Howard Hughes Medical Institute and Division of Biology, 156-29 California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA
* Author for correspondence (e-mail: pws{at}its.caltech.edu)
Accepted 23 September 2003
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SUMMARY |
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Key words: EGF, LIN-3, HLH-2, E-protein/Daughterless, Nuclear hormone receptor, Anchor cell, Vulval induction
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Introduction |
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The LIN-3 protein is synthesized as a transmembrane precursor like other
EGF family growth factors, and an unidentified protease(s) has been proposed
to cleave the precursor to release the extracellular EGF domain that binds to
its receptor, LET-23, in the VPCs (Hill
and Sternberg, 1992). Molecular lesions in the
lin-3-coding region have been identified in seven out of the eight
known lin-3 mutant alleles (Liu
et al., 1999
). The mutation in the eighth allele, e1417,
is not in the coding region of lin-3. As e1417 mutants are
defective only in the vulval development and LIN-3 from the AC is necessary to
induce vulvae, this suggests that the mutation may reside in a regulatory
region that is necessary to specify lin-3 expression in the AC
(Ferguson and Horvitz, 1985
;
Hill and Sternberg, 1992
;
Horvitz and Sulston, 1980
;
Liu et al., 1999
;
Sulston and Horvitz,
1981
).
Although lin-3 was discovered because of its role in vulval
development, lin-3 is also required for growth and viability,
hermaphrodite fertility, male spicule development, and cell fate specification
of the P12 neuroblast and the uterine uv1 cells
(Chamberlin and Sternberg,
1994; Chang et al.,
1999
; Clandinin et al.,
1998
; Ferguson and Horvitz,
1985
; Jiang and Sternberg,
1998
). Studies including laser ablation experiments identified
several cells as sources for the induction of the EGF signaling pathway, such
as the AC for vulval induction (Kimble,
1981
), vulF cells of the primary vulva for uv1 cells specification
(Chang et al., 1999
), and male
F and U cells for spicule development
(Chamberlin and Sternberg,
1994
).
In this study, we identified the molecular lesion in lin-3(e1417) and the regulatory region (59 bp) of lin-3 that drives AC-specific expression. This enhancer region contains two E-box elements and one FTZ-F1 nuclear hormone receptor (NHR) binding site, both of which are necessary for lin-3 expression in the AC. The HLH-2 protein, a basic helix-loop-helix (bHLH) protein and C. elegans homolog of mammalian E-protein and Drosophila Daughterless, binds to both E-box elements. The NHR-25 protein, which is a C. elegans homolog of Drosophila FTZ-F1 NHR, binds to the wild-type form of the NHR-binding site, but not to the e1417 form of the site. Blocking nhr-25 expression using RNAi causes defects in vulval development but does not affect lin-3 expression in the AC, suggesting that NHR-25 in other cells is important for vulval development and that NHRs other than NHR-25 are necessary for lin-3 expression in the AC. Blocking hlh-2 expression using RNAi causes defects in vulval development and also affects lin-3 expression in the AC, suggesting that hlh-2 is required for the expression of lin-3 in the AC.
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Materials and methods |
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Transgenic lines were generated using a standard microinjection protocol
(Mello et al., 1991). Each
gfp construct (100 ng/µl) and a rescue plasmid (50 ng/µl) (pBX,
pMH 86 or pDP#MM016B) were co-injected into pha-1; him-5, dpy-20 or
unc-119 animals. After injection, transgenic animals were obtained by
growing at 20°C (pha-1) or by rescuing a Dpy or Unc phenotype
(dpy-20 or unc-119).
The wild-type strain used in this study is C. elegans var. Bristol strain N2. The following mutant strains of N2 were used: dpy-20(e1282), unc-119(ed4), pha-1(e2123ts); him-5(e1490), dpy-20(e1282) syIs49[zmp-1::gfp; dpy-20(+)] and lin-3(e1417). Information about these alleles can be found through WormBase (http://www.wormbase.org).
