The Caenorhabditis elegans Synthetic Multivulva Genes
Prevent Ras Pathway Activation by Tightly Repressing
Global Ectopic Expression of lin-3 EGF
Adam M. Saffer1,2, Dong Hyun Kim , Alexander van Oudenaarden
31,3, H. Robert Horvitz1,2*
1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2Howard Hughes Medical Institute, Massachusetts
Institute of Technology, Cambridge, Massachusetts, United States of America, 3Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts,
United States of America
The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway
to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene,
establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive
activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA
molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals,
including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal.
Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than
that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest
hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B
synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the
class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in
preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by
preventing broad ectopic expression of EGF-like ligands.
Citation: Saffer AM, Kim D , van Oudenaarden A, Horvitz HR (2011) The Caenorhabditis elegans Synthetic Multivulva Genes Prevent Ras Pathway Activation by
Tightly Repressing Global Ectopic Expression of lin-3 EGF. PLoS Genet 7(12): e1002418. doi:10.1371/journal.pgen.1002418
Editor: Andrew D. Chisholm, University of California San Diego, United States of America
Received June 29, 2011; Accepted October 22, 2011; Published December 29, 2011
Copyright: ? 2011 Saffer 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.
Funding: HRH is the David H. Koch Professor of Biology at MIT and an Investigator of the Howard Hughes Medical Institute. This work was supported by NIH
grant GM24663. AvO was supported by an NIH Pioneer Award (1DP1OD003936). Some nematode strains used in this work were provided by the Caenorhabditis
Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Signaling by epidermal growth factor (EGF) family ligands and
EGF receptor (EGFR) family tyrosine kinases controls many
aspects of mammalian development and can drive cancers:
EGFRs are commonly overexpressed or constitutively activated
by mutations in tumor cells , and EGF-family ligands can be
misregulated in cancer. For example, the EGF-family ligands
heparin-binding EGF-like growth factor, amphiregulin, and TGF-
a are upregulated in cancer cells from many different cancer types
[2,3], and TGF-a overexpression causes widespread epithelial
hyperplasia in mice [4,5]. Growth factors often signal through a
Ras pathway, and approximately 20% of tumors carry a
constitutively active Ras mutation .
In the nematode Caenorhabditis elegans the EGF-family ligand
LIN-3 acts through the EGFR LET-23 and the Ras protein LET-
60 to control many aspects of development, including the
induction of the hermaphrodite vulva [7–10]. In wild-type
animals, of a set of six equipotent cells, three (P5.p, P6.p and
P7.p) adopt vulval cell fates, while the other three (P3.4, P4.p, and
P8.p) adopt non-vulval fates . The expression of vulval cell
fates requires EGF/Ras signaling, and mutations that reduce
EGF/Ras signaling cause a vulvaless (Vul) phenotype in which
none of the six cells adopts vulval cell fates [7–10]. The anchor
cell, located closest to P6.p, is the only cell that both expresses
LIN-3 EGF and is located near the six Pn.p cells , and laser
ablation of the anchor cell results in a Vul phenotype  like that
seen in mutants defective in lin-3 EGF or let-23 EGFR.
Overactivation of the EGF/Ras pathway, by overexpression of
lin-3 EGF or by an activating mutation in either let-23 EGFR or
let-60 Ras, causes a multivulva (Muv) phenotype in which all six
Pn.p cells adopt vulval cell fates [8–10,13].
In vulval development, EGF/Ras signaling is antagonized by
the synthetic multivulva (synMuv) genes. The synMuv genes define
two classes, A and B [14,15]. In synMuv single mutants or in class
A double mutants or class B double mutants, vulval development is
mostly normal. By contrast, animals mutant in both a class A
synMuv gene and a class B synMuv gene exhibit a strong Muv
phenotype. Many class B synMuv genes have homologs that
function in histone modification, chromatin remodeling, and
transcriptional repression. For example, the class B synMuv genes
encode a DP/E2F/Rb complex [16,17], a nucleosome remodeling
and deacteylase (NuRD) complex [18,19], two histone methyl-
transferases [20,21] and a heterochromatin protein 1 homolog
PLoS Genetics | www.plosgenetics.org1 December 2011 | Volume 7 | Issue 12 | e1002418
. Of the three molecularly-characterized class A synMuv
genes, two encode proteins with a zinc-finger-like THAP domain
[23–25]. The expression patterns of three class A synMuv proteins
have been studied, and all three are localized to the nucleus,
suggesting that class A synMuv proteins regulate transcription
The synMuv genes function at least in part by repressing
expression of lin-3 EGF. Loss-of-function mutations in either let-23
EGFR or lin-3 EGF can suppress the synMuv phenotype
[16,27,28], indicating that the synMuv genes act upstream of or
in parallel to lin-3. Furthermore, lin-3 mRNA levels are increased
in synMuv double mutants but not in synMuv single mutants ,
and overexpression of lin-3 EGF causes a Muv phenotype .
Laser ablation of the anchor cell, the source of LIN-3 in wild-
type vulval development, does not fully suppress the Muv
phenotype of synMuv double mutants , indicating that
synMuv genes cannot simply prevent overexpression of lin-3 from
the anchor cell. Mosaic analyses of the class B synMuv gene lin-37
and the lin-15 locus, which contains both a class A and a class B
synMuv gene, did not identify a single site of action. Both
experiments indicated that lin-15 and lin-37 do not act cell-
autonomously in the Pn.P cells and suggested that lin-15 and lin-37
might function in the syncytial hypodermal cell hyp7 [29,30].
Heterologous expression experiments showed that the class B
synMuv gene lin-35 functions in hyp7 to antagonize vulval cell
fates, and tissue-specific RNAi of lin-3 in hyp7 can suppress the
synMuv phenotype, indicating that repression of lin-3 in the
hypoderm is an important function of the synMuv genes [27,31].
Another study using the same heterologous promoters found that
the class B synMuv gene hpl-2 functions in both hyp7 and the Pn.p
cells . However, it is not known where lin-3 is overexpressed in
synMuv mutants, how the synMuv genes control lin-3 expression,
or if the synMuv genes control targets other than lin-3 important
for vulval development.
Here we report the identification of a lin-3 EGF promoter
mutation that causes a dominant class A synMuv phenotype. The
effect of this mutation reveals that the only major role of the class
A synMuv genes in vulval development is to repress lin-3. We find
that lin-3 mRNA is ectopically expressed throughout the animal in
synMuv mutants. Our results show that low levels of ectopic lin-3
expression outside the cells that normally produce and respond to
lin-3 can adversely alter the development of C. elegans, and we
propose that the class A and class B synMuv genes might prevent
ectopic lin-3 expression by distinct mechanisms.
n4441 causes a dominant class A synMuv phenotype
During a screen for new class A synMuv mutations, we
identified a Muv animal in the F1 generation after ethyl
methanesulfonate (EMS) mutagenesis of the class B synMuv
mutant lin-52(n771). We named the mutation that caused this
defect n4441. To seek additional mutations that like n4441
dominantly cause a class A synMuv phenotype, we screened
approximately 492,000 F1 progeny of lin-52(n771) animals
mutagenized by EMS and approximately 89,000 progeny of
animals mutagenized by N-ethyl-N-nitrosourea (ENU), but we did
not identify any additional class A synMuv mutants. As a single
mutant, n4441 animals are wild-type at 20uC and exhibit a low
penetrance Muv defect at 25uC (Table 1), comparable to that of
most class A synMuv mutants . Double mutants between
n4441 and the class B synMuv mutations lin-15B(n744), lin-
52(n771), or lin-61(n3447) exhibit a strong synMuv phenotype.
n4441 causes a fully penetrant Muv defect as a heterozygote in the
class B synMuv mutant background lin-15B(n744), indicating that
n4441 dominantly causes a class A synMuv phenotype. n4441
causes a 97% penetrant synMuv defect in the weak class B synMuv
mutant background lin-61(n3447) at 22.5uC, comparable to the
previously reported phenotype of double mutants between lin-
61(n3447) and the strong class A synMuv mutations lin-15A(n767)
or lin-38(n751) .
