many small-scale studies (1, 5, 10, 12). Instead,
a more variable distribution with isolated polli-
nation events was detected. The multiple polli-
nating agents (wind and insects) of canola and
the large size of the source may contribute to the
randomness of long-distance pollination events.
Varietal differences among canola sink
fields were observed (Fig. 4), but no con-
sistent effect of wind direction on pollen-
mediated gene flow was detected (data not
shown). The variety of canola may be a
contributing factor in random pollination
events at distance. Pollination has been
shown to be affected by crop variety (12).
Varieties have differences in flowering pe-
riod, which will affect pollination events
over such a large scale. Another explana-
tion for these seemingly random events
may also be related to insect behavior.
Roaming insects may target single plants
flowering early or late in a field, resulting
in sporadic pollen movement. However, in-
sects are more likely to remain in a single
field if sufficient resources (e.g., flowers)
are readily available (13).
Gene transfer is a complex process and is
dependent on many factors (14–16), includ-
ing environmental conditions, plant variety,
insect behavior, and plant density. These ob-
servations, coupled with our data on long-
distance pollen movement, indicate that lab-
oratory and small-scale experiments may not
necessarily predict pollination under com-
mercial conditions. This study demonstrates
that cross-pollination between commercial
canola fields occurs at low frequencies but to
References and Notes
1. J. Champolivier, J. Gasquez, A. Messean, M. Richard-
Molard, in Gene Flow and Agriculture—Relevance for
Transgenic Crops, P. Lutman, Ed. (British Crop Protec-
tion Council, University of Keele, Staffordshire, UK,
1999), pp. 233–240.
2. R. Downey, in Gene Flow and Agriculture—Relevance
for Transgenic Crops, P. Lutman, Ed. (British Crop
Protection Council, University of Keele, Staffordshire,
UK, 1999), pp. 109–116.
3. E. Paul, C. Thompson, J. M. Dunwell, Euphytica 81,
4. J. A. Scheffler, R. Parkinson, P. J. Dale, Plant Breed.
114, 317 (1995).
5. G. R. Stringham, R. K. Downey, Can. J. Plant Sci. 58,
6. B. Staniland et al., Can. J. Plant Sci. 80, 521 (2000).
7. L. Hall, K. Topinka, J. Huffman, L. Davis, A. Good,
Weed Sci. 48, 688 (2001).
8. C. Thompson et al., in Gene Flow and Agriculture—
Relevance for Transgenic Crops, P. Lutman, Ed. (British
Crop Protection Council, University of Keele, Staf-
fordshire, UK, 1999), pp. 95–100.
9. C. Preston, S. Powles, Heredity 88, 8 (2002).
10. G. Rakow, D. Woods, Can. J. Plant Sci. 67, 147 (1987).
11. J. Scheffler, R. Parkinson, P. Dale, Transgenic Res. 2,
12. E. Simpson, C. Norris, J. Law, J. Thomas, J. Sweet, in
Gene Flow and Agriculture—Relevance for Transgenic
Crops, P. Lutman, Ed. (British Crop Protection Council,
University of Keele, Staffordshire, UK, 1999), pp.
13. J. Eckert, J. Agri. Res. 47, 257 (1933).
14. J. E. Barton, M. Dracup, Agron. J. 92, 797 (2000).
15. N. C. Ellstrand, H. C. Prentice, J. F. Hancock, Ann. Rev.
Ecol. Syst. 30, 539 (1999).
16. M. Rieger, C. Preston, S. Powles, Aust. J. Agric. Res. 50,
17. We would like to thank P. Salisbury, S. Fisher, and W.
Burton (Agriculture Victoria); S. Sutherland, P. Parker,
P. Mathews, and G. Condon (New South Wales De-
partment of Agriculture); and D. Lorraine-Colwill, B.
Mack, and S. Garvie (Adelaide University) for the
collection of the seed samples. We would also like to
thank all the farmers who participated in this study.
