Genetic Regulation of Unsaturated Fatty Acid
Composition in C. elegans
Trisha J. Brock, John Browse, Jennifer L. Watts*
Institute of Biological Chemistry, Washington State University, Pullman, Washington, United States of America
Delta-9 desaturases, also known as stearoyl-CoA desaturases, are lipogenic enzymes responsible for the generation of
vital components of membranes and energy storage molecules. We have identified a novel nuclear hormone receptor,
NHR-80, that regulates delta-9 desaturase gene expression in Caenorhabditis elegans. Here we describe fatty acid
compositions, lifespans, and gene expression studies of strains carrying mutations in nhr-80 and in the three genes
encoding delta-9 desaturases, fat-5, fat-6, and fat-7. The delta-9 desaturase single mutants display only subtle changes
in fatty acid composition and no other visible phenotypes, yet the fat-5;fat-6;fat-7 triple mutant is lethal, revealing that
endogenous production of monounsaturated fatty acids is essential for survival. In the absence of FAT-6 or FAT-7, the
expression of the remaining desaturases increases, and this ability to compensate depends on NHR-80. We conclude
that, like mammals, C. elegans requires adequate synthesis of unsaturated fatty acids and maintains complex
regulation of the delta-9 desaturases to achieve optimal fatty acid composition.
Citation: Brock TJ, Browse J, Watts JL (2006) Genetic regulation of unsaturated fatty acid composition in C. elegans. PLoS Genet 2(7): e108. DOI: 10.1371/journal.pgen.0020108
Monounsaturated fatty acids (MUFAs) are key components
of membrane phospholipids and triglycerides that play
important roles in diverse cellular processes such as
membrane function, energy storage, and signaling. MUFAs
are synthesized from saturated fatty acids by delta-9 (D9)
desaturases, also known as stearoyl-CoA desaturases (SCDs),
which introduce a double bond between the 9thand 10th
carbon of a saturated fatty acyl chain. Alterations in the ratio
of MUFAs to saturated fatty acids are implicated in heart
disease and cancer , the two leading causes of death in the
United States . The appropriate ratio between MUFAs and
saturated fatty acids is maintained by the activity of the D9
desaturases, which are subject to complex regulation . As a
key control point in metabolic regulation, D9 desaturases
could be therapeutic targets for treatment of obesity,
diabetes, and cardiovascular disease.
The D9 desaturases are ubiquitous enzymes in eukaryotes,
found in organisms from yeast to humans. Yeast have one D9
desaturase, Ole1p, and mutants that lack this activity are not
able to survive without exogenous supplementation of
unsaturated fatty acids . Mice have four D9 desaturases,
each having a unique expression pattern [5,6]. Mutant
analysis has revealed distinct roles for SCD1 and SCD2.
SCD1 is important for adult energy metabolism and lipid
synthesis , while SCD2 is involved in lipid synthesis during
embryonic development . In humans, two SCD isoforms,
hSCD1 and hSCD5, have been described [9,10]. A variety of
environmental and physiological signals affect the expression
of D9 desaturases. Diets rich in unsaturated fatty acids
decrease D9 desaturase expression, while high carbohydrate
consumption increases expression . Decreased temper-
ature leads to increases in D9 desaturase gene expression in
poikilotherms . In addition, endogenous hormones such
as leptin and glucagon cause a decrease in D9 desaturase gene
expression, while insulin has the opposite effect .
Sterol regulatory element binding proteins (SREBPs) and
peroxisome proliferator-activator receptor protein-alpha
(PPARa) have been identified as key transcriptional regu-
lators of SCD1 gene expression in mammals . The SREBP-1
gene encodes a transcription factor that stimulates expres-
sion of genes involved in fatty acid biosynthesis, including
SCD1 , while the SREBP-2 gene product stimulates genes
involved in cholesterol biosynthesis . PPARa is one of a
family of nuclear hormone receptors (NHRs), that, upon
ligand binding, acts as a heterodimer with the retinoid X
receptor to induce transcription of target fat metabolism
genes . PPARa, like all NHRs, contains a hydrophobic
pocket for ligand binding and a DNA binding domain for
interacting with the promoters of target genes. The targets of
PPARa include genes for the b-oxidation enzymes, SCDs, and
other fatty acid desaturases [15,16]. The other members of the
PPAR family, PPARd and PPARc are also involved in
regulation of fat metabolism . These regulators have
unique roles due to differences in their gene expression
patterns and regulatory activities.
Caenorhabditis elegans is becoming recognized as an impor-
tant model for the study of fat metabolism. These animals
synthesize a wide variety of fatty acids using a D12 desaturase,
an D3 desaturase, a D5 desaturase, a D6 desaturase, and three
D9 desaturases [18,19]. C. elegans can also incorporate dietary
Editor: David Valle, Johns Hopkins University School of Medicine, United States of
Received March 22, 2006; Accepted May 30, 2006; Published July 14, 2006
A previous version of this article appeared as an Early Online Release on June 5,
2006 (DOI: 10.1371/journal.pgen.0020108.eor).
Copyright: ? 2006 Brock 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.