To determine how n4441 interacts with other class A synMuv
mutations, we built double mutants between n4441 and an allele of
each known class A synMuv gene. We used the putative null alleles
lin-8(n2731), lin-15A(n767), and lin-56(n2728) and the missense
allele lin-38(n751), since a null allele of lin-38 causes lethality
(A.M.S and H.R.H., unpublished results). At 20uC and 25uC, the
double mutants n4441; lin-15A(n767), lin-38(n751); n4441, and lin-
56(n2728); n4441 were enhanced for the Muv phenotype when
compared to their respective single mutants (Table 1). The lin-
8(n2731); n4441 double mutant was roughly comparable to n4441
alone when scored at 25uC and also exhibited a low penetrance
Muv defect at 20uC, which neither n4441 or lin-8(n2731) did on
their own (Table 1). Thus, mutations in all known class A synMuv
genes can enhance the Muv phenotype of n4441, but the
enhancement is much weaker than the enhancement caused by
class B synMuv mutations.
Several members of a Tip60/NuA4 histone acetyltransferase
complex were previously identified as class C synMuv genes
. Class C synMuv genes are strongly Muv in combination
with class A synMuv mutations and weakly Muv in combination
with class B synMuv mutations and can be considered a subset
of the class B synMuv genes . To test if n4441 might be a
class C synMuv gene, we built a double mutant between n4441
and the partial loss-of-function class C synMuv mutation mys-
1(n3681), as null mutants of mys-1 cannot be maintained as
homozygous strains . The mys-1(n3681); n4441 double
mutant exhibited a 56% penetrant Muv defect at 20uC, which
is much stronger than the 5% penetrant Muv defect of the
n4441; lin-15A(n767) strain at 20uC, despite the fact that lin-
15A(n767) is a null mutation. We conclude that n4441 is not a
class C synMuv mutation.
Extracellular signals that drive cells to divide must be
carefully restricted so that only the correct cells receive
those signals. Failure to properly control the expression of
signaling molecules can lead to aberrant development and
cancer. Studies of vulval development in the nematode
Caenorhabditis elegans have helped define various multi-
step signaling pathways involved in cancer. Here we report
that two groups of proteins that control the EGF/Ras/MAP
kinase pathway of vulval development act by tightly
repressing the spatial expression of the gene lin-3, which
encodes an EGF-like signaling molecule. Using a technique
that detects single mRNA molecules, we show that
inactivation of these proteins causes a low ectopic
expression of lin-3 in many cells. In response, the EGF/
Ras/MAP kinase pathway is activated in cells normally not
exposed to the lin-3 signal, and vulval development is
abnormal. This process is analogous to the cancerous
growth that occurs in humans when mutations cause both
tumor cells and the microenvironment surrounding the
tumor cells to ectopically express factors that drive cellular
proliferation. We propose that mammalian genes analo-
gous to those that repress lin-3 expression in C. elegans
vulval development act as tumor suppressors by prevent-
ing broad ectopic expression of EGF-like ligands.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org2 December 2011 | Volume 7 | Issue 12 | e1002418
n4441 is an allele of lin-3
By performing SNP mapping experiments using the CB4856
polymorphic strain of C. elegans, we mapped the n4441 mutation to
a 661 kb region containing approximately 170 genes between
SNPs dbP6 and uCE4-1148 (Figure 1A). n4441 dominantly causes
a synMuv phenotype and thus might well be a gain-of-function
mutation, so we sought loss-of-function mutations in the gene
affected by n4441. n4441/nT1[qIs51]; lin-15B(n744) animals,
which display a fully penetrant Muv defect, were mutagenized
with EMS. The nT1[qIs51] translocation causes inviability when
homozygous and suppresses recombination across an interval that
includes lin-3 . Approximately 6,800 F1 progeny were
screened, and two animals were identified that were non-Muv
and produced only non-Muv progeny, indicating that they
contained a suppressor mutation tightly linked to n4441. We
named these mutations n4929 and n4951. n4441 n4929; lin-
15B(n744) animals were sterile and exhibited a very low
penetrance Muv defect (Figure 1D). n4441 n4951/nT1[qIs51]
animals were superficially wild-type with no Muv defect, and
n4441 n4951 homozygotes died as L1 larvae with a rod-like
appearance (Figure 1D). The rod-like lethal phenotype is
characteristic of loss-of-function mutations in genes in the EGF/
Ras pathway required for vulval induction . The only known
gene in the EGF/Ras pathway in the genetic interval containing
n4441 is lin-3, which encodes the EGF ligand. Strong loss-of-
function alleles of lin-3 cause a rod-like lethal phenotype, and lin-3
mutations can also cause sterility [36,37]. The n4929 mutant
carries a G-to-A transition in the first nucleotide of exon 8 of lin-3
and is predicted to mutate an arginine to a lysine at amino acid
347 of LIN-3 (Figure 1B). The n4951 mutant carries a G-to-A
transition that results in a nonsense mutation predicted to truncate
LIN-3 after only 26 amino acids, before the EGF domain
(Figure 1B). The lin-3(n1059) nonsense mutation failed to
complement the sterility caused by n4929 and the lethality caused
by n4951, proving that n4929 and n4951 are alleles of lin-3. Since
the lin-3(n4951) nonsense mutation suppressed the n4441 synMuv
defect in cis, but the lin-3(n1059) nonsense mutation did not
suppress the n4441 synMuv defect in trans (Figure 1D), a lin-3 loss-
of-function mutation is a cis dominant suppressor of n4441,
indicating that n4441 is a gain-of-function allele of lin-3.
We determined the sequences of all exons and introns of lin-3
and of approximately 11 kb of upstream DNA in lin-3(n4441)
mutants. The only mutation was a G-to-A transition at nucleotide
30904 of cosmid F36H1, approximately 200 bp upstream of the
lin-3 transcript F36H1.4a (http://www.wormbase.org, release
WS200, 20 Mar 2009) (Figure 1C). To show that the
F36H1(30904) mutation is required for the class A synMuv
phenotype caused by lin-3(n4441), we sought recombinants
between lin-3(n4441) and the lin-3(n4951) nonsense mutation,
which is 5.3 kb downstream of F36H1(30904). We screened
approximately 90,000 progeny from lin-3(n4441 n4951)/+; lin-
15B(n744) animals, identified five independent Muv animals and
established homozygous lines. None of the five lines contained the
lin-3(n4951) mutation, and all five carried the F36H1(30904) G-to-
A mutation. Thus, the lin-3(n4441) mutation that causes the class
A synMuv phenotype must be to the left of lin-3(n4951), because if
it were to the right then the recombinants would not carry the
F36H1(30904) mutation. The 5.3 kb between F36H1(30904) and
lin-3(n4951), as well as 10.8 kb
F36H1(30904), carried no additional mutations in lin-3(n4441)
animals. If the mutation that causes the lin-3(n4441) synMuv
phenotype is not the F36H1(30904) mutation, then the lin-
3(n4441) mutation must be at least 10.8 kb to the left of the
F36H1(30904) mutation. However, in that case, assuming a
constant recombination rate throughout the lin-3 interval, the
likelihood that all five recombination events would have occurred
between F36H1(30904) and lin-3(n4951) is ((5.3)/(5.3+10.8))5, or
,0.004. We conclude that the G-to-A mutation at nucleotide
30904 of cosmid F36F1 is necessary for the class A synMuv
phenotype caused by lin-3(n4441). However, we cannot rule out
the possibility there is a second mutation more than 11 kb
upstream of lin-3 that is also required
F36H1(30904) mutation to cause a class A synMuv phenotype.
