Genetic Basis of Tolerance to O2 Deprivation in Drosophila Melanogaster

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10809–10812, September 1997 Genetics

Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster

GABRIEL G. HADDAD*†,YI-AN SUN†‡,ROBERT J. WYMAN†‡, AND TIAN XU†§

Departments of *Pediatrics, ‡Biology, and §Genetics, and the †Boyer Center for Molecular Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520

Edited by Sherman M. Weissman, Yale University School of Medicine, New Haven, CT, and approved July 28, 1997 (received for review May 15, 1997)

ABSTRACT The ability to tolerate a low-O2 environment (12). This fruit fly, with an O2 consumption rate that exceeds varies widely among species in the animal kingdom. Some even mammalian levels and a total life span of several weeks, animals, such as Drosophila melanogaster, can tolerate anoxia can survive hours in anoxia and, apparently, not suffer tissue for prolonged periods without apparent tissue injury. To injury. Thus, we have taken advantage of this model system to determine the genetic basis of the cellular responses to low O2, study the genetic basis of anoxia tolerance. we performed a genetic screen in Drosophila to identify loci that are responsible for anoxia resistance. Four X-linked, MATERIALS AND METHODS anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mu- Anoxia Testing. A specially designed chamber was used to tagenized flies. The identified recessive and dominant muta- study flies under controlled O2 and temperature conditions tions showed marked delay in recovery from O2 deprivation. (Fig. 1A). Groups of 20–25 adult flies, ages 3–6 days, were In addition, electrophysiologic studies demonstrated that placed in the chamber and exposed to anoxic conditions (O2 polysynaptic transmission in the central nervous system of the concentration ϭ 0% with administration of 100% N2) for 5 min mutant flies was abnormally long during recovery from before allowing recovery in room air (O2 ϭ 20.8%). Although anoxia. These studies show that anoxic tolerance can be Drosophila can survive for a much longer period under very genetically dissected. low O2 conditions, a short period of anoxia of only 5 min was chosen because: (i) mutants defective in protective responses Each year millions of individuals in the United States die or may not survive prolonged periods and (ii) screening a large become morbidly ill because of conditions or diseases that number of flies becomes too impractical if the test time is long. After the introduction of N2 in the chamber, flies became acutely reduce O2 supply to hypoxia-sensitive tissues, such as the myocardium, central nervous system, and kidneys. Over uncoordinated within 25–30 sec, fell to the bottom of the the past several decades, most research has focused on mech- container, stopped moving, and stayed motionless for the rest of the anoxia period. This time (25–30 sec) was deemed to be anisms of vessel disease (e.g., atherosclerosis) that lead to too short to use in a screen. After 2–3 min of reoxygenation, stroke or myocardial infarction and on their prevention. How- flies first exhibited rhythmic movements of the distal parts of ever, little is known about the molecular mechanisms under- their legs; this was followed by an abrupt and discrete change lying the cellular responses to lack of oxygenation and how to from a side or back position to a prone position with purpose- prevent damage once a reduction in O supply has occurred. 2 ful movements such as walking. The time for each fly to reach The study of cell responses to low O is important not only 2 arousal, starting with reoxygenation, was referred to as recov- for understanding disease mechanisms but also for under- ery time. This time (t in Fig. 1B) was deemed to be more standing normal biologic processes. Recent observations have r suitable for a screen and therefore was utilized for all flies. shown, for example, that O deprivation can have profound 2 Genetic Screen for Anoxia-Sensitive Mutants. We focused effects on cell proliferation, differentiation, and tumor growth our screen for mutations on the X chromosome, because the via its effect on cell-cycle check points, including cyclins and mutant phenotype can be observed in the immediate next proteins such as bcl2 and p53 (1–4). When faced with a generation without the need for single-pair matings. Mu- complete lack of O2 (anoxia), shrimp (5) and Drosophila (6) tagenized (x-ray, 4,000 rad) C-S males, which were crossed to embryos stop growing and enter a phase of total developmen- attached-X females [c(1)ywf], transmitted their mutagenized X tal arrest that is relieved only with the reintroduction of O2. chromosome to the male offspring. Suspected mutants were Development can resume even after 2 years of anoxia (6). mated again to attached X females, and their progeny were Different phyla and species have various responses to lack of retested to confirm the transmissibility of the phenotype and O2 (7). Even different organs and different cell types within the ascertain the identity of the inheritance pattern. same organ (e.g., CA1 and dentate cells in the hippocampus) Genetic Mapping. Mapping of the induced mutations was can have marked differences in anoxia tolerance, ranging from performed with X chromosomal markers and complementa- extreme tolerance to high vulnerability. Although there are tion tests. Several markers were used, including y, cv, v, f, car, data describing the changes that occur in cells, especially nerve and su(f). Complementation testing was done on the three cells, of mammalian and nonmammalian species in response to X-linked recessive mutations obtained. a low O2 environment (7, 8–11), the genetic basis for the Central Nervous System Stimulation. Because the giant cellular responses to low O2 has not been elucidated. fiber (GF) system has been well studied (13, 14), we made use We have recently shown that the wild-type Drosophila of it to examine mutant and wild-type flies. Unanesthetized melanogaster [Canton-S (C-S)] is extremely resistant to anoxia flies were placed on a microscope stage in the prone position with the help of soft wax. Stimulating electrodes were placed The publication costs of this article were defrayed in part by page charge through the eye into the brain, and ground electrodes were payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. This paper was submitted directly (Track II) to the Proceedings office. © 1997 by The National Academy of Sciences 0027-8424͞97͞9410809-4$2.00͞0 Abbreviations: DLM, dorsal longitudinal muscle; GF, giant fiber; C-S, PNAS is available online at http:͞͞www.pnas.org. Canton-S.