Abbreviations: GFP, green fluorescent protein; MUFA, monounsaturated fatty
acid; NHR, nuclear hormone receptor; PPAR, peroxisome proliferator-activator
receptor; QPCR, quantitative RT-PCR; RNAi, RNA interference; SCD, stearoyl-CoA
desaturase; SREBP, sterol regulatory element binding protein
* To whom correspondence should be addressed. E-mail: email@example.com
PLoS Genetics | www.plosgenetics.orgJuly 2006 | Volume 2 | Issue 7 | e1080997
fatty acids into lipids, allowing researchers to modify the fatty
acid composition of live animals [20,21]. In an RNAi (RNA
interference) screen, genes were identified that altered fat
storage and many of these genes have mammalian counter-
parts known to function in fat metabolism . In addition,
mutant analysis offers insight into pathways known to
regulate fat storage in both nematodes and mammals such
as the insulin-signaling pathway . A recent study
established a role for NHR-49, as a regulator of lipid
homeostasis . The nhr-49 mutants have increased levels
of the saturated fatty acid 18:0, higher fat accumulation, and a
shorter lifespan than wild-type animals. NHR-49 is also
required for inducing D9 desaturase expression in well-fed
To gain a deeper understanding of fatty acid metabolism in
C. elegans we have characterized the three D9 desaturase
mutants using biochemistry, gene expression, and phenotypic
analysis. While the three D9 desaturase single mutants, fat-5,
fat-6, and fat-7 display few differences from wild type, we show
that they compensate for loss of one isoform by regulated
induction of the remaining D9 desaturase genes. This
induction depends on NHR-80, a novel NHR that we have
identified as a regulator of desaturase expression. Further-
more, the fat-5;fat-6;fat-7 triple mutant is unable to survive,
revealing that endogenous production of monounsaturated
fatty acids is essential for survival under standard growth
conditions. The D9 desaturase genes and their transcriptional
regulators are vital for maintaining optimal fatty acid
unsaturation and proper membrane composition.
Identification of NHR-80 as a Regulator of Fatty Acid
In our search to identify the desaturases and elongases
involved in generation of unsaturated fatty acids in C. elegans,
we performed a genetic screen to identify mutants with
altered fatty acid profiles . In the process of identifying
the molecular nature of one mutation, we used RNAi against
156 genes at the end of Chromosome III to determine the
fatty acid composition of animals when each of these genes
was inactivated. We found an RNAi clone, nhr-80, that caused
C. elegans to accumulate increased levels of 18:0. NHR-80 is a
member of the NHR family of transcription factors in C.
elegans . To further examine this gene we obtained a
deletion allele from the National BioResource Program for
the Experimental Animal C. elegans, Japan. The nhr-80(tm1011)
mutant carries a 446-bp deletion that eliminates approx-
imately half of the nucleotides in the second exon and all of
the third exon (Figure 1). Like the nhr-80(RNAi) worms, these
mutants also showed an accumulation of 18:0 and reduction
of 18:1 D9 (Figure 2) indicating that nhr-80(tm1011) is likely to
be a loss of function mutation. In the nhr-80 mutants, 18:0
accounts for about 10.2 6 0.3% of the total fatty acids and
18:1 D9 accounts for 2.2 6 0.1%, as compared with 6.8 6
0.2% and 3.2 6 0.1%, respectively, in the wild type. The
difference between these fatty acids in the nhr-80 mutants and
wild-type animals is significant, with p , 0.01 for both fatty
acids. The changes in fatty acid composition shown for the
nhr-80 mutants in Figure 2 are similar to those reported for
the nhr-49 mutants. In those mutants the ratio of 18:0 to 18:1
D9 was 4.3 compared to a ratio of 1.9 in wild-type animals
. In our analysis of the nhr-80 mutants the 18:0 to 18:1 D9
ratio was 4.6 compared to the wild-type ratio of 2.2. The nhr-
80 mutants are viable and fertile indicating this change in
fatty acid composition, though significant, does not affect
essential functions of the animal.
Although two NHR mutant lines, nhr-49 and nhr-80, show
increased 18:0 as compared with wild-type worms, not all
NHR mutants cause these changes in fat metabolism .
Both of these transcription factors are proposed to be
derived from the same ancestral gene that also is the
progenitor of the mammalian gene encoding hepatocyte
nuclear factor 4 , which in mammals, binds to fatty acids
as ligands and is a key activator of lipid and cholesterol
metabolism genes .
Figure 1. Diagram of nhr-80, fat-5, fat-6, and fat-7 Genes and Mutations
(A) nhr-80 is composed of a zinc finger domain (green boxes) and a
ligand-binding domain (light blue boxes). nhr-80(tm1011) contains a 446-
bp deletion (light grey bar).
(B) fat-5, fat-6, and fat-7 all contain four trans-membrane domains (dark
blue boxes) and three histidine boxes (red boxes). fat-5(tm420) consists
of a 779-bp deletion (light grey bar). fat-6(tm331) contains a 1,232-bp
deletion (light grey bar), and a 428-bp insertion (purple bar). The fat-7
alleles are point mutations with fat-7(wa36), creating a premature stop
codon and fat-7(wa37) changing a conserved histidine into a tyrosine.
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Regulation of Desaturation by NHR-80
The ratio of saturated to unsaturated fatty acids has a profound
affect on the fluidity and function of cellular membranes. Animals,
plants, and microorganisms regulate the synthesis of unsaturated
fatty acids during changing environmental conditions, as well as in
response to dietary nutrients. In this paper the authors use a
combination of genetic and biochemical approaches to address the
regulation of unsaturated fatty acid synthesis in the roundworm
Caenorhabditis elegans. They identify a new transcription factor,
NHR-80, that activates the expression of genes encoding delta-9
fatty acid desaturases, the enzymes responsible for catalyzing the
insertion of double bonds into saturated fatty acid chains. These
unsaturated fatty acids are critical components of membranes, as
well as fat storage molecules. Experiments presented here
demonstrate that the worms require adequate synthesis of
unsaturated fatty acids for survival and that they maintain intricate
regulation of the three delta-9 desaturase genes in response to
different nutrients. Abnormalities in lipid metabolism lead to obesity
and diabetes in humans; this study contributes to our under-
standing of the regulation of this metabolic pathway.