Sequence analysis of lin-3 genomic region
To identify the lin-3(e1417) mutation, 11.4 kb of lin-3
genomic region including 6 kb of upstream sequence was amplified from N2 and
lin-3(e1417) animals using PCR. The PCR products were directly
sequenced using several internal DNA sequencing primers and the dideoxy chain
termination method with a ABI PRISM cycle sequencing kit (Applied Biosystems,
Foster City, CA), and the DNA sequencing chromatograms of N2 and
e1417 were compared. When ambiguous sequence differences were
observed, sequencing was repeated with another sequencing primer or using the
other strand as a template.
Construction of lin-3::gfp enhancer assay reporters
lin-3::gfp constructs containing different lengths of
lin-3 were prepared by fusing gfp after the transmembrane
domain of an inactive form of lin-3, in which nucleotides encoding
two cysteine residues in the EGF domain were changed to those encoding serine
residues (Hill and Sternberg,
1992). Two of the constructs contain either 10 kb or 3 kb of
5' upstream sequences from the first lin-3 exon. The other two
contain either 4 kb or 0.2 kb of 5' upstream sequences from the putative
second promoter in the fourth intron of lin-3. The 59 bp of the ACEL
(Anchor cell-specific enhancer of lin-3) DNA fragments were
PCR-amplified from N2 and lin-3(e1417) genomic DNA, and the PCR
products were cloned into a
pes-10::gfp enhancer assay vector
(Fire et al., 1998
). PCR
amplification was also used to generate deletion and site-directed mutations
in the ACEL. All of the mutations were confirmed by DNA sequencing.
Preparation of recombinant proteins
The entire open reading frames of hlh-2 and luciferase
were N-terminally tagged with a FLAG epitope by insertion into a pCMVTag2
vector (Stratagene, La Jolla, CA). The FLAG-cDNAs were then inserted into a
pFastBacHT vector (Life Technologies, Gaithersburg, MD) in which a
(His)6 tag is located at the N terminus to facilitate rapid
purification of recombinant proteins. Recombinant proteins were expressed in
Sf9 insect cells and purified using nickel agarose according to the
manufacturer's instructions (Life Technologies). Purified proteins were stored
at 80°C in aliquots.
The NHR-25 proteins were synthesized by transcribing - and
ß-nhr-25 cDNAs in pCMV-Tag2 vectors from a T3 promoter with T3
RNA polymerase and then translating the mRNA in rabbit reticulocyte lysates in
the presence of 35S-methionine or cold methionine according to the
manufacturer's instructions (Promega Madison, WI).
The proteins were analyzed by SDS-PAGE and immunoblotting as described
previously (Hwang et al.,
1999). Briefly, proteins were resolved by SDS-PAGE, transferred to
a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA), and probed
with mouse anti-FLAG immunoglobulin G (IgG) antibodies (1:1000 dilution) and
then with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies
(1:1,000 dilution; Vector Laboratories, Burlingame, Calif.). Antibody binding
was detected by enhanced chemiluminescence (ECL reagents; Amersham, Little
Chalfont, Buckinghamshire, UK).
Electrophoretic mobility shift assay
The DNA fragment that contains one E-box near the 5' end, the other
E-box in the middle, and a FTZ-F1 binding site between the two E-boxes, was
used as the wild-type ACEL probe. The three binding sites were systematically
mutated in other probes. Each probe was labeled with
32P--dCTP and Klenow DNA polymerase as described
(Hwang et al., 1999
). Two
different buffers were used to form protein-DNA complexes: buffer A (12 mM
HEPES, pH 7.9, 60 mM KCl, 5 mM MgCl2, 4 mM Tris-HCl, 0.6 mM EDTA, 1
mM DTT, 12% (v/v) glycerol) for HLH-2; and buffer B (10 mM HEPES, pH 7.7, 100
mM NaCl, 2 mM DTT, 12% glycerol) for NHR-25.
The binding reaction was initiated by adding proteins in 10 µl of a
reaction mixture that contains 1 ng of 32P-labeled probe, 1 µg
of BSA and 1 µg of poly (dI-dC)-(dI-dC) to mask the effects of non-specific
DNA-binding proteins. After incubating on ice for 30 minutes, the mixture was
resolved by non-denaturing polyacrylamide gel electrophoresis in 0.3xTBE
at 4°C, and the gel was dried and exposed to a phosphoimager. Anti-FLAG
(M2; IBI-Kodak, New Haven, Conn.) antibodies were used in a mobility
supershift assay of protein-DNA complex as described previously
(Hwang et al., 1999). Briefly,
proteins were pre-incubated on ice for 10 minutes with the FLAG antibodies,
incubated on ice for 30 minutes with DNA probe, and then resolved by
nondenaturing gel electrophoresis.