WAHRI is an initiative of the Grains Research &
8 March 2002; accepted 13 May 2002
Regulation of Hypoxic Death in
C. elegans by the Insulin/IGF
Receptor Homolog DAF-2
Barbara A. Scott,1Michael S. Avidan,1C. Michael Crowder1,2*
resistant (Hyp) mutants in Caenorhabditis elegans and found that specific
(IGF) receptor (INR) homolog gene, were profoundly Hyp. The hypoxia resis-
tance was acutely inducible just before hypoxic exposure and was mediated
through an AKT-1/PDK-1/forkhead transcription factor pathway overlapping
with but distinct from signaling pathways regulating life-span and stress re-
sistance. Selective neuronal and muscle expression of daf-2(?) restored hy-
poxic death, and daf-2(rf) prevented hypoxia-induced muscle and neuronal cell
death, which demonstrates a potential for INR modulation in prophylaxis
against hypoxic injury of neurons and myocytes.
Although genetically tractable model organ-
isms have made longstanding contributions to
and recently to identification of molecular
mechanisms of hypoxic adaptation and sensing
(2, 3), direct genetic screens for hypoxia-resis-
tant mutants have been relatively unexplored.
To identify genes that regulate hypoxic cell
death, we screened new and existing mutant
strains for animals that survived exposure to
either hypoxia or sodium azide (4), an electron-
transport chain inhibitor used as a chemical
surrogate for hypoxia. High-level resistance to
hypoxia or azide was an uncommon phenotype.
We identified only two new mutants and a few
existing ones that had significantly improved
survival. We found the strongest Hyp strains
among existing mutants with reduced activity
of the insulin/IGF receptor (INR) signaling
pathway. daf-2(e1370), which carries a rf mu-
tation in the homolog of the human insulin/IGF
receptor (5), was markedly azide resistant com-
pared with wild-type strain N2 (13.2 ? 1.8%
dead versus 80.8 ? 5.9%; P ? 0.0001). Sub-
sequent hypoxic incubation demonstrated that
daf-2(e1370) was indeed Hyp (Fig. 1, Table 1).
Genetic mapping confirmed the e1370 muta-
daf-2(e1370) not only survived but fully
recovered normal locomotion behavior after as
long as 20 hours of hypoxic incubation (Fig.
1A, movies S1 and S2). N2 displayed signifi-
cant locomotion defects after recovery from a
6.5-hour incubation. Hypoxic sensitivity was
not stage or age specific with the exception of
N2 dauers (a long-lived alternative larval
stage), which were Hyp (Fig. 1C). The Hyp
phenotype of daf-2(e1370) was markedly sen-
sitive to temperature; e1370 animals were less
1Department of Anesthesiology, and2Department of
Molecular Biology and Pharmacology, Washington
University School of Medicine, St. Louis, MO 63110,
*To whom correspondence should be addressed. E-
Table 1. daf-2 allelic variation for hypoxia resis-
tance (Hyp). Animals were raised at 20°C except
sa187, e1369, e979, which were raised at 15°C
then shifted to 20°C 2 days before testing. Percent
dead is reported as means ? SEM per trial. Adults
2 days post L4 were exposed to ?0.3% oxygen at
28°C for 20 hours then scored after a 24-hour
recovery period. Each trial was a completely inde-
pendent experiment done on a different day.
95.5 ? 1.2
3.6 ? 1.3*
4.6 ? 2.4*
8.6 ? 3.8*
46.4 ? 15.6*
47.0 ? 3.6*
53.3 ? 6.3*
55.6 ? 12.4*
59.0 ? 11.7*
77.3 ? 7.7
80.1 ? 10.6
87.9 ? 6.7
90.9 ? 6.7
95.8 ? 2.4
*P ? 0.01 versus wild type by Mann-Whitney test.