There are no known consensus transcription factor binding sites
that include the site of the lin-3(n4441) mutation (Transfac
database of known transcription binding sites; http://www.gene-
regulation.com). The region surrounding the lin-3(n4441) muta
tion is moderately conserved in the related nematodes C. briggsae
and C. remanei (data not shown).
lin-3(n4441) might be a class A synMuv specific allele of lin-3.
Alternatively, lin-3(n4441) might cause weak overexpression of lin-
3 if weak overexpression of lin-3 behaves like a class A synMuv
mutation. To differentiate between these alternatives, we overex-
pressed lin-3 weakly using the syIs12 integrated transgene. syIs12
expresses the EGF domain of lin-3 under the control of a heat-
shock promoter . At 20uC in the absence of heat-shock, syIs12
ofDNA upstream of
Table 1. lin-3(n4441) causes a dominant class A synMuv
% multivulva ± s.d. (n)a
N2060 (1422)060 (1368)
lin-3(n4441)060 (954)160.4 (1105)
060 (621)162 (830)
lin-15B(n744)060 (1058)060 (642)
lin-3(n4441); lin-15B(n744)10060 (729)10060 (248)
lin-52(n771)060 (1029)060 (1233)
lin-3(n4441); lin-52(n771)9860.1 (974)10060 (1244)
lin-61(n3447)060 (1039)060 (986)
lin-61(n3447); lin-3(n4441)17613(778)10060 (1201)
mys-1(n3681); lin-3(n4441)5669 (640) Lvad
lin-8(n2731)060 (902)060 (910)
lin-8(n2731); lin-3(n4441)0.460.5(1093)162 (788)
lin-15A(n767) 0.160.2 (1022)261 (1009)
lin-3(n4441); lin-15A(n767)563 (831)60617(609)
lin-38(n751)060 (947)161 (1307)
lin-38(n751); lin-3(n4441)162 (1028)1263 (889)
lin-56(n2728)060 (932)161 (733)
lin-56(n2728); lin-3(n4441) 0.360.3 (930)664 (843)
aAnimals were scored as Muv if any ventral ectopic protrusions were observed.
For each strain, animals were scored on three separate days, and % multivulva
shown is the average for those three days. SD, standard deviation. n, total
number of animals scored.
bThese animals were also heterozygous for dpy-17(e164) and unc-32(e189) and
descended from lin-3(n4441) homozygous parents.
cThese animals were also heterozygous for dpy-5(e61) and descended from dpy-
5(e61); lin-3(n4441); lin-15B(n744) parents.
dLva, larval arrest. Animals arrested as larvae, precluding the assaying of vulval
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org3 December 2011 | Volume 7 | Issue 12 | e1002418
did not cause a Muv phenotype (Table 2). syIs12; lin-15B(n744)
animals were mostly wild-type, with only a 1% penetrant Muv
defect, whereas syIs12; lin-15A(n767) animals exhibited a Muv
defect with 40% penetrance (Table 2). Thus, weak overexpression
of lin-3 from the syIs12 transgene was enhanced by a class A
synMuv mutation but not by a class B synMuv mutation. By
contrast, lin-3(n4441) was enhanced much more strongly by class B
synMuv mutations than by class A synMuv mutations (Table 1).
We conclude that lin-3(n4441) is a class A synMuv specific allele of
lin-3 and does not simply cause weak overexpression of lin-3.
lin-3(n4441) specifically prevents repression of lin-3
The class A and B synMuv genes redundantly repress
expression of lin-3 mRNA . To test if the lin-3(n4441)
mutation affects lin-3 mRNA levels similarly to other class A
synMuv mutations, we assayed lin-3 mRNA levels using real-time
RT-PCR. As previously reported, the class B synMuv mutant lin-
15B(n744) has wild-type lin-3 levels (Figure 2B). The class A
synMuv mutants lin-15A(n767) and lin-3(n4441) both had slightly
increased levels of lin-3 mRNA. The synMuv double mutants lin-
15AB(e1763) and lin-3(n4441); lin-15B(n744) had substantially
increased lin-3 mRNA levels (Figure 2B). Therefore, the lin-
3(n4441) mutation behaves as a class A synMuv mutation with
respect to lin-3 mRNA repression.
The lin-3(n4441) mutation is located 211 bp upstream of lin-3
and is also 465 bp upstream of F36H1.12, which is upstream of lin-
3 in the opposite orientation (Figure 2A). To determine if lin-
3(n4441) or other synMuv mutations also affect expression of
F36H1.12, we assayed F36H1.12 mRNA levels by real-time RT-
PCR. F36H1.12 mRNA levels were roughly equivalent to those of
the wild type in all possible single and double mutant
combinations involving lin-15A(n767), lin-3(n4441), and lin-
15B(n744) (Figure 2C). Therefore, the synMuv proteins specifically
repress lin-3 and do not establish a broad domain of repression.
Expression pattern of lin-3 in wild-type animals and
Although lin-3 is overexpressed in synMuv double mutants 
(also, Figure 2), it is not known where this overexpression occurs.
GFP- and LacZ-tagged lin-3 repetitive transgene arrays have been
used as reporters for lin-3 expression [10,39,40], but these
reporters might not be appropriate for determining lin-3
expression in synMuv mutants: first, the level of ectopic lin-3
expression might be too low to visualize using a GFP reporter;
Figure 1. n4441 is an allele of lin-3. (A) Genetic map showing n4441 on LGIV. n4441 was localized between dpy-13 and unc-30 by three-factor
mapping. SNP mapping using polymorphisms present in the CB4856 strain further localized n4441 to a 661 kb region between the SNPs dbP6 at
10909553 and uCE4-1148 at 11570158 of LGIV (data not shown). (B) The lin-3 locus. The lin-3a isoform (Wormbase web site, http://www.wormbase.
org, release WS200, Mar 20 2009) is shown. Solid boxes, exons; open boxes, UTRs. The start of the transcript is indicated by an arrow. Arrowheads
indicate the locations of mutations. n4951 is a nonsense mutation that truncates LIN-3 after 26 amino acids, and n4929 is a missense mutation that
converts an arginine to a lysine at amino acid 347. (C) n4441 is a G-to-A mutation at nucleotide 30904 of cosmid F36H1, 211 bp upstream of the lin-3
transcript. No other mutations were present in n4441 mutants in the region shown in (B). (D) A lin-3 loss-of-function mutation suppresses the n4441
synMuv phenotype in cis but not in trans. lin-3(n1059) is a nonsense mutation in lin-3 . lin-3(n4441 n4951) and lin-3(n4441 n4929) heterozygotes
also carried the nT1[qIs51] translocation. All animals were grown at 20uC. Animals were scored as Muv if any ventral ectopic protrusions were
observed. n, total number of animals scored.