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average of 314 Ϯ 52.5 sec. Recovery time was reproducible not only for fly populations but also for individual flies because the same flies tested repeatedly and individually (e.g., consecutive days) had very similar recovery times. This tight spread of the wild-type distribution (Fig. 2A) and repeatability of recovery times provided an excellent opportunity to detect individual mutant flies that deviated from the normal. A total of 10,818 flies, carrying mutagenized chromosomes, were screened. A threshold of 480 sec, which is close to the 96th percentile of the wild-type distribution, was used to identify and isolate mutants. Six mutations, four of which were X- linked, were identified and found to alter profoundly the distribution of recovery times. The marked delay in recovery after anoxia displayed by these mutant flies suggested that they were much more sensitive to a lack of O2. We therefore have termed these mutants hypnos to describe (i) sensitivity to O2 deprivation (hypoxia, anoxia, sensitive) and (ii) the phenotype of delayed recovery and ‘‘sleepiness,’’ because Hypnos is the personification of sleep in Greek mythology. Fig. 2 B and C shows the recovery time distributions of male flies with the X chromosome mutations hypnos-1N and hypnos- 2P, respectively. The distribution of their recovery times has little overlap with and is wider than the C-S distribution. For both mutants, the average fly takes twice as long to recover as the wild-type fly (means Ϯ SD ϭ 653.4 Ϯ 134.8 and 644 Ϯ 147.2 sec). The other two mutations have less severe phenotypes and their means Ϯ SD are 509 Ϯ 100 (hypnos-2L) and 390 Ϯ 118.2 sec (hypnos-3I). The cumulative frequency of recovery times for each of the four X chromosomal mutant flies is shown in Fig. 2E along with the wild-type response for comparison. Three of the mutations are recessive, because the heterozygote females have recovery distributions similar to N FIG.1. (A) Specially designed testing chamber for adult Drosophila those of the wild type. The mutant hypnos-1 has a dominant flies. The fly chamber (volume, 275 ml) is surrounded by a jacket effect, because the heterozygote females have a recovery time through which temperature-controlled water (25°C) circulates from a close to that in the males with this mutation (Fig. 2 D and E). water bath. Temperature and O2 are continuously measured in the fly The delay in recovery from anoxia in these mutants appears chamber. Illumination is used to attract flies to the top of the chamber. to have resulted from an enhanced sensitivity to low O2, Flies are introduced in the chamber from a top opening. The hatched because these mutants also lose motor control faster than container is a removable part used for fly collection. (B) Schematic wild-type flies when exposed to anoxia. C-S flies start to lose diagram showing both the quick descent and ascent in O2 concentration in the fly chamber, both of which occurred within 2 min. With the control at an O2 concentration of 2.5–3% during anoxia administration of N2, flies lose coordination, fall onto the floor of the induction, but the mutant ones are affected at a concentration chamber (at O2 ϭ 2.5%), and