R E P O R T S
28 JUNE 2002 VOL 296 SCIENCEwww.sciencemag.org
Hyp when raised at 15°C than at 20°C (table
extended from early larval through adult stages,
and switching to the restrictive temperature just
before hypoxic incubation induced hypoxia re-
sistance. Similar temperature elevation had the
opposite effect in wild-type animals; warmer
animals were more sensitive.
daf-2(rf) alleles including e1370 have three
well-characterized phenotypes. They were orig-
inally isolated based on their dauer constitutive
(Daf-c) phenotype, forming dauer larvaewhen
wild type normally does not. Adult daf-2(rf)
mutants also have a prolonged life-span. Final-
ly, daf-2(rf) adults are resistant to various en-
vironmental stresses, and this resistance corre-
lates with prolonged life-span (6). To assess
whether the hypoxia resistance of daf-2(e1370)
is a consequence of the mechanisms producing
its Daf-c, Age, and/or stress-resistance pheno-
types, we tested 12 other daf-2(rf) alleles with
various phenotypic severities (Table 1). Two
additional alleles were strongly Hyp, five were
weakly but significantly Hyp, and five others
were non-Hyp. The Hyp phenotypes did not
correlate well with life-span (r ? 0.32; P ?
0.36). Eight weak or non-Hyp alleles had sig-
nificantly increased median life-spans as long
as or longer than e1370 (7). Similarly, four
weak Hyp alleles (e1369, sa187, e979, e1391)
have stronger Daf-c phenotypes than e1370,
and non-Hyp e1368 and sa229 are as Daf-c as
e1370 (5, 7). As for stress resistance, seven
e1365, e1371) are significantly resistant to ther-
Thus, the hypoxia resistance of daf-2(rf) is
highly allele specific and does not appear to be
span, dauer formation, or stress resistance.
Seeking a molecular explanation for the al-
lelic differences, we sequenced select Hyp al-
leles (4). m579, a strong Hyp, contained a
missense mutation (Arg437to Cys) in a highly
conserved residue in the cysteine-rich ligand-
binding domain. The other strong alleles, e1370
and sa219, carry missense mutations in con-
served residues in the tyrosine kinase domain
(5). Two weakly Hyp alleles had mutations in
less well-conserved residues in the cysteine-
rich region: m596 (Gly547to Ser) and e979
(Gly383to Glu), whereas sa187 and e1391 have
mutations in highly conserved residues in the
cysteine-rich and kinase domains, respectively
(5). The non-Hyp alleles have mutations in
poorly conserved residues in the ligand-binding
domain (5). The location and nature of the
mutations do not explain their phenotypic se-
verities, but the strong Hyp mutations suggest
that DAF-2 regulates hypoxic death through
ligand-mediated activation of a downstream ki-
To define the daf-2 hypoxic death pathway,
we tested mutants in genes previously found to
lie downstream of daf-2’s regulation of life-
span and dauer formation (Table 2) (4). age-1
codes for a phosphatidylinositol 3-kinase ho-
molog that is a major output for the daf-2
signaling cascade (8). Homozygous age-
1(mg44), a likely null, has a maternally rescued
Daf-c phenotype and a zygotically prolonged
ic homozygous mg44) animals were weakly
Hyp, which suggests either that daf-2(e1370)’s
strong Hyp phenotype was mediated in part
through an age-1-independent pathway or that
there was maternal rescue of mg44. mg44 can
be propagated as a homozygote in the presence
of gain-of-function mutations in akt-1 and pdk-
1, both of which suppress the Daf-c but not the
long life-span phenotype of mg44 (9, 10). Nei-
ther pdk-1(gf) nor akt-1(gf) suppressed the
Hyp phenotype of age-1(mg44) (Table 2). In-
deed, the double mutants were more hypoxia
resistant than age-1(mg44) alone, which indi-
cates that the weak Hyp phenotype of mg44
Fig. 1. Behavioral and lethal effects of hypoxia in
2(e1370rf) (open circles). All animals were scored
24 hours after recovery from incubation in a
hypoxic chamber (?0.3% O2at 28°C). (A) Per-
cent dead of N2 and daf-2(e1370) animals as a
function of hypoxic incubation time. Animals
without spontaneous or evoked body or pharyn-
geal movement were scored as dead. (B) Loco-
motion rate quantified as body bends per minute
(mean ? SEM; 10 animals) after recovery from
various durations of hypoxic incubation. Locomo-
tion of N2 was significantly reduced at the 6.5-
hour time point and thereafter (P ? 0.01; one-
tailed t test); daf-2(e1370) remained unchanged.