Table 2. lin-3 overexpression is enhanced more strongly by a
class A synMuv mutation than by a class B synMuv mutation.
genotype % multivulva (n)a
syIs12; lin-15A(n767)38 (105)
syIs12; lin-15B(n744) 1 (100)
aAnimals were scored as Muv if any ventral ectopic protrusions were observed.
n, total number of animals scored.
bsyIs12 is an integrated transgene expressing the lin-3 EGF domain under the
control of a heat-shock promoter . Animals were assayed in the absence of
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org4 December 2011 | Volume 7 | Issue 12 | e1002418
second, many synMuv mutations affect the expression of repetitive
transgene arrays, potentially confounding interpretation of the
expression pattern of such reporters . Instead, we assayed lin-3
expression using a fluorescence in situ hybridization (FISH)
technique that has sufficient sensitivity to detect single mRNA
molecules . We used 48 non-overlapping probes against lin-3
(Table S1), each conjugated to a single fluorophore, to label
individual mRNA molecules brightly enough to be visible as
distinct fluorescent spots. Because there are 48 probes that bind
independently to the target mRNA, any single probe that binds
non-specifically should not cause a false-positive signal. The
distribution of intensities of the spots in any given animal was
unimodal, consistent with each spot’s representing a single mRNA
molecule (Figure S1). Furthermore, by comparing the spot
intensities in different tissues and mutants, we found that the level
of expression in a given cell or tissue was independent of the
intensity of the spots in that cell or tissue, and if the number of
spots in a cell was altered then the average intensity of spots in that
cell was unchanged (Figure S2). If each spot represented multiple
mRNA molecules, then as the expression level in a given cell
increased the average number of mRNA molecules in each of
those spots would also be expected to increase, leading to greater
intensity. Because the intensity of each spot was independent of the
level of expression, we conclude that each spot is likely to represent
a single mRNA molecule. We also found that all tissues are
accessible to FISH probes, as probes directed against ama-1 and eft-
2 robustly detected mRNA in all cells (data not shown). However,
we cannot know if we are detecting every mRNA molecule; it is
possible that some mRNA molecules are not accessible to the
oligonucleotide probes or are not detected for some other reason.
We first determined the expression pattern of lin-3 in wild-type
animals at the late L2 to early L3 stage when vulval induction
occurs. Previous studies found that at the early L3 stage lin-3 is
expressed in the anchor cell and in the pharynx [10,40]. We
indeed observed robust expression of lin-3 in the anchor cell and
throughout the pharynx. We also saw expression of lin-3 in the
germline (Figure 3A and Figure S3). In some wild-type animals we
also observed a few copies of lin-3 mRNA in one or more cells in
the tail, on the ventral side slightly anterior to the anus. In
addition, a few copies of lin-3 mRNA were seen on the ventral side
of the animal, slightly behind the posterior gonad arm. We imaged
several animals that were slightly older, in the late L3 stage, and
observed expression of several copies of lin-3 mRNA in the region
where P6.p and its descendants are located (data not shown),
consistent with previous reports of expression of lin-3 in the
descendants of P6.p by the L4 stage . We did not consistently
detect any lin-3 mRNA in other tissues, although in some animals
we observed a single lin-3 mRNA molecule elsewhere. For
example, in the animal shown in Figure 3A a single lin-3 mRNA
molecule was observed in or near an intestinal cell close to the
anchor cell. Overall, other than for those tissues that highly
expressed lin-3 there was very tight repression of lin-3. The
numbers of copies of lin-3 mRNA we observed in each tissue in
individual animals are listed in Table S2 and are summarized in
Table 3. The expression pattern we observed for lin-3 is consistent
with that seen using GFP- and LacZ-tagged lin-3 reporters
[10,39,40] and with functional studies of lin-3 , indicating that
most if not all of the mRNA spots identified by this technique are
likely to represent actual lin-3 mRNA molecules.
lin-15AB(e1763) animals expressed lin-3 in the pharynx, germ-
line, and anchor cell at levels grossly similar to those of wild-type
animals (Figure 3D and Table 3). In addition there was
widespread ectopic expression of lin-3, with an average of
approximately 1100 ectopic copies of lin-3 mRNA observed per
animal (Figure 3D and Table 3). This ectopic expression was
much weaker than the normal expression in the anchor cell;
whereas an average of 29 copies of lin-3 mRNA was seen in the
anchor cell in wild-type animals (Table 3), only one or a few copies
of lin-3 mRNA were observed in most cells in lin-15AB(e1763)
mutants. Because we could not see cell boundaries, we could not
determine if every cell ectopically expressed lin-3, but there were
no tissues that appeared to lack ectopic lin-3 mRNA (Figure S4).
Cells around the perimeter of the animal expressed lin-3 in the lin-
15AB(e1763) mutant, consistent with ectopic expression in the
Figure 2. The lin-3(n4441) mutation specifically prevents
repression of lin-3. (A) The lin-3(n4441) mutation is located 211 bp
upstream of the lin-3 transcript. The gene F36H1.12 is upstream of lin-3
in the opposite orientation, and lin-3(n4441) is located 465 bp from the
predicted F36H1.12 transcript. Solid boxes, exons; open boxes, UTRs. (B)
lin-3 mRNA levels in lin-3(n4441) single and double mutants. As
reported previously , lin-3 mRNA levels are substantially increased in
lin-15AB double mutants but not in lin-15A or lin-15B single mutants.
Like other class A synMuv mutations, lin-3(n4441) caused a substantial
increase in lin-3 mRNA levels only in a class B synMuv mutant
background. Realtime RT-PCR experiments were performed using RNA
harvested at the late L2 or early L3 stage from each strain shown.
Relative lin-3 mRNA levels were normalized to the levels of mRNA
encoding the ribosomal protein subunit rpl-26 using the DDCt method
. The means and standard deviations of relative lin-3 mRNA levels
from two independent trials are shown. The lin-15A(n767), lin-15B(n744)
and lin-15AB(e1763) alleles were used in this experiment. (C) F36H1.12
mRNA levels in synMuv single and double mutants. No combination of
lin-3(n4441), lin-15A, and lin-15B mutations affected F36H1.12 mRNA
levels. Realtime RT-PCR experiments were performed using RNA
harvested at the late L2 or early L3 stage from each strain shown.
Relative F36H1.12 mRNA levels were normalized to the levels of mRNA
encoding the ribosomal protein subunit rpl-26 using the DDCt method.
The means and standard deviations of relative F36H1.12 mRNA levels
from two independent trials are shown.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org5 December 2011 | Volume 7 | Issue 12 | e1002418
hypodermis (Figure 3D and Figure S4). There were also many
ectopic lin-3 mRNA copies that clearly were not in the hypodermis
We also determined lin-3 expression in lin-15A(n767) and lin-
15B(n744) single mutants. lin-15B(n744) animals had a lin-3
expression pattern similar to that of wild-type animals (Figure 3C).
In lin-15B(n744) mutants there was an extremely low level of
ectopic lin-3 expression, with an average of six ectopic lin-3 mRNA
molecules detected per animal (Table 3), but lin-3 was still tightly
repressed outside of the germline, anchor cell, and pharynx. lin-
15A(n767) animals exhibited broad ectopic expression of lin-3, but
at a much lower level than that of lin-15AB(e1763) animals
(Figure 3B). An average of 64 copies of lin-3 mRNA were seen
outside of the pharynx, germline, and anchor cell in lin-15A(n767)
Figure 3. The synMuv genes prevent widespread ectopic expression of lin-3 mRNA. FISH of lin-3 mRNA in late L2 to early L3 animals. Each
dot represents a single mRNA molecule . lin-3 mRNAs are shown in red, and 49,6-diamidino-2-phenylindole (DAPI) staining of nuclei is shown in
blue. The images shown are maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated by an arrowhead in each panel.