become motionless until room air (O2 of about 5% when the same N2 flow rate is used. ϭ 20.8%) is re-admitted. The 5-min period of anoxia is measured Mutation Mapping. Fig. 3 shows the cumulative distribution starting from when O2 concentration reaches 0.9%. tr, or recovery of recovery times for the three recessive mutations in females. time, usually is more than 3.5 min, with a mean of about 5.5 min in The heterozygote females (hypnos͞ϩ; Fig. 3A) have a distri- wild-type flies. bution similar to C-S males or females (see above). However, transheterozygotes of hypnos-2P and hypnos-2L have a very placed in the abdomen. Dorsal longitudinal muscle (DLM)- abnormal distribution with a mean Ϯ SD of 621 Ϯ 238 sec, recording electrodes were inserted in the thorax medial to the suggesting that they belong to the same locus. In contrast, Fig. anterior dorsocentral bristles. Latency was measured with an 3B shows that the third mutation (hypnos-3I) complements oscilloscope (Tektronix 5111) and was defined as the period both hypnos-2P and hypnos-2L. Results of genetic mapping from the end of the stimulus artifact to the beginning of the using multiply marked chromosomes [y, cv, v, f, car, su(f)] are voltage deflection of the evoked response. Low-voltage pulses consistent with these complementation tests. Both hypnos-2P to the brain gave rise to long latency responses (on the order and hypnos-2L have been mapped to the region between y and of 4 msec) when recording from the DLM. With increasing cv, whereas hypnos-3I is between v and f. The dominant stimulus strength, shorter latencies were obtained, reaching a mutation (Hypnos-1N) has also been mapped and is located minimum of 1.3 msec at the DLM. The shortest latency distal to f. These data show that the X-linked mutants belong responses were shown to be the result of the direct stimulation to three different loci. of the GF neuronal system (15). hypnos Mutations Affect Central Nervous System Recovery from Anoxia. The behavioral testing, which showed delayed RESULTS AND DISCUSSION recovery from anoxia, led us to believe that the hypnos mutations affected the central nervous system. To further our Anoxic Response of Wild-Type Flies and Isolation of Anox- understanding of the mutations we obtained, we directly ia-Sensitive Mutants. To find optimal conditions for the examined the effect of these mutations on central nervous isolation of anoxia-sensitive mutants, we first studied the system function. The only identified neurons that can be wild-type (C-S) response to anoxia. Fig. 2 A and D shows the studied electrophysiologically in Drosophila are those of the wild-type male and female recovery times. The recovery time GF system. Voltage pulses given in the brain directly to the GF distribution for males had a relatively narrow spread around a neurons exhibited the typical short latency responses (Ͻ1.3 mean of 331 sec (SD ϭϮ61.1 sec; n ϭ 91), and the female msec) of a direct GF stimulation. DLM spikes followed distribution (n ϭ 77) was similar to that of the male, with an stimulation of GF neurons up to 100 Hz in both wild type and Downloaded by guest on September 25, 2021 Genetics: Haddad et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10811