RNAi experiments
RNAi was performed by soaking synchronized animals in hlh-2 or
nhr-25 dsRNA solutions (Tabara et
al., 1998). RNA was synthesized in vitro using a Ambion MEGAscript
kit (Ambion, Austin, TX). Equal amounts of sense and anti-sense strand RNA
were denatured at 80°C for 5 minutes, mixed and slowly cooled to room
temperature to generate dsRNA.
Animals were grown on 10 cm special NGM plates
(Brenner, 1974; but with
peptone at 2% (w/v) and cholesterol at 20 mg/l). When most animals were young
gravid adults they were collected and treated with hypochlorite. Eggs were
then allowed to hatch in M9 and transferred to special NGM plates after 17 to
22 hours (Lewis and Fleming,
1995
). Synchronized L1 animals were harvested from the plates at
different stages. The larvae were then either soaked immediately in
hlh-2 or nhr-25 dsRNA solutions, or grown on regular plates
for 10-16 hours and then soaked in the dsRNA solutions. After the soaking,
animals were transferred to regular plates and phenotypes were scored at
several developmental stages.
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Results |
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A point mutation in lin-3 abolishes its expression in the AC
As the lin-3(e1417) mutation causes only a defect in vulval
development and the molecular lesion of the lin-3(e1417) allele was
not identified in the coding region of lin-3
(Liu et al., 1999), we
searched for the e1417 mutation in the non-coding region of
lin-3, expecting that the e1417 mutation might lead to the
identification of an AC-specific enhancer element for lin-3. We
compared genomic DNA sequences of lin-3 from wild-type and
e1417 mutant animals, and identified a G to A change located just
5' to the second promoter (labeled`G to A' in
Fig. 2A). The
lin-3::gfp construct that expresses GFP only in the AC
(Fig. 1A, construct 1) also
includes the region of the`G to A' change. The 5' end of the
lin-3 sequence in the lin-3::gfp construct 1 was 54 bp
upstream from this lesion. Both findings suggested that an enhancer for the
AC-specific expression of lin-3 is located just 5' to the
second promoter.
|
To test the potential for this conserved region to function as an
AC-specific enhancer, wild-type and e1417 forms of the putative
enhancer region (59 bp in C. elegans) were fused with a
pes-10::gfp enhancer assay vector
(Fig. 2A). The
pes-10::gfp vector itself expresses gfp in cells near
the tail, thus providing a way to identify transgenic animals containing
extra-chromosomal arrays. When the wild-type form of the putative enhancer was
fused with the
pes-10::gfp, 83% of the animals expressed
gfp in the AC. By contrast, gfp expression in the AC was not
observed in any of the animals that contain extrachromosomal arrays of either
pes-10::gfp itself (15 animals) or the e1417 form of
the putative enhancer fused with
pes-10::gfp (33 animals)
(Fig. 2A). Therefore, this 59
bp region, ACEL (AC-specific enhancer of lin-3), is an AC-specific
enhancer, and the e1417 change of G to A in this region inactivates
the enhancer activity.
E-box and FTZ-F1 nuclear hormone receptor binding sites in the ACEL are necessary for lin-3 expression in the AC
Deletion and site-directed mutagenesis studies suggest that three
cis-acting elements in the ACEL are necessary to express
lin-3::gfp in the AC (Fig.
3). Mutation of the FTZ-F1 binding site (e1417 mutation)
as well as deletions on either side of the FTZ-F1 binding site eliminated
gfp expression in the AC (Fig.
3A). Consistent with the deletion analysis, mutation of the E-box
(`CACCTG' to`CACCAA') on either side of the ACEL impaired the ability of the
ACEL to express gfp in the AC
(Fig. 3B, constructs 9 and 11).