(C) Hypoxic death of N2 and e1370 as a function
of developmental age. Hypoxic incubations were
20 hours for all stages. L1, L2, L3, and L4 are
successive larval stages followed by adulthood.
The dauer stage is an alternative third larval form
tested only for N2. N2 dauers were Hyp com-
pared with N2 adults but less hypoxia resistant
than daf-2(e1370) adults (P ? 0.05; ?2statistic).
Table 2. Hyp phenotype of mutants in daf-2 life-span pathway. All animals were raised at 20°C. Adults
2 days post L4 were exposed to ?0.3% oxygen at 28°C for 20 hours then scored after a 24-hour recovery
period. The genotype age-1(mg44)m?z?is zygotic homozygous mg44 from mg44/? mothers.
GenotypeMutationPercent deadAnimals (n)
95.5 ? 1.2
78.8 ? 10.3†
46.2 ? 3.6*†
79.3 ? 9.3†
98.4 ? 1.0†
42 ? 4.4*†
3.6 ? 1.3*
7.5 ? 3.5*
77.5 ? 3.8†
12.5 ? 5.1*
44.4 ? 5.6*†
100 ? 0†
97.2 ? 0.9†
27.6 ? 13.3*†
100 ? 0†
100 ? 0†
96.0 ? 0.8†
86.8 ? 5.3†
79.8 ? 11.3†
3.0 ? 0.6*
P ? 0.01 by Fisher’s exact test:* Versus wild type;
† Versus e1370.
R E P O R T S
www.sciencemag.orgSCIENCEVOL 29628 JUNE 2002
homozygotes was due to maternal rescue. The
lack of suppression of age-1(null) offers the
possibility of an akt-1/pdk-1 independent sig-
naling pathway for hypoxic death. The weak
Hyp phenotype of pdk-1(sa680rf), a strong,
perhaps null, allele is consistent with this alter-
native pathway (Table 2). The Hyp phenotype
of daf-2(e1370) was strongly suppressed by
both akt-1(gf) and pdk-1(gf), which suggests
that DAF-2 signals exclusively through AKT-1
to PDK-1 to regulate hypoxic death or that the
e1370 mutation is not severe enough to reveal
the alternative pathway suggested by age-
some 10) that functions to turn over phospha-
tidylinositol-1,4,5-trisphosphate and thereby in-
hibit the DAF-2/AGE-1 signaling cascade (11–
13). daf-18(null) fully suppresses e1370’s Hyp
phenotype (Table 2). However, daf-18(e1375),
a weaker allele that fully suppresses the long
life-span of daf-2(e1370) (14), only weakly
suppresses Hyp. Thus, long life-span is neither
necessary as shown here nor sufficient as
shown by the long-lived non-Hyp daf-2 alleles
to confer Hyp. daf-16 codes for a forkhead/
FKHRL1 transcription factor homolog, and
daf-16(rf) mutants suppress the Daf-c and Age
phenotypes of daf-2(rf) (15, 16). daf-16(rf)
also completely suppresses the Hyp phenotype
of daf-2(e1370) (Table 2); therefore, daf-
tion to produce hypoxia resistance. Finally, old-
1, a receptor tyrosine kinase, has been proposed
to function downstream of daf-16 based on
regulation of its expression by daf-16 (17).
old-1(gf) mutants are long-lived and stress re-
sistant, whereas old-1(null) is short-lived and
fully suppresses the long life-span and stress
resistance of daf-2(e1370). However, old-
1(null) does not suppress the hypoxia resistance
of e1370 (Table 2). Thus, while daf-2 signals
through daf-16 for regulation of aging, stress
resistance, and hypoxic death, the mechanisms
downstream of daf-16 diverge (fig. S1).
To search for evidence of cell death protec-
tion by daf-2(rf), we examined the cell number
and morphology of animals surviving hypoxia
(Fig. 2). Compared with controls, hypoxia-ex-
posed animals contained multiple strikingly
swollen, necrotic-looking cells (Fig. 2, A and
B). These necrotic cells were seen among mul-
tiple cell types and organs including pharynx,
body wall muscle, gonad primordium, and oth-
er unidentified cells (Fig. 2B) (18). daf-
2(e1370) significantly reduced the number of
necrotic cells (Fig. 2, C and D); weaker daf-2
alleles were also protective but less so than
e1370. daf-16(rf) completely suppressed the
cell-protective effect of e1370.
