Regulation of Hypoxic Death in C. elegans by the Insulin/IGF Receptor Homolog DAF-2 Barbara A. Scott, Michael S. Avidan, C. Michael Crowder

Hypoxic and azide incubations. Synchronous NGM agar cultures of adult C. elegans, 2 days post L4 stage, were transferred with 1 ml M9 to 1.5-ml polypropylene tubes. For azide incubations, animals were pelleted by gravity, and the M9 was exchanged 3 times with 1 ml of 0.5 M freshly made sodium azide (Sigma) in M9. The animals were incubated in azide solution for 1.5 hours, pelleted, rinsed 3 times with 1 ml M9, and transferred to one or more NGM agar plates. For hypoxic incubations, animals in 1.5-ml tubes were placed in a hypoxia chamber (Forma Scientific, model 1025) filled with anoxic gas (5% CO₂, 10% H₂, 85% N₂), and the M9 was replaced 5 times with 1 ml of M9 that had been vigorously bubbled for 30 min with anoxic gas. Except where noted, animals were incubated in the hypoxic chamber at 28°C in 100 l of M9 for 20 hours then transferred back to agar plates and allowed to recover in room air. Oxygen tension in the hypoxia chamber was measured by an O₂ meter and electrode (Microelectrodes; OM4 meter, MI730 electrode) and was always <0.3%. Both azide- and hypoxia-treated animals were scored after a 24-hour recovery period for spontaneous or, if necessary, evoked movement (touching with a platinum wire) or pharyngeal pumping. Animals not moving or not pumping were scored as dead. In pilot experiments, we found that animals recovered to their full extent by 8 hours. We also found that the volatile anesthetic isoflurane induced uncoordinated locomotion to the same degree and with the same rapid time course in N2 and daf-2(e1370) adults (Fig. S2), indicating that the cuticle of both strains was similarly gas permeant.

Mutagenesis screen. The F₂ progeny of animals exposed to 50 mM EMS in M9 for 4 hours were screened for survival following exposure to either azide (70,000 F₁ genomes) or hypoxia (7000 F₁ genomes) as described above. Only two Hyp mutants, which complemented one another and daf-2(rf), were isolated. daf-2(e1370) was discovered to be Hyp in a screen through 100 selected existing mutant strains.

Mapping of Hyp phenotype to the daf-2 locus. The Hyp phenotype of daf-2(e1370) was mapped relative to dpy-1 and unc-32, which flank daf-2, 9.5 and 9.9 cM away, respectively. 52/52 Unc Dpy animals segregating from dpy-1(e1) daf-2(e1370) unc-32(e189)/+ heterozygotes were Hyp. 7/18 Dpy non-Uncs and 12/20 Unc non-Dpys segregating from the above heterozygote were Hyp. These mapping data link the Hyp locus to the dpy-1 unc-32 interval about halfway between the two genes near daf-2. daf-2 alleles were obtained from various sources, and all had already been outcrossed at least once. To confirm linkage of the Hyp-conferring locus to daf-2, e1370, sa219, m579, e1369, m596, sa187, e979, and e1391 were all outcrossed at least two more times and re-homozygosed for their Daf-c phenotype. All outcrossed strains retained their Hyp phenotypes.

daf-2 sequencing. For each daf-2 allele sequenced, animals were lysed, their DNA was prepared, and the entire daf-2 coding sequence was PCR-amplified then sequenced by using dye-terminators. Any difference from the published sequence was confirmed by sequencing the opposite strand. Only one mutation was found in each mutant.

Source of double mutants. Constructions of double mutants were previously published, and the strains obtained from those labs except for Pmyo-3::GFP;e1370, Pmec-4::GFP;e1370, and Plin-11::GFP;e1370 strains, which were generated by crossing daf-2(e1370)/+ males into each of the integrated GFP-array bearing strains. F₂ dauer progeny segregating 100% green progeny were kept, and their Daf-c phenotype was confirmed.

Test of cell-type specific rescue of Hyp phenotype of daf-2(e1370). daf-2(e1370) transformed with extrachromosomal arrays containing wild-type daf-2 driven by the neuronal, muscle, or intestinal cell type-specific promoters (S1) and the dominant transformation marker rol-6(su1006) were separated from their non-Rol siblings and were scored for hypoxia induced death along with wild-type animals and untransformed daf-2(e1370). Hypoxic incubations were the standard 20-hour exposure at 28°C in the hypoxia chamber as described above.