In addition to the change in fatty acid composition, the nhr-
49 mutants display an increase in fat storage based on
staining of whole worms with the lipophilic dye Nile red .
In the nhr-80 mutants, we observed no increase in Nile red
staining as compared to wild type (unpublished data)
indicating no increase in fat storage. To confirm this, we
tested fat storage in the nhr-80 mutants by measuring the
percent triglycerides in the total lipids. In the nhr-80 mutants
triglycerides comprised 44 6 1% of the total lipids as
compared to 45 6 1% in wild type signifying no increase in
fat storage. Thus the increased 18:0 accumulation and
increased 18:0 to 18:1 D9 ratio does not cause increased
triglyceride synthesis. However, the altered fatty acid profile
of nhr-80 mutants indicates a role for NHR-80 in the
regulation of fatty acid metabolism in C. elegans.
NHR-80 Is Required for Normal Expression of D9
As NHR-80 is a transcription factor expressed in the
intestine , the major site of fat metabolism in C. elegans,
the increased 18:0 accumulation in the nhr-80 mutants may be
due to a reduced expression of the D9 desaturase genes. To
test this we used quantitative RT-PCR (QPCR) to measure
gene expression with primers designed to amplify fat-5, fat-6,
and fat-7, along with the control genes tbb-2 (b-tubulin) and
ubc-2 (ubiquitin-conjugating enzyme, E2). Relative expression
of these genes was examined in wild-type and nhr-80 mutant
adult populations and we found that expression of all three
D9 desaturases was decreased in the nhr-80 mutants relative to
wild type for eight experimental replicates (Figure 3). On
average, fat-5 and fat-6 expression were reduced by 66% and
22% respectively, while fat-7 expression was almost com-
pletely eliminated in the nhr-80 mutants.
To determine if the expression pattern of nhr-80 over-
lapped with the expression pattern of the D9 desaturases we
created two green fluorescent protein (GFP)-fusion express-
ing lines for each of the D9 desaturase genes. Like nhr-80, all
three D9 desaturase genes were expressed in the intestine in
adult worms (Figure 3B), and in all four larval stages
(unpublished data). The fat-5 promoter::GFP expressing lines
showed additional expression in the pharynx and tail cells
after hatching and throughout the lifespan. The fat-6 whole
gene::GFP expressing lines displayed additional expression in
the hypodermis in all life stages. The overlapping intestinal
expression for all three D9 desaturase genes indicates possible
functional redundancy. The potential role for fat-5 in the
pharynx and fat-6 in the hypodermis remain to be deter-
mined; however, the constitutive expression of these genes in
the intestine is consistent with a central role for D9
desaturation in normal C. elegans function.
To confirm the regulation of the D9 desaturases by NHR-
80, lines expressing the GFP fusions were grown on nhr-
80(RNAi) bacteria. Transformed adults were allowed to lay
eggs on nhr-80(RNAi) and control bacteria. The adults were
removed and about 20 of the progeny were examined for GFP
expression after 4 d of growth. Representative samples are
shown in Figure 3B. Expression of fat-7 whole gene::GFP was
completely eliminated by the RNAi treatment. Expression of
fat-5 promoter::GFP was decreased but only in the intestine, not
in the pharynx. Expression of fat-6 whole gene::GFP was also
slightly decreased. The reduction of fat-5 and fat-6 expression
and the elimination of fat-7 expression likely accounts for the
changes in fatty acid composition observed in the nhr-80
mutant. Similar to the nhr-80 mutants, the nhr-49 mutants
exhibited an increased level of 18:0 accumulation and a
decrease in expression of the D9 fatty acid desaturase genes
by QPCR with fat-5 and fat-7 as the most reduced .
However, the nhr-49 mutants have increased fat storage,
which is not seen in the nhr-80 mutants, and show decreased
expression of two genes that encode proteins that participate
in the mitochondrial b-oxidation pathway, an enoyl-CoA
Figure 2. Fatty Acid Composition of nhr-80
Relative abundance of selected fatty acid species expressed as
percentage of total fatty acid as determined by gas chromatography
analysis. The nhr-80 mutants have significantly higher levels of 18:0 and
lower levels of 16:0 and 18:1 D9 than wild type. Error bars represent the
standard error. *significant differences between wild type and nhr-80
mutant, p , 0.01.
Figure 3. Expression of the D9 Desaturase Genes in nhr-80
(A) Gene expression by QPCR in the nhr-80 mutant reveals a decrease in
expression of the D9 desaturase genes relative to wild type. Error bars
represent standard error.
(B) Transformed lines expressing D9 desaturase gene GFP fusions grown
to adulthood on empty vector control bacteria or nhr-80(RNAi) bacteria.
Exposure times for photographs were adjusted due to different GFP
expression in the three genes, although the exposure time for the two
treatments was kept the same for each genotype. The exposure time for
the fat-5::GFP worms was 1/4 s, for the fat-6::GFP worms was 1/30 s, and
for the fat-7::GFP worms was 1/8 s. After 4 d, there is a dramatic
reduction in D9 desaturase gene expression in the intestine for fat-5::GFP
and fat-7::GFP lines grown on nhr-80(RNAi).