To focus on neuronal and muscle cell types,
we used cell-type-specific promoters driving
green fluorescent protein (GFP) expression in
neurons and muscle (Fig. 2, E to K). Hypoxia
Fig. 2. Hypoxia-induced morphologic cell de-
fects and death blocked by daf-2(rf). L1 ani-
mals were exposed to 18 hours of hypoxia;
after a 24-hour recovery, surviving animals
were scored for cell morphology and death. All
GFP reporter genes were stably integrated.
Scale bars ? 20 ?m. (A) Pharyngeal cells of
untreated wild-type animal. (B) Hypoxia-treated wild-type animal with swollen necrotic
pharyngeal cells (arrows). (C) Hypoxia-treated daf-2(e1370) with no evidence of necrotic cell
death. (D) Necrotic cells per animal (mean ? SEM). Number of animals scored ? 20 per strain
except daf-16(mgDf50), daf-16(mgDf47); daf-2(e1370) (n ? 10). Asterisk indicates ? wild
type (P ? 0.01; one-tailed t test). e1370 ? m596 ? e1371 (P ? 0.01). (E) Nuclear-localized
pmyo-3::GFP reporter gene expression (strain PD4251) (25) in body wall muscle nuclei of
untreated animals. (F) Hypoxia-treated PD4251. Nuclear GFP expression was fragmented in-
to twoflanking (arrow)ormultiple (arrowhead)
pmyo-3::GFP;daf-2(e1370) animal with preservation of nuclear morphology. (H) GFP expres-
sion in untreated wild-type touch cell sensory neuron body and axon using pmec-4::GFP
reporter gene. (I) pmec-4::GFP expression after hypoxia with axonal beading (arrow). (J)
Normal pmec-4::GFP expression in daf-2(e1370) after hypoxia. (K) Remaining GFP-expressing
neurons and muscle cells after hypoxia as a percent of untreated controls in daf-2(?) (shaded
bars) and daf-2(e1370) (solid bars) backgrounds. A plin-11::GFP reporter gene (26) was used
to score a subset of neurons in the head and tail ganglia. PD4251 was used to score muscle
cells. GFP-positive neurons and muscle cells were reduced in daf-2(?) animals versus daf-
2(e1370) and untreated animals (P ? 0.01; one-tailed t test).
R E P O R T S
28 JUNE 2002VOL 296SCIENCEwww.sciencemag.org
produced striking nuclear fragmentation in es- Download full-text
sentially all myocytes (Fig. 2, E and F). The
fragmentation typically was not random; in-
stead, the nuclear GFP was segregated into two
satellite fragments flanking a shrunken or even
myocytes, the GFP was diffusely fragmented,
with no nucleus apparent by fluorescence or
Normarski microscopy (Fig. 2F, arrowhead).
The number of GFP-positive muscle nuclei was
significantly reduced, which is consistent with
cell death (Fig. 2K). daf-2(e1370) protected
myocytes from both nuclear fragmentation and
death (Fig. 2, H and K). In neurons with a
cytoplasmic GFP marker, hypoxia induced a
dramatic axonal beading morphology (Fig. 2I).
Hypoxia also reduced the number of GFP(?)
neurons. e1370 mutants did not show neuronal
loss and axonal pathology (Fig. 2, J and K).
Through which cells is daf-2 regulating hy-
poxic death? Using cell type-specific promoters,
Wolkow et al. showed that daf-2(?) expression
in neurons, but not in muscle or intestine, could
rescue the long life-span and dauer formation
phenotypes of daf-2(e1370) (19). We used these
strains to determine the cell types involved in
daf-2-mediated organismal death (4). Pan-neu-
ronal expression of daf-2(?) in a daf-2(e1370)
background significantly increased hypoxia-in-
duced death (65.0 ? 5.7 % dead; P ? 0.01
versus e1370; Mann–Whitney nonparametric
test) compared with daf-2(e1370) alone (4.0 ?
