Drosophila, a golden bug, for the dissection of the genetic basis of tolerance and susceptibility to hypoxia

Abstract

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.

Figure 1

Results of forward genetic screen: dADAR and dMRP4. A) The prolonged recovery of hypnos-2^P (ADAR^−/− null) flies can be fully rescued by a wild-type dADAR transgene (○ : hypnose-2^P-dADAR; : ● w¹¹¹⁸; □: hypnose-2^P; ■: hypnose-2^P-32B). B) The dADAR transcripts are expressed mainly in adult heads, and the deletion in hypnos-2P is an in-frame deletion. C) The expression of dADAR can be detected by RT-PCR in C-S embryos (6–8 hours). No full-length dADAR transcripts can be detected in hypnos-2^P embryos and adults. St, low mass DNA marker; Hyp, hypnos-2^P; E, embryos (6–8 hours); A, adults. D) Localization of dADAR transcripts in adult fly heads (left panel; arrowheads). Sense dADAR RNA probe gave no positive signals in the same tissues (right panel). E) Misexpression of dMRP4 reduces viability in Drosophila. Viability was shown as percentage of first instar larvae surviving to pupae, referring to numbers of yw pupae from the normoxic groups as 100%. (□: 21% O₂; : 4% O₂). F) dMRP4 mRNA expression in pupae. Semi-quantitative RT-PCR was used to determine the expression levels of dMRP4 mRNA from 24B>EP(3)3177 pupae exposed to 4% O₂ for 10 days (line 2) or reared at 21% O₂ (line 3), yw flies were used as control (line 1). (Adapted with permission from Ma E, et al., J Clin Invest 107:685–693, copyright © 2001, by The American Society for Clinical Investigation, and Huang H and Haddad GG, Physiol Genomics 29:260–266, copyright © 2007 by the American Physiological Society).

Figure 2

Phenotypic changes in hypoxia-selected Drosophila melanogaster. A) Survival rate of parental Drosophila melanogaster population in hypoxic conditions. The parental population survived well at 8% O₂ but their survival rate was reduced at 6%; and 4% O₂ was lethal. B) No significant differences were found in lifespan between naïve flies (Δ: NF) and hypoxia-adapted/selected flies (□: AF). The mean lifespan for NF is 33.7±6.2 days (n=180) and the mean lifespan for AF is 33.7±3.9 days (n=220). C) and D) Body size and weight in AF. The body size and weight of AF were significantly reduced, as compared to NF. Data were presented as means ± SD (* p<0.05, **p<0.01; student’s t-test). (Adapted with permission from Zhou D et al., PLoS Genet 4:e1000221, copyright © 2008).

Figure 3

Suppression of genes encoding glycolytic, TCA cycle enzymes and components of the respiratory chain complexes. A) Schematic illustration of the alterations in genes encoding glycolytic and TCA cycle enzymes (Green: down-regulated genes; Black: not significantly changed genes; Red, up-regulated genes). B) Cluster map of changes in genes encoding glycolytic enzymes in NF and AF larvae. C) Cluster map of changes in genes encoding TCA cycle enzymes in NF and AF larvae. D) Cluster map of changes in gene expression encoding components of respiratory chain complexes. Each cluster contained 9 hybridizations of NF and 8 hybridizations of AF samples. E) Summary of alterations in each respiratory chain complex measured in larvae. F) Comparison of gene expression changes in each respiratory chain complex between larvae and adult. (Adapted with permission from Zhou D et al., PLoS Genet 4:e1000221, copyright © 2008).

Figure 4

Effect of the transcriptional suppressor, hairy, on TCA cycle gene expression and hypoxia tolerance. A) Genomic localization of hairy binding elements in the cis-regulatory region of genes encoding TCA cycle enzymes (Arrowheads, consensus hairy binding sites; Arrows, transcriptional start sites; Bold, down-regulated genes; Regular, not significantly changed or up-regulated genes). B) Significant up-regulation of hairy expression in hypoxia-selected AF populations (□: NF; : AF) (**p<0.01). C) Abolished suppression of genes encoding TCA cycle enzymes in hairy loss-of-function mutants (■: AF; □: h¹ mutant; : h^1j3 mutant). D) Significant reduction in hypoxia survival in hairy loss-of-function mutants (open bar: normoxia; gray bar: 6% O₂) (**p<0.01). (Adapted with permission from Zhou D et al., PLoS Genet 4:e1000221, copyright © 2008).

Figure 5

Left upper panel: Cell viability assay after exposure to 1% O₂ for 4 days. There were four times more dead cells in HEK-v than in HEK-dtps1. Right upper panel: Apoptosis assay of the cells after hypoxic treatment for 2 days. Annexin V-FITC and propidium iodide stained on average 13.4 ± 3.2% and 6.4 ± 0.3% of the HEK-v cells but a much lower proportion of HEK-dtps1 cells (1.4 ± 0.4%, p < 0.001 and 0.3 ± 0.2%, p<0.0001). Left lower panel: A) Western signals of Na⁺-K⁺ ATPase and β-actin. B) The Na⁺-K⁺ ATPase amount present in the insoluble proteins fraction increases dramatically in HEK-v cells after 2 and 3 days of exposure (■: HEK-v; □: HEK-dtps1) (*p<0.05). Right lower panel: Intracellular trehalose reduces ubiquitinated proteins during hypoxia (■: HEK-v;□: HEK-dtps1) (*p<0.05). (Adapted with permission from Chen Q et al., J Biol Chem 278:49113–49118, copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.).