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Regulation of Desaturation by NHR-80
hydratase gene (C29F3.1, ech-1) and an acyl-CoA synthetase
gene (F28F8.2, acs-2). We tested the expression of ech-1 and
acs-2 in the nhr-80 mutants by QPCR and found that there was
no change in expression levels relative to wild-type expres-
sion. This is consistent with the normal level of fat storage
seen in the nhr-80 mutants. Though both NHR-49 and NHR-
80 are required for D9 desaturase expression, their effects on
fatty acid metabolism in C. elegans are not identical; NHR-49
appears to regulate a wider range of lipid homeostasis
nhr-80 Mutants Do Not Die Early like nhr-49 Mutants
It has been suggested that shifts in the ratio of saturated
fatty acids to MUFAs in C. elegans may lead to a decreased
lifespan. For example, the change in the ratio of 18:0 to 18:1
D9 from 1.9 in wild type to 4.3 in nhr-49 mutants has been
proposed to cause a substantial reduction in lifespan from
15–18 d in wild type, to 6–8 d in nhr-49 mutants . We
examined the lifespan of the nhr-80 mutants (Figure 4) and
found that they may have slightly shorter lifespans than wild
type but live considerably longer than nhr-49 mutants despite
having a similar fatty acid composition. In this experiment,
the average lifespan of the nhr-80 mutant was 12.5 6 0.5 d as
compared to 13.9 6 0.4 d in wild-type animals and 8.2 6 0.2 d
in nhr-49 mutants when grown at 25 8C. These data indicate a
10% decrease in mean lifespan between wild type and nhr-80
mutants, the difference between wild type and nhr-49 mutants
is much greater with a 41% reduction in mean lifespan. The
early death of the nhr-49 does not seem to be caused solely by
an elimination of fat-7 expression or an increase in the ratio
of 18:0 to 18:1 D9 since nhr-80 mutants also show these
characteristics but do not have a dramatically shortened
lifespan. It is possible that the shorter lifespan of the nhr-49
mutants is caused by metabolic changes due to other targets
of NHR-49 regulation.
C. elegans D9 Desaturases Are Redundant under Standard
Previous studies revealed that the three C. elegans D9
desaturase isozymes display different substrate specificities.
While FAT-6 and FAT-7 preferentially desaturate stearic acid
(18:0), similar to most of the characterized SCDs, FAT-5
prefers palmitic acid (16:0) and has little or no activity on
stearic acid . We obtained D9 desaturase single mutants to
further characterize the roles of these three desaturases. We
obtained fat-5(tm420) and fat-6(tm331) deletion alleles from the
National BioResource Program for the Experimental Animal
C. elegans, Japan (Figure 1B). The fat-5 allele has a 779-bp
deletion early in the coding sequence that eliminates two of
the conserved histidine boxes and two of the trans-membrane
domains. The fat-6 allele has a 1,232-bp deletion and a 428-bp
insertion. The deletion is early in the coding sequence and
also eliminates twoof the conserved histidine-rich regions and
two trans-membrane domains. Both of these mutations are
likely null. The fat-7(tm326) deletion allele is available but
molecular analysis of this allele led us to believe that a more
extensive genetic disruption had occurred that affects other
genes in addition to fat-7. Alternative fat-7 alleles were isolated
by TILLING (Targeting Induced Local Lesions IN Genomes)
 and are single base pair changes (Figure 1B). The fat-
7(wa36) allele is a C to T mutation that leads to a premature
stop codon that eliminates two trans-membrane domains and
one of the conserved histidine boxes required for activity of
the rat SCD enzyme , indicating that this allele is, at a
minimum, a strong reduction-of-function allele. The fat-
7(wa37) allele is a C to T mutation that replaces a conserved
histidine with tyrosine . Because these histidines are
expected to be required for D9 desaturase activity we
expressed this allele in mutant yeast that lack D9 desaturase
activity (ole1 mutants). The mutant fat-7(wa37) did not support
growth of the ole1 mutant yeast, whereas expression of wild-
type fat-7 did allow growth . Phenotypic characterization
including fatty acid composition and lifespan with fat-7(wa37)
showed no difference from fat-7(wa36) therefore only data
from fat-7(wa36) are reported here.
The C. elegans D9 desaturase mutants show subtle differ-
ences from wild type in their fatty acid profile when grown on
an Escherichia coli lawn on NGM plates at 20 8C (Figure 5).
Compared to wild type (4.1 6 0.2%), the fat-5 mutants display
decreased 16:1 D9 (3.4 6 0.1%), which is the product of FAT-
5 desaturation based on the substrate specificity exhibited in
yeast . The fat-6 mutants exhibit a significant increase in
their accumulation of the predicted substrate of FAT-6, 18:0
(9.6 6 0.2%), over wild type (7.0 6 0.2%).
Figure 4. Aging of Adult Mutant Populations
(A) Life span of nhr-49, nhr-80, and wild type at 25 8C. The nhr-80 and wild
type display a considerably longer life than the nhr-49 mutants. All
lifespan data are presented as mean lifespan 6 standard error (total
number of animals scored). Wild type: 13.9 6 0.4 (83); nhr-49: 8.2 6 0.2
(80); nhr-80: 12.5 6 0.5 (70).
(B) Life span of fat-5, fat-6, fat-7, and wild type at 25 8C. The fat-5, fat-6,
and fat-7 mutants exhibit a lifespan similar to wild type. Wild type: 13.9 6
0.4 (83); fat-5: 15.9 6 0.6 (82); fat-6: 14.2 6 0.5 (82); fat-7: 15.0 6 0.5 (75).