(A) Wild type. lin-3 mRNA is expressed in the pharynx, anchor cell, and germline and is tightly repressed elsewhere. (B) lin-15A(n767). There is a low
level of ectopic lin-3 expression, with approximately 60 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline. (C) lin-
15B(n744). There is a very low level of ectopic lin-3 expression, with approximately 10 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor
cell, and germline. The large bright spot at the edge of the head is outside the animal. (D) lin-15AB(e1763). lin-3 is ectopically expressed throughout
the animal, with approximately 900 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org6 December 2011 | Volume 7 | Issue 12 | e1002418
animals (Table 3). Unlike in lin-15AB(e1763) animals, in any given
lin-15A(n767) animal most cells did not display ectopic lin-3
expression. However, we observed no obvious cell or tissue
specificity to the ectopic expression among several lin-15A(n767)
animals. Rather, it appeared that in lin-15A(n767) animals lin-3 is
globally derepressed, but at a very low level.
The numerous class B synMuv genes have highly similar
although not identical effects on vulval development .
However, the class B synMuv genes have widely differing effects
on other aspects of growth and development. For example, PGL-
1, which is normally expressed in the germline, is misexpressed in
the somatic cells of mutants of many class B synMuv genes,
including lin-15B, but not in mutants of some other class B
synMuv genes, including lin-36, lin-52, and lin-53 [44,45]. We
therefore investigated the role of the class B synMuv genes lin-36,
lin-52, and lin-53 in controlling lin-3 expression. We determined
the expression pattern of lin-3 in lin-36(n766); lin-15A(n767), lin-
52(n771); lin-15A(n767), and lin-53(n833); lin-15A(n767) mutants.
All three double mutants exhibited ubiquitous ectopic expression
of lin-3, with lin-3 mRNA observed in most if not all tissues
(Figure 4). There were no obvious differences in the spatial pattern
of lin-3 expression in lin-36(n766); lin-15A(n767), lin-52(n771); lin-
15A(n767), and lin-53(n833); lin-15A(n767) double mutants as
compared to lin-15AB(e1763) mutants.
The lin-3(n4441) mutation could cause global derepression of
lin-3 similarly to lin-15A(n767), or it could affect lin-3 expression in
a subset of tissues. We examined the expression of lin-3 mRNA in
lin-3(n4441) and lin-3(n4441); lin-15B(n744) animals. lin-3(n4441)
animals had widespread but weak ectopic expression of lin-3,
similar to lin-15A(n767) animals (Figure 5A and Table 3). lin-
3(n4441); lin-15B(n744) animals exhibited ectopic lin-3 expression
in most cells and were indistinguishable from lin-15AB(e1763)
animals (Figure 5B and Table 3).
Identifying the biologically relevant targets of transcriptional
regulators that control development is a challenging problem. The
synMuv genes encode putative transcriptional repressors that
prevent ectopic vulval development. Mutating a synMuv binding
site in a target gene might relieve repression of that target, and if
that repression were essential to prevent ectopic vulval develop-
ment could cause a dominant synMuv phenotype. We isolated a
mutation in the lin-3 EGF gene that derepresses lin-3 transcription
and causes a dominant class A synMuv phenotype. This finding
establishes that lin-3 is a functionally important target of the class
A synMuv genes, consistent with a previous report that lin-3
expression is repressed by the synMuv genes and that double-
stranded RNA directed against lin-3 can suppress the synMuv
phenotype . Importantly, the lin-3(n4441) mutation fully
recapitulates the class A synMuv phenotype with regard to vulval
development and lin-3 expression and causes a class A synMuv
phenotype equivalent to that caused by strong alleles of class A
synMuv genes. If the class A synMuv genes repressed multiple
targets to prevent ectopic vulval development, then a mutation
that abolished class A synMuv-mediated repression of lin-3 would
recapitulate only partially the class A synMuv phenotype. We
conclude that lin-3 is likely to be the only key biologically relevant
target of the class A synMuv genes in vulval development.
The simplest interpretation of the effect of the lin-3(n4441)
mutation is that this mutation abolishes a binding site for a
transcriptional repressor consisting of or controlled by class A
synMuv proteins. However, the effect of the lin-3(n4441) mutation
is slightly enhanced by mutations in all other class A synMuv
genes. If the lin-3(n4441) mutation completely inactivated a
binding site that responds to only one of the known class A
synMuv proteins, then mutation of that class A synMuv gene
should not enhance the synMuv phenotype caused by lin-3(n4441).
One possibility is that a complex consisting of multiple class A
synMuv proteins binds to the lin-3 promoter, the lin-3(n4441)
mutation strongly reduces but does not completely eliminate that
binding, and removing any one class A synMuv protein does not
fully abrogate the ability of the complex to bind to the lin-3 locus
and repress transcription. This model is consistent with the
observation that most class A synMuv mutations, including lin-
3(n4441), are enhanced by class A synMuv mutations in other
genes . Alternatively, the class A synMuv genes might
indirectly repress lin-3 by regulating the expression or activity of
or by binding to another protein that binds to the lin-3 promoter to
prevent ectopic transcription. Because a mutation in the lin-3
promoter can cause a class A synMuv phenotype, the class A and
class B synMuv pathways must be integrated at the point of lin-3
repression, and hence it is unlikely that the class A and B synMuv
genes redundantly control a transcriptional regulator which in
turn controls lin-3 expression.
lin-3 expression in the germline had not been previously
observed, likely because the reporters used to assay lin-3 expression
were either silenced in the germline  or lacked distant
regulatory regions necessary to drive germline expression.
Mutations in the FOG and FBF translational inhibitor RNA-
binding proteins cause a germline-dependent Muv phenotype, and
the FBF proteins can bind to the 39 UTR of lin-3 in vitro, suggesting
that germline lin-3 mRNA is translationally repressed during the
larval stage when vulval induction occurs . In many class B
Table 3. Quantification of lin-3 expression.
N26 2965314669 284648161
lin-15A(n767)7 2365 279646 451692 64621
lin-15B(n744)6 2164 1996573806134662
lin-15AB(e1763)7 2264 231654 503615410876211
lin-3(n4441)8 2664 34066238766871628
lin-3(n4441); lin-15B(n744)5 2167 3606109578670 11566111
The average number of copies of lin-3 mRNA observed in each tissue and the standard deviation between animals is shown.
an, total number of animals assayed.