FIG. 2. Distribution and cumulative frequency of recovery times for wild-type (C-S) and mutant flies. The Hypnos-1N, hypnos-2P,L, and hypnos-3I mutants are abbreviated by N, P, L, or I on the plots. Y is used to indicate Y chromosome in males. (A–C) Distributions of the wild-type and the two more severe mutant male populations. Note the major difference in their distributions with almost complete lack of overlap between the wild-type and mutant populations. Female flies heterozygous for mutations hypnos-2L,P and hypnos-3I have a phenotype similar to that of wild-type flies (D–F). In F, the phenotype of Hypnos-1N heterozygotes is also shown.

hypnos mutants. Within 15–25 sec after the onset of anoxia, an allele of this locus (hypnos-2L) or with the other severe this evoked DLM response was lost, and it continued to be mutation (Hypnos-1N)(nϭ25) had a baseline long latency absent during the 5-min period of anoxia. After the reintro- recording that was similar to that of C-S flies (n ϭ 10). duction of room air, the response returned within 1–2 min, with However, during recovery, whereas the DLM of wild-type flies no difference between C-S and mutant flies in the recovery started to respond after 2 min into recovery (Fig. 4A), flies time of the GF system. These data indicate that the hypnos with these two mutations had a much longer time to first mutations do not eliminate any gene products that might be evoked potential, with some mutant flies requiring up to 25–30 necessary for axonal conduction or synaptic function between min for the first evoked response to occur (Fig. 4 A–C). As a the GF neurons and the DLM. measure of a further stage of recovery, we studied the time it Because no defect was detected in the GF system, we took before the flies could respond to consecutive stimuli at examined the effect of anoxia on the interneurons in the 0.05 Hz without failure (Fig. 4D). Both hypnos-2L and Hyp- Drosophila brain that are presynaptic to GF. Stimulation of eye nos-1N mutants took about three times as long as wild types to neurons, using low voltages, evoked spikes in the DLM with a recover to this degree. These data strongly suggest that the long latency (4 msec) (13). Experiments were carried out in mutation has a profound effect on synaptic transmission in the three of the four mutations (hypnos-2P,L, Hypnos-1N) and on central nervous system, most likely in neurons proximal to the wild-type flies. In one mutant (hypnos-2P), which had a severe GF system. Furthermore, we believe that the defects in the phenotype, long-latency evoked potentials could not be ob- mutants are not general ones that affect all synapses. The tained, irrespective of the voltage and duration used. Flies with reason for this is that our studies on shaking B mutants (13, 14),

FIG. 3. Cumulative frequency of recovery times for heterozygote and transheterozygote females. The hypnos-2P͞hypnos-2L transheterozygote females (P͞L) had very delayed recovery times (A), whereas double heterozygotes between mutations hypnos-3 and hypnos-2 complemented each other (B). Downloaded by guest on September 25, 2021 10812 Genetics: Haddad et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 4. Recovery from anoxia of DLM evoked potential in wild-type and mutant flies. DLM-evoked potentials, which are present during baseline in both wild type and mutant Hypnos-1N, are abolished within 20–25 sec of the start of the 5-min anoxic period. (A) A series of consecutive traces of evoked potentials in a single wild-type fly. Bottom trace is recorded at the end of anoxia or 0 min into reoxygenation, and evoked potential is still absent (bottom arrow). First evoked potential during recovery occurs at 2 min after the start of reoxygenation. Subsequent stimulation at 0.05 Hz shows persistent responsiveness. (B) A series of consecutive traces in a single hypnos-1N mutant fly with a very severe phenotype. Even after 23 min and 40 sec into reoxygenation, no response was seen, and the first evoked potential elicited occurred at 25 min, followed at least once (ૺ) by a failed response to stimulation. (C) Means Ϯ SE of the time to first evoked potential (EP) in wild-type (C-S males) and Hypnos-1N mutants. (D) Means Ϯ SE of time to repetitive EP (10 consecutive stimuli at 0.05 Hz without failure) for the same two groups of flies. Stimulation parameters were 6 V, 0.3-msec duration at 0.05 Hz. [Bars ϭ 1 msec (horizontal) and 10 mV (vertical).]

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