch, thus playing a crucial role in hypoxia-tolerance. Citation: Zhou D, Xue J, Lai JCK, Schork NJ, White KP, et al. (2008) Mechanisms Underlying Hypoxia Tolerance in Drosophila melanogaster: hairy as a Metabolic Switch. PLoS Genet 4(10): e1000221. doi:10.1371/journal.pgen.1000221 Editor: Eric Rulifson, University of California San Francisco, United States of America Received April 3, 2008; Accepted September 10, 2008; Published October 17, 2008 Copyright: 2008 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by NIH grants to GGH (RO1NS-037756 and PO1HD-032573); grants from the W.M. Keck Foundation, the Beckman Foundation and the NIH to KPW; a fellowship grant from Parker B. Francis foundation to JX; and a grant from American Heart Association to DZ. Competing Interests: The authors have declared that no competing interests exist. * E-mail: 2zhou@ucsd.edu (DZ); ghaddad@ucsd.edu (GGH) Introduction Mammalian tissues experience a reduction in oxygen delivery at high altitude or during certain disease states, such as myocardial infarction and stroke. In order to survive, cells, tissues and organisms have developed various strategies to adapt to such O2 limited condition. There are indeed major differences between different organisms and cells in their ability to survive reduced environmental O2. For example, turtle neurons are very tolerant to low oxygen and can survive without O2 for hours and days [1,2]. In contrast, mammalian neurons are very sensitive to reduced oxygen and cannot survive for even minutes under similar conditions. However, the mechanisms underlying survival in such extreme hypoxic conditions are not clear at present, in spite of the fact that there have been a number of interesting observations in this regard in the past few decades. For instance, it has been demonstrated that a number of hypoxia-tolerant animals (e.g. Pseudemys scripta and Crucian Carp) reduce their O2 consumption during hypoxia in such a way to minimize the mismatch between O2 supply and demand [3–5]. Similar phenomena was also observed in Drosophila melanogaster [6,7] and in newborn mammals [8,9]. Many questions, however, remain unsolved. For instance, we do not have an adequate understanding of the mechanisms that are responsible for reducing metabolic rate during low O2 conditions; and similarly, the mechanisms that are responsible for coordinating the suppression of these metabolic processes are still largely unknown. In the early 1990s, we discovered that the fruit fly, Drosophila melanogaster, is tolerant to acute anoxia (zero mmHg O2). Flies can sustain such environment for a few hours without any evidence of injury [6]. Since a) Drosophila has been demonstrated to be a powerful genetic model for human diseases [10–14] and b) many biochemical and genetic pathways are highly conserved between Drosophila and mammals, we used Drosophila in the current study to explore the mechanisms underlying tolerance to long-term hypoxia. We first generated a Drosophila melanogaster strain that can live perpetually (i.e., from generation to generation) in severe, normally lethal, hypoxic conditions. To better understand the mechanisms underlying this remarkable hypoxia tolerance, we used cDNA microarrays containing 13,061 predicted or known genes (,90% genes in the genome) to examine the differences in gene expression profiles between the hypoxia-selected (AF) and na¨ıve control (NF) flies [15–17]. We performed these studies in both larvae and adults to determine gene expression as a function PLoS Genetics | www.plosgenetics.org 1 October 2008 | Volume 4 | Issue 10 | e1000221