blin-3 mRNA observed outside of the normal domain of expression of lin-3.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org7 December 2011 | Volume 7 | Issue 12 | e1002418
synMuv mutants, somatic cells express normally germline-specific
genes [19,44,45]. Given our finding that lin-3 is normally
expressed in the germline, one possibility is that the class B
synMuv genes repress ectopic lin-3 expression in somatic cells as a
consequence of their role in ensuring that somatic cells do not
inappropriately adopt germline-like fates. The class B synMuv
genes might all directly repress lin-3 in somatic cells. Alternatively,
as there are a large number of class B synMuv genes and their
effects on vulval development are not identical, perhaps at least
some class B synMuv genes indirectly repress lin-3 by preventing
the ectopic adoption of germline-like fates. In class B synMuv
single mutants, the somatic cells adopt a more germline-like fate
that would include lin-3 expression except that the class A synMuv
genes still tightly repress lin-3, mostly preventing ectopic lin-3
expression. In class A synMuv single mutants, lin-3 is not tightly
repressed, but most somatic cells are not fated to express lin-3, so
there is only a low level of leaky ectopic lin-3 expression. However,
in class AB synMuv double mutants, the somatic cells adopt a
germline-like fate that includes lin-3 expression, and there is no
class A synMuv mechanism that tightly represses lin-3, resulting in
widespread and substantial ectopic lin-3 expression. In short, we
suggest that the synthetic Muv phenotype caused by mutations in
the synMuv genes might be a consequence of two distinct
functions of the class A and class B synMuv genes: the class A
synMuv genes either directly or indirectly tightly repress ectopic
lin-3 transcription, and the class B synMuv genes prevent somatic
expression of germline-expressed genes, which include lin-3; only if
both functions are lost will somatic cells ectopically express
sufficient lin-3 mRNA to cause ectopic vulval induction. These
findings raise the possibility that the development of some human
tumors might require the loss of one tumor suppressor gene that
prevents cells from adopting a fate that is permissive for the
expression of a growth factor and the loss of a second tumor
suppressor gene that specifically represses the expression of that
A subset of the class B synMuv genes is required to prevent the
somatic misexpression of normally germline-restricted P-granule
proteins such as PGL-1 [44,45]. We found that lin-15B mutants,
Figure 4. Multiple class B synMuv mutations have similar effects on lin-3 mRNA expression. FISH of lin-3 mRNA in late L2 to early L3
animals. Each dot represents a single mRNA molecule . lin-3 mRNAs are shown in red, and 49,6-diamidino-2-phenylindole (DAPI) staining of nuclei
is shown in blue. The images shown are maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated by an arrowhead in
each panel. Each mutant displayed ectopic lin-3 mRNA expression throughout the animal in most if not all tissues. (A) lin-36(n766); lin-15A(n767). (B)
lin-52(n771); lin-15A(n767). (C) lin-53(n833); lin-15A(n767).
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org8 December 2011 | Volume 7 | Issue 12 | e1002418
which do exhibit somatic PGL-1 expression, and lin-36, lin-52, and
lin-53 mutants, which do not exhibit somatic PGL-1, all have
highly similar effects on lin-3 expression. These results indicate
that different germline genes are broadly repressed in the soma by
different sets of transcriptional repressors. The class B synMuv
genes define one such group of repressors and are classified
together because they have comparable effects on the germline
gene lin-3, resulting in similar vulval phenotypes. Many such
partially-overlapping groups of transcriptional repressors, includ-
ing various subsets of the class B synMuv genes, are likely to be
required for the repression in the soma of other germline-restricted
Whereas lin-3 expression in most cells is tightly repressed by the
synMuv genes, the anchor cell and germline exhibit robust lin-3
expression that is not substantially affected by the synMuv genes.
While it has not been reported whether or not any synMuv genes
are expressed in the anchor cell, several synMuv genes are
expressed in the germline [17,22,26,33], and we are not aware of
studies that have conclusively shown any synMuv genes not to be
expressed in the germline. In most cells, the synMuv genes reduce
lin-3 expression from an average of one to two copies per cell to
nearly zero copies per cell. The synMuv genes clearly do not have
a similar fold effect on lin-3 expression in the anchor cell and
germline. However, it is possible that the synMuv genes repress a
similar absolute number of leaky lin-3 mRNA molecules in all cells;
given the animal-to-animal variability in lin-3 expression we likely
would not have been able to detect such a small increase in the
anchor cell or germline. Alternatively, the synMuv genes might
not repress lin-3 in the anchor cell or germline. The strong
activator(s) of lin-3 that drive expression in those tissues could
override the activity of the synMuv genes, or one or more synMuv
genes might not be expressed in those tissues, thereby compro-
mising synMuv repression of lin-3.
In lin-15AB mutants, lin-3 is ectopically expressed throughout
the animal in a broad range of cells and tissues. Site-of-action
experiments have shown that the synMuv genes function at least in
large part in the hyp7 hypodermal syncytium to prevent ectopic
vulval development . The expression pattern of lin-3 in
synMuv mutants does not directly identify the site-of-action of
synMuv genes in regulating vulval development but does show
that the synMuv genes function throughout the animals to keep
lin-3 very tightly repressed in numerous cells and tissues. lin-3 EGF
regulates non-vulval cell fates in C. elegans development, and at
least some of these fates, such as the P11/P12 fate, are also
regulated by the synMuv genes in a manner analogous to that of
vulval development . In short, the synMuv genes act
throughout the animal to prevent ectopic lin-3 expression, which
can cause a variety of developmental abnormalities. Mutants with
a displaced anchor cell show that lin-3 can act at a distance , so
a cell ectopically expressing lin-3 could affect fates in both nearby
and distant cells. We suggest that for any given cell-fate decision,
the site of action of the synMuv genes is likely to be spread across
multiple cells and determined by the size and proximity of those
cells to the cell being regulated by lin-3. In the case of vulval
development, hyp7 plays the major role, given its large size and
close proximity to the Pn.p cells, with likely lesser contributions
from many other cells. The site at which the synMuv genes repress
lin-3 to ensure proper vulval development is therefore probably a
Figure 5. The lin-3(n4441) mutation causes widespread ectopic expression of lin-3 mRNA. FISH of lin-3 mRNA in late L2 to early L3 animals.
Each dot represents a single mRNA molecule . lin-3 mRNAs are shown in red, and 49,6-diamidino-2-phenylindole (DAPI) staining of nuclei is shown
in blue. The images shown are maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated by an arrowhead in each panel.
(A) lin-3(n4441). there is a low level of ectopic lin-3 expression, with approximately 45 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor
cell, and germline. (B) lin-3(n4441); lin-15B(n744). lin-3 is ectopically expressed throughout the animal, with approximately 1000 ectopic copies of lin-3
mRNA seen outside the pharynx, anchor cell, and germline.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org9December 2011 | Volume 7 | Issue 12 | e1002418
combination of the Pn.p cells themselves and neighboring cells
that do not normally either express or respond to lin-3. This
situation is similar to that in which both tumor cells and the
microenvironment surrounding the tumor provide factors that
drive tumor development . We suggest that analogously to the
synMuv genes some tumor suppressor genes function by repressing
growth factor expression in both tumor cells and the surrounding
In synMuv double mutants, lin-3 was ectopically expressed but
at a much lower level than at its major normal site of function, the
anchor cell. synMuv double mutants might ectopically express as
few as one to two copies of lin-3 mRNA per cell. Thus, normal C.
elegans development requires lin-3 to be exceedingly tightly
repressed outside of a few cells, and only slight expression of lin-
3 throughout the animal can cause abnormal cell-fate transfor-
mations. Such low levels of ectopic expression would likely be
missed by most techniques used to assay gene expression. We
suggest it could be important to examine the expression of EGF-
family ligands in tumors using highly sensitive techniques with
single-molecule resolution to determine if broad low-level
misexpression of EGF-family ligands plays a role in oncogenic
growth. In C. elegans, the tight repression of lin-3 EGF requires
both the class A synMuv gene pathway and the class B gene
synMuv pathway, which includes homologs of known tumor
suppressor genes, such as lin-35 Rb. Therefore, some tumor
suppressor genes in mammals might function by tightly repressing
low-level ectopic expression of EGF-family ligands in many cells,
possibly in both the tumor and the microenvironment surrounding
Materials and Methods
Strains and genetics
C. elegans strains were cultured by standard methods on OP50
bacteria . All animals were grown at 20uC, except where
otherwise noted. The wild-type strain was N2, except in SNP
mapping experiments in which the polymorphic CB4856 strain
was also used . The following mutations were used in this
LGI: dpy-5(e61), lin-61(n3447), lin-53(n833)
LGII: lin-8(n2731), lin-56(n2728), lin-38(n751), syIs12
LGIII: dpy-17(e164), lin-36(n766), unc-32(e189), lin-52(n771)
LGIV: lin-3(n4441), lin-3(n4929), lin-3(n4951), lin-3(n1059)
LGX: lin-15A(n767), lin-15B(n744), lin-15(e1763)
The balancer strain nT1[qIs51] IV:V  was used; qIs51 is a
GFP-expressing transgene integrated onto the nT1 translocation.