Thus, both of the E-boxes on either side of the FTZ-F1 binding site were
necessary to express lin-3::gfp in the AC. By contrast, site-directed
mutagenesis of the predicted POU binding sites did not affect the AC-specific
expression (Fig. 3B, constructs
2-7). Furthermore, RNAi against all three POU homeodomain genes in C.
elegans (ceh-6, ceh-18 and unc-86) did not affect the
lin-3::gfp expression in the AC or interfere with vulval induction
(data not shown). Thus, we exclude the possibility POU proteins from playing a
role in the AC-specific lin-3 expression. Mutations in other
non-conserved regions did not affect the lin-3::gfp expression in the
AC (Fig. 3B).
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To circumvent the effect of hlh-2 RNAi on early gonadal development, we carried out the RNAi experiments with L2 and L3 animals. Animals treated with hlh-2 dsRNA at these later stages showed normal gonadal morphology and were fertile. However, mid- to late L2 stage animals soaked in the dsRNA induced a partial vulvae (Fig. 5F) and did not show lin-3::gfp expression in the gonad. As hlh-2 RNAi was initiated at the stages in which lin-3::gfp was already expressed in the AC, the incomplete vulval induction most probably reflects a functional reduction of lin-3 expression in the AC (roughly similar to ablation of the AC during vulval induction). When the hlh-2 RNAi was performed with early L3 stage animals, 45% (17/38) of the animals induced VPCs normally but showed defects in the vulval-uterine connection (Fig. 5G), 18% (7/38) induced a partial vulvae, and 37% induced a normal vulvae and vulval-uterine connection. About 40% (6/15) of the animals with normal vulval induction showed little or no expression of lin-3::gfp in the AC (Fig. 5B), suggesting that the AC-specific gfp expression had been eliminated by hlh-2 RNAi after vulval induction. In control RNAi experiments, only one out of 1 more than 100 animals examined did not express lin-3::gfp in the AC, suggesting a low frequency of the mosaic loss of lin-3::gfp expression in the AC. Taken together, the hlh-2 RNAi experiments demonstrate that the continuous expression of hlh-2 is necessary to maintain the expression of lin-3 in the AC.
|
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To determine whether nhr-25 is the NHR necessary to express
lin-3 in the AC, we performed a RNAi experiment similar to that
performed with hlh-2 as the null mutant of nhr-25 is also
not viable (Asahina et al.,
2000). RNAi with L1 larvae resulted in a severe defect in gonadal
development and a complete failure to induce vulvae. RNAi using later stages
of animals caused defects in vulval induction and in the vulvaluterine
connection (24/32 animals) similar to the RNAi against hlh-2
(Fig. 5H-J). However,
nhr-25 RNAi did not eliminate lin-3::gfp expression in the
AC, even in the animals that had defects in vulval induction. We cannot rule
out the possibility that nhr-25 RNAi slightly decreases the
lin-3::gfp expression. Failure of the nhr-25 RNAi to
eliminate the LIN-3::GFP signal in the AC can be interpreted in two different
ways. First, the NHR-25 protein in the AC may be too stable to be completely
eliminated by RNAi. Alternatively, as there are about 270 nhr genes
in the C. elegans genome, another NHR protein(s) may be involved in
expressing lin-3 in the AC
(Sluder and Maina, 2001
).
Because nhr-25 is also expressed in the VPCs
(Asahina et al., 2000
;
Gissendanner and Sluder, 2000
)
and its null mutant has not been rescued, most probably because NHR-25 plays
an important role in germ line development
(Asahina et al., 2000
), we
cannot determine by mosaic analysis whether the defects in vulval induction by
nhr-25 RNAi result from a partial reduction of lin-3
expression in the AC or from inhibiting a function of NHR-25 in the VPCs.
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Discussion |
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The spatially and temporally regulated cell-specific expression of the EGF
ligand lin-3 reflects an activation mechanism for the EGF receptor
signaling pathway in C. elegans which is distinct from that observed
in Drosophila. In Drosophila, the activation of EGF receptor
signaling is regulated by cleavage of the ligands in specific cells rather
than by cell-specific ligand expression
(Freeman, 1997;
Gabay et al., 1997
;
Ghiglione et al., 2002
;
Urban et al., 2002
). The main
activating EGF ligand, Spitz, is expressed in most tissues during fly
development (Rutledge et al.,
1992
), including all developing photoreceptors
(Tio et al., 1994
). However,
its processing is tightly controlled by Rhomboid and Star proteins, expression
of which is restricted to specific cells
(Freeman et al., 1992
;
Heberlein et al., 1993
).