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Regulation of Desaturation by NHR-80
The D9 desaturase mutants are indistinguishable from wild
type in other characteristics tested including growth rate,
reproduction, and behavior. The lack of phenotype indicates
that subtle changes in fatty acid composition have no
apparent effect and that the desaturases are functionally
redundant. To determine if gene expression changes are
involved in compensating for the lack of one isozyme, we
examined expression of the D9 desaturase genes in the fat-5,
fat-6, and fat-7 mutants (Figure 6). In the fat-6 mutants, fat-7
expression is increased approximately 4-fold over wild type
and fat-5 expression is increased 2–3-fold over wild type. In
the fat-7 mutant, expression of fat-6 and fat-5 is also slightly
increased over wild type. The fat-5 mutant shows little
difference from wild type in fat-6 and fat-7 expression.
Axenic Growth Reveals Substrate Specificity of the D9
The standard strain of E. coli on which C. elegans are
maintained in the laboratory contains palmitic (16:0),
palmitoleic (16:1 D9), and vaccenic (18:1 D11), but not oleic
acid (18:1 D9) or polyunsaturated fatty acids . When
worms eat these bacteria they incorporate the fatty acids in
their lipids. To test the fatty acid composition of the D9
desaturase mutants grown on a different food source we grew
the C. elegans strains in axenic media devoid of bacteria. This
liquid media provides amino acids, vitamins, growth factors,
and heme . Our measurements reveal that the axenic
Figure 5. Fatty Acid Composition of the D9 Desaturase Mutants
(A?C) There is little change in fatty acid composition for fat-5 (A), fat-6 (B), and fat-7 (C) mutants compared to wild-type worms when grown under
standard growth conditions with OP50 E. coli as the sole food source.
(D?F) Axenic growth conditions for wild-type worms and fat-5 (D), fat-6 (E), and fat-7 (F) mutants reveal major changes in fatty acid composition for fat-5
and fat-6 mutants compared to wild-type worms. In all graphs relative abundance of selected fatty acid species is expressed as percentage of total fatty
acid as determined by gas chromatography analysis. Error bars represent the standard error. *significant difference from wild type, p , 0.01
Figure 6. Expression of the D9 Desaturase Genes in the fat-5, fat-6, and
Gene expression by QPCR in the fat-5, fat-6, and fat-7 mutants relative to
wild type reveals an increase in D9 desaturase gene expression in the fat-
6 and fat-7 mutants, relative to wild type. Error bars represent standard
error of 7–12 experiments.
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Regulation of Desaturation by NHR-80
media contains palmitic, palmitoleic, oleic and linoleic acids,
but no vaccenic acid (unpublished data). Wild-type worms
grow considerably more slowly under the axenic growth
conditions, and the fatty acid profile is also dramatically
different. In axenic culture, wild-type worms accumulate
higher levels of 16:0, 18:0, and 18:1 D9, while they produce
lesser amounts of 20:5 (Figure 5A and 5D).
The D9 desaturase mutants show greater differences in
fatty acid composition when grown axenically than when
grown on E. coli plates (Figure 5D–5F). Comparing the fatty
acid composition of the fat-5 mutant with wild type we
observe an increase in 16:0 (19 6 1% versus 12 6 1%) and a
decrease in 16:1 D9 (1.1 6 0.4% versus 3.0 6 0.3%) and 18:1
D11 (3.8 6 ?0.1% versus 17 6 1%) in the fat-5 mutants. The
fat-6 mutants also display dramatic differences from wild
type, with an increase in 18:0 (16.7 6 0.8% versus 10.9 6
0.7%) and a decrease in 18:1 D9 (11.3 6 0.6% versus 21.8 6
0.5%) in the fat-6 mutants. The fatty acid composition of fat-7
mutants does not differ significantly from wild type, indicat-
ing that fat-6 can completely compensate for fat-7 in axenic
culture and therefore that FAT-7 does not play an important
role in maintaining proper fatty acid composition under
axenic conditions. The dramatic reduction of 16:1 D9 and
18:1 D11 fatty acids in fat-5 mutants and 18:1D9 in fat-6
mutants grown in axenic culture is the first evidence that
these enzymes have the same substrate specificity in C. elegans
as they do when expressed in yeast .
To determine whether the levels of D9 desaturase gene
expression are modulated in response to diet we examined
the expression of fat-5, fat-6, and fat-7 genes in axenic media
and on E. coli seeded plates using QPCR. We found that
compared to worms grown on E. coli, fat-5 expression
increases about 6-fold in axenic media. In contrast, fat-6
expression is maintained at similar levels while fat-7 expres-
sion is dramatically decreased in axenic media (Figure S1).