Table S3 lists all strains used in this study.
Quantitative PCR assay
Synchronized animals were harvested at or near the L2-to-L3
larval transition, when vulval induction occurs. N2 animals were
harvested 33 hours after starved L1 larvae were placed on plates
with food. Some mutants grew more slowly and were harvested
after 39 hours. Quantitative RT-PCR for lin-3 was performed as
previously described . lin-3 was amplified using the primers
Fluorescence in situ hybridization
Animals were grown to the L2-to-L3 transition as in the
quantitative RT-PCR experiments. Fixation and hybridization
were performed as described previously , except that worms
were fixed for one hour instead of 45 minutes. The lin-3 probes
(Biosearch Technologies, Inc) were conjugated to the fluorophore
Cy5 using the Amersham Cy5 Mono-reactive Dye pack (GE
Healthcare). DNA was visualized using 49,6-diamidino-2-pheny-
lindole (DAPI). The probe sequences used are shown in Table S1.
Figure 2 and Figure 3 are maximum intensity projections of a Z-
stack of images processed with the Find Edges and Smooth
operations in ImageJ. lin-3 mRNA spots were computationally
identified with manually determined thresholds as previously
described . The number of molecules within each tissue were
then manually counted. The anchor cell was identified based on
position. lin-3 mRNA expression in the anchor cell appeared as a
tight cluster of spots; molecules within that cluster were considered
to be in the anchor cell. To determine which lin-3 mRNA
molecules were in the pharynx, we noted the outline of the
pharynx that was clearly visible as a dark boundary in the Cy5
channel of the image stacks (see Figure S3 and Figure S4). The
boundaries of the germline were estimated from the positions of
DAPI-labeled germline nuclei.
Histograms of FISH spot intensity for six images from six different
animals are shown. Each image included the anchor cell and most
or all of the germline. Intensities were calculated by taking the
maximum intensity of a spot and subtracting the average
background intensity of a four-pixel radius surrounding the spot.
Unimodal distribution of FISH spot intensity.
level. The mean spot intensity was calculated for mRNAs
expressed in the anchor cell, germline, or ectopically. For each
animal, the intensity of each spot was normalized to the mean
intensity of the spots in the anchor cell, which was set to 1. The
mean and standard deviation for the expression in the germline
or ectopically from 7–10 animals for each genotype are shown.
lin-3(e1417) had roughly wild-type numbers of lin-3 FISH spots in
the germline but had approximately 4.5-fold fewer lin-3 FISH
spots in the anchor cell (Table S2). In both wild-type and lin-
15AB(e1763) animals the density of spots in the anchor cell was
noticeably higher than in the germline or elsewhere (e.g.
Figure 3A and 3D).
FISH spot intensity is independent of expression
of lin-3 mRNA in a wild-type animal. The animal shown is the
same as in Figure 3A. Each frame is a different plane in the Z-axis.
lin-3 mRNA was expressed in the pharynx, anchor cell, and
germline and was tightly repressed elsewhere.
lin-3 mRNA expression in a wild-type animal. FISH
expression of lin-3 mRNA. FISH of lin-3 mRNA in a lin-
15AB(e1763) mutant animal. The animal shown is the same as in
Figure 3D. Each frame is a different plane in the Z-axis. lin-3 was
ectopically expressed throughout the animal, with approximately
900 ectopic copies of lin-3 mRNA seen outside of the pharynx,
anchor cell, and germline.
The synMuv genes prevent widespread ectopic
Oligonucleotides in lin-3 FISH probe.
Quantification of lin-3 expression.
List of strains used in this study.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org 10December 2011 | Volume 7 | Issue 12 | e1002418
Acknowledgments Download full-text
We thank Dave Harris for helpful comments concerning this manuscript;
Beth Castor, Elissa Murphy, and Rita Droste for technical assistance; and
Conceived and designed the experiments: AMS DHK AvO HRH.
Performed the experiments: AMS DHK. Analyzed the data: AMS
DHK HRH. Wrote the paper: AMS HRH.
1. Normanno N, De Luca A, Bianco C, Strizzi L, Mancino M, et al. (2006)
Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366: 2–16.
2. Massague J (1990) Transforming growth factor-alpha. A model for membrane-
anchored growth factors. J Biol Chem 265: 21393–21396.
3. Yotsumoto F, Yagi H, Suzuki SO, Oki E, Tsujioka H, et al. (2008) Validation of
HB-EGF and amphiregulin as targets for human cancer therapy. Biochem
Biophys Res Commun 365: 555–561.
4. Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, et al. (1990) TGF
alpha overexpression in transgenic mice induces liver neoplasia and abnormal
development of the mammary gland and pancreas. Cell 61: 1137–1146.
5. Sandgren EP, Luetteke NC, Palmiter RD, Brinster RL, Lee DC (1990)
Overexpression of TGF alpha in transgenic mice: induction of epithelial
hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61:
6. Downward J (2003) Targeting RAS signalling pathways in cancer therapy. Nat
Rev Cancer 3: 11–22.
7. Aroian RV, Koga M, Mendel JE, Ohshima Y, Sternberg PW (1990) The let-23
gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase
of the EGF receptor subfamily. Nature 348: 693–699.
8. Beitel GJ, Clark SG, Horvitz HR (1990) Caenorhabditis elegans ras gene let-60 acts
as a switch in the pathway of vulval induction. Nature 348: 503–509.
9. Han M, Sternberg PW (1990) let-60, a gene that specifies cell fates during C.
elegans vulval induction, encodes a ras protein. Cell 63: 921–931.
10. Hill RJ, Sternberg PW (1992) The gene lin-3 encodes an inductive signal for
vulval development in C. elegans. Nature 358: 470–476.
11. Sternberg PW, Horvitz HR (1986) Pattern formation during vulval development
in C. elegans. Cell 44: 761–772.
12. Kimble J (1981) Alterations in cell lineage following laser ablation of cells in the
somatic gonad of Caenorhabditis elegans. Dev Biol 87: 286–300.
13. Katz WS, Lesa GM, Yannoukakos D, Clandinin TR, Schlessinger J, et al. (1996)
A point mutation in the extracellular domain activates LET-23, the Caenorhabditis
elegans epidermal growth factor receptor homolog. Mol Cell Biol 16: 529–537.
14. Ferguson EL, Horvitz HR (1989) The multivulva phenotype of certain
Caenorhabditis elegans mutants results from defects in two functionally redundant
pathways. Genetics 123: 109–121.
15. Andersen EC, Saffer AM, Horvitz HR (2008) Multiple levels of redundant
processes inhibit Caenorhabditis elegans vulval cell fates. Genetics 179: 2001–2012.