AC-specific lin-3 transcription
We have identified a 59 bp enhancer element (ACEL) that directs
lin-3 expression in the AC, consisting of two E-boxes (bHLH protein
binding sites) and one FTZ-F1 nuclear hormone receptor-binding site. We found
that the C. elegans E-protein homolog, HLH-2, binds to the enhancer
element to activate the lin-3 transcription, and that NHR-25 also
binds to the enhancer. E-protein/Daughterless proteins generally recognize
target DNA sequences as a heterodimer with other bHLH proteins
(Massari and Murre, 2000).
However, we prefer the model that a HLH-2 homodimer activates lin-3
transcription in the AC as purified HLH-2 proteins alone recognize the E-box
(Zhang et al., 1999
), and
hlh-2 is expressed in the AC but not in the VU cells
(Karp and Greenwald, 2003
).
The nhr-25 gene is expressed in the AC
(Gissendanner and Sluder,
2000
), and its protein binds to the wild-type form but not to the
e1417 form of the NHR binding site in the ACEL
(Fig. 6). However,
nhr-25 appears not to be the NHR that activates lin-3
transcription in the AC as RNAi against nhr-25 did not eliminate
lin-3::gfp expression in the AC. About 270 nhr genes were
predicted in C. elegans and most of them are not pseudogenes
(Sluder and Maina, 2001
); this
contrasts with 21 nhr genes in Drosophila and 50 in human
(Sluder and Maina, 2001
). All
of the C. elegans NHRs are orphan receptors for which ligands have
not been identified, but evidence indicates the presence of unidentified
ligands such as steroids, metabolic intermediates and external materials from
the environment (Sluder and Maina,
2001
). Furthermore, although the amino acid sequences of the
ligand-binding domains in C. elegans NHRs are evolutionarily less
conserved than those of the DNA-binding domains
(Clarke and Berg, 1998
),
structural modeling indicates that many of the C. elegans ligand
binding domain sequences are compatible with the X-ray crystal structures of
the known ligand-binding domains
(Francoijs et al., 2000
),
suggesting they may bind to ligands.
The two transcriptional regulatory activities necessary for lin-3
expression in the AC (Fig. 7)
might reflect distinct regulatory inputs that program the appropriate time,
place and level of lin-3 expression. The presence of a NHR-binding
site in the ACEL makes it conceivable that unidentified NHR ligand(s)
responding to physiological conditions and environments might control vulval
development by activating lin-3 expression in the AC. As
lin-12/Notch signaling is involved in the fate determination of the
AC (Seydoux and Greenwald,
1989; Wilkinson et al.,
1994
), it is interesting to know how or whether the
lin-12 signaling is coupled to the AC-specific lin-3
expression. Several pieces of evidence suggest such a coupling. The expression
pattern of lag-2, a lin-12 ligand that is involved in the
AC/VU cell fate determination, overlaps with that of lin-3 in the
somatic gonad. The lag-2 gene is also expressed in the pre-AC/VU
cells before the cell fate determination, but only in the AC after the fate
determination (Wilkinson et al.,
1994
). It was shown that this kind of lag-2 expression
pattern is established by the interaction between lag-2 and
lin-12 during the AC/VU cell fate determination
(Seydoux and Greenwald, 1989
;
Wilkinson et al., 1994
). As
the AC-specific lin-3 expression is established at the time of the
cell fate determination, it is likely that lin-12 signaling is also
involved in establishing the AC-specific lin-3 expression. The
mechanism that establishes the lag-2 or lin-3 expression in
the AC is not understood well, but interestingly a well-known Notch downstream
pathway, which is involved in the specification of sensory organ precursors in
the Drosophila peripheral nervous system, involves the binding of
bHLH proteins to E-boxes (Heitzler et al.,
1996
; Kunisch et al.,
1994
; Parks et al.,
1997
). Therefore, the findings that E-boxes in the ACEL are
necessary for the AC-specific lin-3 expression and that the
expression patterns of lag-2 and lin-3 overlap in the
somatic gonad suggest that a similar kind of Notch downstream pathway may
exist to specify lag-2 or lin-3 expression in the C.
elegans AC.
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ACKNOWLEDGMENTS |
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