Single D9 Desaturase Mutants Have No Early-Death
Previous studies investigating the C. elegans D9 desaturases
have used RNAi to deplete fat-7 expression and have
suggested that fat-7 expression is required to maintain a
normal lifespan [23,24]. Based on these results, it was
proposed that the reduced expression of fat-7 was the cause
of the short lifespan in the nhr-49 mutants . However, the
fat-7mutants used in our experiment as well as the other D9
desaturase mutants, fat-5 and fat-6, do not exhibit an early
death phenotype (Figure 4B). The average lifespan of the fat-5
mutants is 15.8 6 0.6 d, the fat-6 mutant is 14.2 6 0.5 d, and
the fat-7 mutant is 15.0 6 0.5 d, as compared with a lifespan
of 13.9 6 0.4 d in wild-type animals. In this experiment, the
fat-5 mutant displayed a slight but significant (p , 0.01)
increase in lifespan over wild type, while the fat-6 and fat-7
mutants were not significantly different from wild type in
Our experiments with the fat-7 mutant do not support the
requirement for fat-7 for normal lifespan as proposed from
studies using fat-7(RNAi) [23,24]. Additionally, fat-7(RNAi)
revealed major changes in fatty acid composition and a
reduction of fat storage  that was not observed in the fat-7
mutants. The RNAi phenotype observed could be due to
transitive secondary RNAi effect  as fat-7 has 84%
nucleotide identity with fat-6 including eight regions of 21–
44 nucleotides with 100% identity. Van Gilst et al. report that
fat-7(RNAi) did not reduce fat-6 expression when measured by
QPCR ; however, we observe an elimination of fat-6
expression when fat-6 whole gene::GFP lines were grown on fat-
7(RNAi) (unpublished data). In addition, it is possible that
compensation by the third D9 desaturase, fat-5, is inhibited in
the fat-7(RNAi). Because the fat-7 loss-of-function mutant is
wild type for fatty acid composition and lifespan it must be
concluded that fat-7(RNAi) is having off-target effects on the
D9 Desaturase Activity and Monounsaturated Fatty Acids
Are Required for Survival
Because the D9 desaturase genes appear to compensate for
each other, we constructed a fat-5;fat-6;fat-7 triple mutant
lacking all three D9 desaturases. We expected these mutants
would be unable to survive under standard growth con-
ditions, so we supplemented the worms with a combination of
18:1 D9, 18:2 x6, and 20:5 x3 dietary fatty acids. After
identifying the fat-5;fat-6;fat-7 triple mutant, we moved the
worms to plates without fatty acid supplementation and
found that indeed these worms could not survive. Larvae that
hatch from eggs laid on unsupplemented plates arrest in the
L1 stage, while L3 and L4 stage larvae that are moved from
supplemented to unsupplemented plates develop into thin,
sterile adults with reduced movement and early death. The
MUFAs provided by the standard E. coli diet are not sufficient
for survival in the fat-5;fat-6;fat-7 triple mutant. Thus C. elegans
have a requirement for a certain level of D9 desaturation that
cannot be met by the standard E. coli diet. The yeast D9
desaturase mutant, ole1, is also unable to grow without
supplementation . The fat-5;fat-6;fat-7 triple mutant is the
first multicellular organism generated that lacks all endoge-
nous D9 desaturase activity.
To examine genetic interaction between nhr-80 and fat-6,
the most highly expressed D9 desaturase, we constructed the
fat-6;nhr-80 double mutant using plates supplemented with
dietary fatty acids. When we removed the fat-6;nhr-80 double
mutants to unsupplemented plates we found that these
worms also did not survive. Since the nhr-80(RNAi) phenotype
resembles the nhr-80 mutants, we used RNAi in combination
with the D9 desaturase mutants to study this interaction
further. The fat-6 mutants, when grown on nhr-80(RNAi) from
eggs, become thin, slow growing, and reproductively inviable
after 4 d of growth (Figure 7). They also accumulate very high
levels of 18:0 (Figure 7B). The fat-6 mutants grown on nhr-
80(RNAi) accumulate 34 6 1% of their fatty acids as 18:0 as
compared to 9.1 6 0.1% when fat-6 is grown on control
bacteria or 14 6 2% when wild-type worms are grown on nhr-
80(RNAi) bacteria. Although 18:0 also accumulates in the fat-5
and fat-7 mutants grown on nhr-80(RNAi), the extent of 18:0
accumulation is not as dramatic as observed in fat-6 (Figure
7B) and they do not show a synthetic lethality (Figure 7A).
One explanation for the synthetic lethality of fat-6;nhr-80
double mutants is that NHR-80 is required for the increased
fat-5 and fat-7 expression in the fat-6 mutant. To test this we
examined the expression of the D9 desaturase genes in the fat-
5, fat-6, and fat-7 mutants grown on nhr-80(RNAi). We found
that expression of fat-7 in the fat-6;nhr-80(RNAi) is less than
10% of the expression of fat-7 in the fat-6 mutants grown on
control bacteria, consistent with the notion that NHR-80 is
required to induce the expression of fat-7 (Figure 7C). We
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Regulation of Desaturation by NHR-80
graphed the relative expression values, setting fat-6 expres-
sion in wild-type worms grown on control bacteria as 100%.
In wild-type worms on control bacteria fat-6 is the most highly
expressed D9 desaturase gene and fat-5 and fat-7 are
expressed at 3.6 6 0.2% and 6.5 6 0.6% of the level of fat-6
respectively. When the wild-type worms are grown on nhr-
80(RNAi) we observe a similar relative decrease in D9
desaturase gene expression seen in the nhr-80 mutants (Figure
3A). Comparing the fat-5 and fat-7 mutants grown on control
with those grown on nhr-80(RNAi) reveals a decrease in D9
desaturase gene expression. However, the biggest difference
is seen in the fat-6 mutants. When these animals are grown on
control bacteria fat-7 is increased in expression 37-fold over
wild type. When fat-6 is grown on nhr-80(RNAi) the fat-7
relative expression is only a 3-fold increase over wild type.
The overall amount of D9 desaturase gene expression is
approximately equal for all worms grown on nhr-80(RNAi),
but only fat-6 displays synthetic lethality with nhr-80. This
could be due to the composition of the D9 desaturase gene
expression. When wild type, fat-5, or fat-7 are grown on nhr-
80(RNAi) fat-6 is the major gene expressed suggesting its
central importance for D9 desaturation activity. The fat-6
mutants lack fat-6 expression and compensate by substantially
increasing fat-7 expression when grown on control bacteria. It
is noteworthy that under these conditions fat-7 expression is
increased 37-fold, perhaps indicating that fat-7 is not as
effective at D9 desaturation as fat-6 due to differences in
tissue specific expression, translation efficiency or protein
stability. When the fat-6 mutants are grown on nhr-80(RNAi)
they are unable to compensate with an increase in fat-7
expression to an appropriate level and this may cause their
reduced survival. Thus NHR-80 is required for increasing fat-
7 expression in situations where higher fat-7 levels are
necessary and consequently defines a critical regulator of
fatty acid metabolism.