16. Lu X, Horvitz HR (1998) lin-35 and lin-53, two genes that antagonize a C. elegans
Ras pathway, encode proteins similar to Rb and its binding protein RbAp48.
Cell 95: 981–991.
17. Ceol CJ, Horvitz HR (2001) dpl-1 DP and efl-1 E2F act with lin-35 Rb to
antagonize Ras signaling in C. elegans vulval development. Mol Cell 7: 461–473.
18. Solari F, Ahringer J (2000) NURD-complex genes antagonise Ras-induced
vulval development in Caenorhabditis elegans. Curr Biol 10: 223–226.
19. Unhavaithaya Y, Shin TH, Miliaras N, Lee J, Oyama T, et al. (2002) MEP-1
and a homolog of the NURD complex component Mi-2 act together to maintain
germline-soma distinctions in C. elegans. Cell 111: 991–1002.
20. Andersen EC, Horvitz HR (2007) Two C. elegans histone methyltransferases
repress lin-3 EGF transcription to inhibit vulval development. Development 134:
21. Poulin G, Dong Y, Fraser AG, Hopper NA, Ahringer J (2005) Chromatin
regulation and sumoylation in the inhibition of Ras-induced vulval development
in Caenorhabditis elegans. Embo J 24: 2613–2623.
22. Couteau F, Guerry F, Muller F, Palladino F (2002) A heterochromatin protein 1
homologue in Caenorhabditis elegans acts in germline and vulval development.
EMBO Rep 3: 235–241.
23. Huang LS, Tzou P, Sternberg PW (1994) The lin-15 locus encodes two negative
regulators of Caenorhabditis elegans vulval development. Mol Biol Cell 5: 395–411.
24. Clark SG, Lu X, Horvitz HR (1994) The Caenorhabditis elegans locus lin-15, a
negative regulator of a tyrosine kinase signaling pathway, encodes two different
proteins. Genetics 137: 987–997.
25. Davison EM, Saffer AM, Huang LS, Demodena J, Sternberg PW, et al. (2011)
The LIN-15A and LIN-56 Transcriptional Regulators Interact to Negatively
Regulate EGF/Ras Signaling in Caenorhabditis elegans Vulval Cell-Fate Determi-
nation. Genetics 187: 803–815.
26. Davison EM, Harrison MM, Walhout AJ, Vidal M, Horvitz HR (2005) lin-8,
which antagonizes Caenorhabditis elegans Ras-mediated vulval induction, encodes a
novel nuclear protein that interacts with the LIN-35 Rb protein. Genetics 171:
27. Cui M, Chen J, Myers TR, Hwang BJ, Sternberg PW, et al. (2006) SynMuv
genes redundantly inhibit lin-3/EGF expression to prevent inappropriate vulval
induction in C. elegans. Dev Cell 10: 667–672.
28. Ferguson EL, Sternberg PW, Horvitz HR (1987) A genetic pathway for the
specification of the vulval cell lineages of Caenorhabditis elegans. Nature 326:
29. Hedgecock EM, Herman RK (1995) The ncl-1 gene and genetic mosaics of
Caenorhabditis elegans. Genetics 141: 989–1006.
30. Herman RK, Hedgecock EM (1990) Limitation of the size of the vulval
primordium of Caenorhabditis elegans by lin-15 expression in surrounding
hypodermis. Nature 348: 169–171.
31. Myers TR, Greenwald I (2005) lin-35 Rb acts in the major hypodermis to oppose
ras-mediated vulval induction in C. elegans. Dev Cell 8: 117–123.
32. Schott S, Ramos F, Coustham V, Palladino F (2009) HPL-2/HP1 prevents
inappropriate vulval induction in Caenorhabditis elegans by acting in both HYP7
and vulval precursor cells. Genetics 181: 797–801.
33. Ceol CJ, Horvitz HR (2004) A new class of C. elegans synMuv genes implicates a
Tip60/NuA4-like HAT complex as a negative regulator of Ras signaling. Dev
Cell 6: 563–576.
34. Siegfried KR, Kidd AR, 3rd, Chesney MA, Kimble J (2004) The sys-1 and sys-3
genes cooperate with Wnt signaling to establish the proximal-distal axis of the
Caenorhabditis elegans gonad. Genetics 166: 171–186.
35. Han M, Aroian RV, Sternberg PW (1990) The let-60 locus controls the switch
between vulval and nonvulval cell fates in Caenorhabditis elegans. Genetics 126:
36. Ferguson EL, Horvitz HR (1985) Identification and characterization of 22 genes
that affect the vulval cell lineages of the nematode Caenorhabditis elegans. Genetics
37. Liu J, Tzou P, Hill RJ, Sternberg PW (1999) Structural requirements for the
tissue-specific and tissue-general functions of the Caenorhabditis elegans epidermal
growth factor LIN-3. Genetics 153: 1257–1269.
38. Katz WS, Hill RJ, Clandinin TR, Sternberg PW (1995) Different levels of the C.
elegans growth factor LIN-3 promote distinct vulval precursor fates. Cell 82:
39. Chang C, Newman AP, Sternberg PW (1999) Reciprocal EGF signaling back to
the uterus from the induced C. elegans vulva coordinates morphogenesis of
epithelia. Curr Biol 9: 237–246.
40. Hwang BJ, Sternberg PW (2004) A cell-specific enhancer that specifies lin-3
expression in the C. elegans anchor cell for vulval development. Development
41. Hsieh J, Liu J, Kostas SA, Chang C, Sternberg PW, et al. (1999) The RING
finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate
context-dependent gene silencing in Caenorhabditis elegans. Genes Dev 13:
42. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008)
Imaging individual mRNA molecules using multiple singly labeled probes. Nat
Methods 5: 877–879.
43. Thompson BE, Lamont LB, Kimble J (2006) Germ-line induction of the
Caenorhabditis elegans vulva. Proc Natl Acad Sci U S A 103: 620–625.
44. Wang D, Kennedy S, Conte D, Jr., Kim JK, Gabel HW, et al. (2005) Somatic
misexpression of germline P granules and enhanced RNA interference in
retinoblastoma pathway mutants. Nature 436: 593–597.
45. Petrella LN, Wang W, Spike CA, Rechtsteiner A, Reinke V, et al. (2011)
synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans
survival. Development 138: 1069–1079.
46. Kelly WG, Xu S, Montgomery MK, Fire A (1997) Distinct requirements for
somatic and germline expression of a generally expressed Caernorhabditis elegans
gene. Genetics 146: 227–238.
47. Jiang LI, Sternberg PW (1998) Interactions of EGF, Wnt and HOM-C genes
specify the P12 neuroectoblast fate in C. elegans. Development 125: 2337–2347.
48. Thomas JH, Stern MJ, Horvitz HR (1990) Cell interactions coordinate the
development of the C. elegans egg-laying system. Cell 62: 1041–1052.
49. Hu M, Polyak K (2008) Microenvironmental regulation of cancer development.
Curr Opin Genet Dev 18: 27–34.
50. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
51. Wicks SR, Yeh RT, Gish WR, Waterston RH, Plasterk RH (2001) Rapid gene
mapping in Caenorhabditis elegans using a high density polymorphism map. Nat
Genet 28: 160–164.
52. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the
comparative C(T) method. Nat Protoc 3: 1101–1108.
SynMuv Genes Repress Ectopic lin-3 EGF Expression
PLoS Genetics | www.plosgenetics.org11December 2011 | Volume 7 | Issue 12 | e1002418