Supplemental Figure 1. Proposed hypoxic death pathway. The primary pathway for DAF-2/AGE-1 signaling is through PDK-1/AKT-1. An alternative undefined pathway is shown because of incomplete suppression by pdk-1(gf) and akt-1(gf) of age-1(null) along with a weak Hyp phenotype of pdk-1(rf).

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Supplemental Figure 2. The speed of locomotion was measured just before the addition of the volatile anesthetic isoflurane (time = 0) and at various times thereafter (given relative to the time of addition of isoflurane) as described previously (S2). Both strains were exposed to 2.7 vol% isoflurane as described previously (S3). Data points represent the means ± SEM of 10 animals. There are no significant differences between the rate constants of the two curves.

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Supplemental Table 1. Temperature sensitivity of wild-type and daf-2(e1370) hypoxic death. For percent dead, n is the number of animals tested; in all cases the number of trials was 3. The P value by Student's t test is calculated by comparing percent dead with the row immediately above it. NA, not applicable.
Genotype	Initial temp	Temp shift	Stage of shift	Hypoxic temp	Recovery temp	Percent dead (n)	P value
+/+	15	none	NA	28	15	79.4 ± 1.9 (374)
"	15	25	adult	28	15	96.3 ± 0.9 (598)	<0.01
"	15	none	NA	28	25	83.2 ± 8.3 (331)
"	15	25	adult	28	25	96.4 ± 1.3 (453)	<0.01
"	20	none	NA	28	20	96.7 ± 1.8 (97)
"	20	none	NA	26	20	74.3 ± 2.3 (139)	<0.01
"	20	none	NA	24	20	51.3 ± 4.6 (455)	<0.01
"	20	none	NA	22	20	21.3 ± 6.4 (316)	<0.01
"	20	none	NA	18	20	0 ± 0 (375)	<0.01
daf-2(e1370)	15	none	NA	28*	20	94.7 ± 1.9 (497)
"	20	none	NA	28*	20	3.2 ± 1.1 (765)	<0.01
"	15	20	L1-L2	28*	20	2.7 ± 2.1 (173)	NS
"	15	20	L3	28*	20	0 ± 0 (122)	NS
"	15	20	L4	28*	20	0 ± 0 (111)	NS
"	20	15	L1-L2	28*	20	99.5 ± 0.3 (345)
"	20	15	L3	28*	20	97.6 ± 1.2 (243)	NS
"	20	15	L4	28*	20	95.8 ± 2.2 (151)	NS
"	15	none	NA	28	15	13.2 ± 3.6 (218)
"	15	25	adult	28	15	0.3 ± 0.3 (237)	<0.01
"	15	none	NA	28	25	42.2 ± 6.3 (253)	<0.01
"	15	25	adult	28	25	1.5 ± 0.7 (261)	<0.01
"	20	none	NA	28	20	0 ± 0 (86)
"	20	none	NA	26	20	0 ± 0 (47)	NS
"	20	none	NA	24	20	0 ± 0 (135)	NS
"	20	none	NA	22	20	1 ± 1 (47)	NS
"	20	none	NA	22	20	0 ± 0 (270)	NS
*Denotes hypoxic incubation duration for 24 hours to increase the death of daf-2(e1370); all other hypoxic times were 20 hours. P < 0.01 vs percent dead two rows above.

References

S1. C. A. Wolkow et al. Science 290, 147 (2000).

S2. C. M. Crowder et al. J. Biol. Chem. 276, 44369 (2001).

S3. C. M. Crowder et al. Anesthesiology 85, 901 (1996).

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• Movie 1
  Locomotion behavior of N2 after a 24-hour recovery from a 20-hour hypoxic incubation. Post-hypoxic but alive animals were placed on an agar pad, and movement over a 60-s period was recorded. The behavior of these animals was typical of the few live N2 animals; the majority of N2 were dead. This 60-s movie at 4 s/frame was made with Scion image software using a Dage MTI CCD camera mounted to a Nikon dissecting microscope. The speed of the movie is 1 frame/s. The magnification is identical in S1 and S2.

• Movie 2
  Locomotion behavior of daf-2(e1370) after a 24-hour recovery from a 20-hour hypoxic incubation. Immediately after removal from hypoxic conditions both N2 and daf-2(e1370) were paralyzed, but e1370 recovered the ability to move completely over the next 2 to 5 hours. This 60-s movie at 4 s/frame was made with Scion image software using a Dage MTI CCD camera mounted to a Nikon dissecting microscope. The speed of the movie is 1 frame/s.