Our characterization of the novel NHR-80 and the family
of C. elegans D9 desaturase mutants enhances our under-
standing of the regulation of lipid homeostasis. Maintaining
appropriate fatty acid composition is essential and without
sufficient D9 desaturase activity both the fat-5;fat-6;fat-7 triple
mutants and the fat-6;nhr-80 double mutants are unable to
survive. The integration of endogenous and environmental
signals by NHRs such as NHR-80 precisely regulates the
expression of the D9 desaturase genes and the production of
monounsaturated fatty acids leads to optimal membrane
fluidity and fat storage.
Materials and Methods
Culture of nematodes. Unless otherwise noted, C. elegans were
grown on nematode growth media (NGM) plates with OP50 strain of
E. coli as a food source . The wild-type strain used is strain N2.
Mutant strains obtained from Shohei Mitani and Edwin Cuppen were
outcrossed at least four times to the N2 strain. The nhr-80(RNAi)
construct, as well as the others used in the screen of Chromosome III,
are from the Ahringer RNAi library  and were used as described
. As a control for RNAi experiments, nematodes were grown on
NGM plates with the HT115 strain of E. coli transformed with
pPD129.36 (L4440) empty vector plasmid. The axenic culture media
consisted of 3% soy peptone, 3% yeast extract, 0.5 mg/ml hemoglobin
in 1M KOH, and 20% ultra-high temperature pasteurized skim milk
. Worms were grown in this liquid culture at room temperature
(22–23 8C) with constant shaking. To make plates supplemented with
dietary fatty acids a 0.1 M stock solution of fatty acid sodium salts
(NuCheck Prep, Elysian, Minnesota, United States) in water was
prepared fresh for each supplementation experiment. The fatty acid
stock was added slowly to NGM containing 0.1% tergitol. Plates were
poured, covered and allowed to dry in the dark at room temperature
overnight. The OP50 strain of E. coli was added to each plate and
allowed to dry for at least one night .
Fatty acid and lipid analysis. For fatty acid analysis, adult
nematodes were washed from plates and allowed to settle. The excess
water was removed from the worm pellet and 1 ml of 2.5%
methanolic H2SO4was added and incubated at 80 8C for 1 h to
generate fatty acid methyl esters, which were extracted by adding 1.5
ml water and 0.2 ml hexane. The hexane was sampled for
determination of fatty acid composition by gas chromatography on
an SP-2380 fused silica capillary column (Supelco, Bellefonte,
Pennsylvania, United States) using an Agilent (Palo Alto, California,
United States) 6890 series gas chromatograph .
For lipid analysis, about 0.5 ml of adult nematodes were collected
in a glass tube and frozen. Lipids were extracted by incubation in (1:1)
Figure 7. Effects of nhr-80(RNAi) in the D9 Desaturase Mutant
(A) Photographs showing adult worms after 4 d of growth on nhr-
80(RNAi) and empty vector control bacteria. The fat-6 mutants grown on
nhr-80(RNAi) are thin, pale, and produce no viable progeny.
(B) Relative abundance of 18:0 expressed as a percentage of total fatty
acid as determined by gas chromatography analysis. The fat-6 mutants
grown on nhr-80(RNAi) (n¼5) accumulate much higher levels than fat-6
mutants grown on control (n ¼ 7) and wild type grown on nhr-80(RNAi)
(n¼6). *significant differences from growth on control bacteria, p , 0.01.
(C) Effects of nhr-80 on D9 desaturase gene expression in fat-5, fat-6, and
fat-7 mutants. QPCR in fat-5, fat-6, fat-7, and wild type for worms grown
on empty vector control bacteria (ev) and nhr-80(RNAi) (80i) (n ¼ 6).
Values are expressed relative to fat-6 expression in wild-type worms
grown on control bacteria. For all graphs error bars represent standard
PLoS Genetics | www.plosgenetics.org July 2006 | Volume 2 | Issue 7 | e1081003
Regulation of Desaturation by NHR-80
chloroform/methanol overnight at ?20 8C. The samples were washed
with 2.2 ml Hajra’s solution (0.2M H3PO4, 1M KCl) and the
chloroform phase containing the lipids was isolated. The silica gel
HL plates (Analtech, Newark, Delaware, United States) were activated
by incubation at 110 8C for 1 h and 15 min. The samples were loaded
onto the thin layer chromatography plates along with lipid standards
(Sigma, St. Louis, Missouri, United States). The plates were run with a
65:43:3:2.5 chloroform/methanol/water/acetic acid solvent mixture
until the solvent front was three-fourths of the way up the plate. The
plate was dried, a new solvent mixture of 80:20:2 hexane/diethyl ether/
acetic acid was added, and the plate was run until the solvent front
reached the top of the plate. The marker lanes were visualized using
iodine vapor and the corresponding bands for triglycerides and
individual phospholipids in the silica gel were scraped into individual
tubes. To quantitate, 50 lg of 15:0 free fatty acid was added to each
tube as an internal standard and fatty acid analysis was performed by
gas chromatography as described above .