0.6% dead) . However, unlike its other pheno-
types, e1370’s Hyp phenotype was also rescued
by muscle expression of daf-2(?) (82.8 ? 7.9%
dead; P ? 0.01 versus e1370). Intestinal expres-
sion did not increase hypoxic death after the
standard 20-hour incubation (10.0 ? 4.3 %
dead) but it did after longer incubations (41-
hour incubation: 100% dead versus 29.7% of
e1370; P ? 0.01). The potent rescue of Hyp by
neuronal and muscle daf-2(?) expression con-
firms the assignment of the Hyp phenotype to
daf-2. Consistent with the direct observation of
these data also suggest that daf-2(?) expression
induces hypoxic death of muscle and neuronal
cell types, whose death, perhaps along with
other cell types, then kills the organism. Alter-
natively, neuronal and muscle expression could
induce death of other cell types responsible for
organismal death. Indeed, cell nonautonomous
effects of daf-2 have been observed for both
aging and dauer formation (19, 20).
How might DAF-2 INR so potently regu-
late hypoxic death? We initially examined
daf-2 mutants because the INR signaling cas-
cade had been found to regulate apoptosis of
vertebrate cells. However, vertebrate INR
cascades antagonize apoptosis (21); thus, re-
duction of DAF-2 signaling should, if any-
thing, increase cell death. Hypoxic cell death
is not, however, exclusively apoptotic, and
after severe insults, it may be almost entirely
necrotic (22). Given the role of the insulin
receptor in regulating glucose utilization, al-
terations in metabolism by daf-2(rf) do pro-
vide an appealing mechanism for its hypoxia
resistance. daf-2(e1370) has been found to
have lower O2consumption than wild type,
perhaps prolonging the time needed for de-
pletion of energy stores and subsequent cell
death (23). However, these results have been
questioned on methodologic grounds and not
all findings by van Voorhies are consistent
with a metabolic mechanism for daf-2’s reg-
ulation of hypoxic sensitivity (24). Identifi-
cation of additional Hyp mutants and genes
downstream of daf-16 should clarify the
mechanisms underlying daf-2’s regulation of
hypoxic cell death.
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and M. Driscoll for providing pmec-4::GFP reporter
gene. Some strains were provided by the Caenorhab-
ditis Genetics Center funded by the National Insti-
tutes of Health National Center for Research Re-
sources. We thank M. Nonet for help with microsco-
py and K. Kornfeld for critique of the manuscript.
Supporting Online Material
Materials and Methods
Figs. S1 and S2
Movies S1 and S2
27 March 2002; accepted 24 May 2002
Published online 13 June 2002;
Include this information when citing this paper.
Jason P. Eiserich,1,2,3*† Stephan Baldus,2,3*‡ Marie-Luise Brennan,6
Wenxin Ma,4Chunxiang Zhang,4Albert Tousson,5
Laura Castro,2,3Aldons J. Lusis,6William M. Nauseef,7
C. Roger White,3,4Bruce A. Freeman2,3†
Myeloperoxidase (MPO) is an abundant mammalian phagocyte hemoprotein
thought to primarily mediate host defense reactions. Although its microbicidal
functions are well established in vitro, humans deficient in MPO are not at
unusual risk of infection. MPO was observed herein to modulate the vascular
signaling and vasodilatory functions of nitric oxide (NO) during acute inflam-
mation. After leukocyte degranulation, MPO localized in and around vascular
endothelial cells in a rodent model of acute endotoxemia and impaired endo-
thelium-dependent relaxant responses, to which MPO-deficient mice were
resistant. Altered vascular responsiveness was due to catalytic consumption of
NO by substrate radicals generated by MPO. Thus MPO can directly modulate
vascular inflammatory responses by regulating NO bioavailability.
Vascular endothelial dysfunction is an estab-
lished feature of acute inflammation (1, 2)
and is typified by compromised function of
the endothelium-derived signaling molecule
nitric oxide (NO), which serves to stimulate
relaxation of vascular smooth muscle cells.
Neutrophils contribute to endothelial dys-
function and altered NO signaling during in-
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www.sciencemag.orgSCIENCEVOL 29628 JUNE 2002