QPCR analysis. Adult nematodes were harvested and frozen in
liquid nitrogen. RNA was prepared using TRIzol Reagent (Invitrogen,
Carlsbad, California, United States). A DNA-FREE RNA kit (Zymo
Research, Orange, California, United States) was used for Dnase
treatment and purification. After quantification, 1 lg of RNA was
used in a reverse-transcription reaction with SuperScriptIII (Invi-
trogen) to generate cDNA. Primer sequences for the D9 desaturase
genes and the reference genes were designed using PrimerQuest
software at http://www.idtdna.com. Other primer sequences were
obtained from Dr. Marc Van Gilst . Primer sequences are listed in
Table S1. The PCR mixture consisted of 0.3 lM primers, cDNA, ROX,
and 13 SYBR green mix (Invitrogen Platinum SYBR green qPCR
Supermix UDG). The QPCR was run and monitored on a MX3000P
(Stratagene, La Jolla, California, United States). Relative abundance
was determined using the DDCt method and an average of the
expression of the reference genes tbb-2 and ubc-2 to control for
template levels .
Construction of GFP fusions and microinjection. Fusion PCR was
used to create translational fat-5, fat-6, and fat-7 GFP constructs. The
promoters and coding sequences of fat-6 and fat-7 and the promoter
and first exon of fat-5 were amplified from genomic DNA. The
upstream regulatory region for fat-5 was 4 kb, for fat-6 was 2.6 kb, and
for fat-7 was 3.0 kb. GFP was amplified from the Fire vector pPD95.75
including the entire coding sequence and a termination sequence.
These PCR products were fused together in a final PCR using nested
primers . These fusions were microinjected into lin-15 mutant C.
elegans along with a rescuing plasmid, pJM23, containing the wild-type
lin-15 gene [39,40]. Multiple independent lines of nematodes without
the lin-15 phenotype were selected and examined for GFP expression
using fluorescence microscopy on an Olympus IX70 microscope.
Lifespan analysis. Aging experiments were performed on adult
nematodes grown at 25 8C. Worms were moved to plates containing
5-fluoro-29-deoxyuridine (Sigma) at the fourth larval stage of
development (L4). Live animals were assayed for movement in
response to touch every 1–2 d .
Generation of fat-5;fat-6;fat-7 triple mutants and fat-6;nhr-80 double
mutants. The fat-6(tm331);fat-7(wa36) hermaphrodites were crossed
with fat-5(tm420);fat-7(wa36) males on plates supplemented with 18:1
D9. The F1 generation was moved to new 18:1 D9 supplemented
plates and their progeny were moved to plates supplemented with a
combination of 18:1 D9, 18:2 x6, and 20:5 x3. After the F2 generation
reproduced, the adults were harvested for single worm PCR to
determine the genotype . The fat-5 and fat-6 mutations were
monitored using the difference in amplicon size between wild-type
and mutant alleles due to the large deletions. The wild-type products
were 1,100 bp for fat-5 and 1,457 bp, for fat-6 compared with the
mutant products of 321 bp and 652 bp, respectively. All cross-
progeny were homozygous for the fat-7 single base pair mutation.
To generate nhr-80;fat-6 double mutants we crossed fat-6 males with
nhr-80 hermaphrodites on 18:1 D9 supplemented plates and isolated
the F1 generation onto new supplemented plates. The F2s were
moved to fresh 18:1 D9 supplemented plates and allowed to
reproduce then single worm PCR was used to identify nhr-80;fat-6
double mutants. The nhr-80 wild-type allele generated a PCR product
of 745 bp, whereas the nhr-80(tm1011) mutant allele generated a
product 298 bp in length.
Figure S1. Expression of D9 Desaturase Genes in Wild-Type Worms
Grown on E. coli (OP50) Seeded Plates or Axenic Liquid Media
The percent expression shown is relative to fat-6 expression on E. coli
plates, which is set at 100%. In wild-type worms grown in axenic
culture the expression of fat-5 is increased and the fat-7 expression is
nearly eliminated relative to expression in wild-type worms grown on
E. coli (OP50) plates. Relative to fat-6 expression, fat-5 and fat-7
expression is higher in wild-type worms grown on E. coli (OP50)
compared to wild-type worms grown on E. coli (HT115) (Figure 7C).
Error bars are SEM, n ¼ 3 replicates for plate grown and n ¼ 6
replicates for axenic cultured nematodes.
Found at DOI: 10.1371/journal.pgen.0020108.sg001 (56 KB TIF).
Table S1. Sequence of DNA Primers Used in These Studies
Found at DOI: 10.1371/journal.pgen.0020108.st001 (34 KB DOC).
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession num-
bers for genes used in this study are nhr-80 (H10E21.3) (AY204179),
fat-5 (W06D12.3) (AF260242), fat-6 (VZK822L.1) (AF260244), and fat-7
We thank Dr. Shohei Mitani (Tokyo Women’s Medical University,
Japan) and the National BioResource Project for the Nematode
(Japan) for providing the nhr-80(tm1011), fat-5(tm420), fat-6(tm331), and
fat-7(tm326) mutants. We are also grateful to Dr. Edwin Cuppen
(Hubrecht Laboratory, The Netherlands) who screened his TILLING
library for the fat-7(wa36 and wa37) mutants. We thank Dr. Andy Fire
(Stanford University, California) for vectors and Dr. Marc Van Gilst
(Fred Hutchinson Cancer Research Center, Washington) for the
primer sequences for the metabolism genes used in QPCR and for
comments on the manuscript.
Author contributions. TJB, JB, and JLW conceived and designed
the experiments. TB performed the experiments and analyzed the
data. TJB, JB, and JLW wrote the paper.
Funding. This work was supported by the National Institutes of
Health grant R01-DK074114 and the Agricultural Research Center,
Washington State University. Additional funding for TJB was
provided by the National Institutes of Health Biotechnology Training
Program at WSU.
Competing interests. The authors have declared that no competing
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