1 Department of Molecular, Cellular and Developmental Biology, University of
California, Santa Barbara, CA 93106, USA
2 Department of Biological Sciences, University of Maryland Baltimore County,
Baltimore, MD 21250, USA
* Present address: Department of Genetics, University of Pennsylvania,
Philadelphia, PA 19104, USA
Author for correspondence (e-mail:
rothman{at}lifesci.ucsb.edu)
Accepted 9 August 2002
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SUMMARY |
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Key words: Cell fusion, Vulva, GATA factor, Hox, C. elegans
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INTRODUCTION |
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Development of the vulva in C. elegans hermaphrodites is
controlled by the intersection of several conserved signaling pathways,
including the Ras, Wnt, Notch and Rb-related pathways; it has therefore served
as a useful model system with which to study the function of these pathways
(for reviews, see Greenwald,
1997; Kornfeld,
1997
; Wang and Sternberg,
2000
). During the L1 larval stage, 12 cells (P1.p-P12.p) are born
along the ventral midline. While Pn.p cells in the anterior and posterior body
regions (P1.p-P2.p and P9.p-P11.p, respectively) fuse with the surrounding
syncytial epidermis shortly after their birth, the central Pn.p cells
(P3.p-P8.p), or vulval precursor cells (VPCs), remain unfused and are
competent to generate cells of the vulva. In response to a Rasmediated
inductive signal from the gonadal anchor cell during the L3 stage, three
central VPCs, P5.p, P6.p, and P7.p, undergo three rounds of cell division,
adopting the secondary, primary and secondary vulval fates, respectively. P4.p
and P8.p divide once and then fuse with the surrounding syncytium, adopting
the tertiary fate, and P3.p either fuses without dividing (adopting the `F'
fate) or adopts the tertiary fate.
The C. elegans Hox gene, lin-39, plays a pivotal role in
the development of the mid-body region, and controls several aspects of vulval
development (Clark et al.,
1993; Clandinin et al.,
1997
; Maloof and Kenyon,
1998
; Wang et al.,
1993
). The LIN-39 protein is expressed in mid-body cells,
including the VPCs, and is required to prevent the VPCs from fusing with the
surrounding syncytium (Clark et al.,
1993
; Wang et al.,
1993
). LIN-39 performs an additional function in induction of
vulval cell fates by the anchor cell-activated Ras signaling pathway. Recent
studies have demonstrated that several regulatory inputs control
lin-39 expression in the developing vulva. The Ras, Wnt, and
Rb-related pathways coordinately regulate lin-39 in the VPCs
(Chen and Han, 2001
;
Eisenmann et al., 1998
).
Moreover, the SEM-4 transcription factor also regulates lin-39 in the
VPCs during the L2 and L3 stages (Grant et
al., 2000
) and lin-39 itself is required to upregulate
lin-39 expression in response to Ras signaling
(Maloof and Kenyon, 1998
).
Although the regulatory inputs into lin-39 expression have been
characterized, the downstream targets of this Hox gene and how it executes its
regulatory functions in the vulva are unknown.
We report that a pair of GATA-type transcription factors, ELT-5 and ELT-6,
previously shown to be essential for regulation of epidermal seam cell fusion
and differentiation (Koh and Rothman,
2001), are essential regulators of cell fates and fusion during
vulval development. Our results indicate that ELT-5 is encoded by the
egl-18 gene, previously identified in screens for mutants with vulval
and egg-laying defects (Eisenmann and Kim,
2000
; Trent et al.,
1983
), and that EGL-18 (ELT-5) and ELT-6 are likely to be direct
targets of lin-39 in the developing vulva.
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MATERIALS AND METHODS |
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Molecular identification of egl-18
Rescue of an egl-18 mutation by the cloned elt-5 gene
(Koh and Rothman, 2001) was
tested using a GFP-tagged elt-5 transgene containing
3.4 kb of
upstream sequence and all intronic sequences (pKK52)
(Koh and Rothman, 2001
).
Although 100% (n>200) of egl-18(n162) mutant animals
exhibited Lumpy and Uncoordinated phenotypes at hatching, most (73%,
n=66) of the egl-18(n162) animals carrying the
elt-5 transgene (as determined by the presence of GFP) had a
wild-type morphology and movement. The elt-5 transgene also partially
rescued the vulval defects, or abnormal vulval invaginations, at the L4 stage
(30% wild type, n=79, without the elt-5 transgene versus 60%
wild type, n=109 with the elt-5 transgene;
2(1)=16.7; P<0.0001).
We sequenced the entire elt-5-coding region and intron-exon boundaries of the four previously identified egl-18 alleles (ga97, n162, n474 and n475), as well as a deletion allele (ok290), recently isolated by the Genome Knockout Consortium, from two independent PCR reactions of genomic DNA each. For the latter, we identified an 816 bp deletion that removes sequences from a region of exon 2 through to a region of exon 4 (corresponding to base pairs 698-1513 relative to the egl-18 ATG).
RNA-mediated interference (RNAi)
egl-18 or elt-6 dsRNA (2 mg/ml), prepared as
described (Koh and Rothman,
2001
), was injected into young hermaphrodites
(Fire et al., 1998
) and
progeny laid at least 12 hours after injection were analyzed. Injection of
egl-18 dsRNA into N2 adults resulted in fully penetrant lethality of
their progeny. When egl-18 dsRNA was injected into wEx1070
animals, in which ELT-6 is driven by a partial (
3.4 kb) promoter of
egl-18 (pKK47) (Koh and Rothman,
2001
), the progeny developed to fertile adults but exhibited
variable vulval defects. We report data pooled from multiple injected animals
whose progeny were affected to varying degrees.
Characterization of lethal and vulval phenotypes
To determine the penetrance of the lethal phenotypes, embryos from
individual hermaphrodites were collected over sequential 12-24-hour periods
and the embryos and larvae counted. Three days after the initial count, the
live adults were counted. To characterize vulval phenotypes of
egl-18(RNAi) animals rescued for lethality, wEx1070
hermaphrodites injected with egl-18 dsRNA were allowed to lay eggs
and develop at 15, 20 or 25°C. F1 animals were scored at the L4
stage by Nomarski microscopy to determine the number of VPCs that had adopted
induced cell fates and contributed to the vulva. In addition, F1
progeny of injected animals were allowed to develop into adults and their
vulval phenotypes examined. Lineage analysis was performed as described
(Eisenmann and Kim, 2000).
To determine the time of VPC fusion, egl-18 RNAi was performed on
worms carrying both wEx1070 and jcIs1 (ajm-1::GFP)
(Koppen et al., 2001;
Mohler et al., 1998
). The
presence of the adherens junction GFP expression surrounding a VPC indicated
that it had not yet fused or divided, while the absence of expression was
taken as evidence of fusion.
Reporter constructs and germ-line transformation
DNA constructs were made according to standard methods
(Sambrook et al., 1989). Two
transcriptional constructs, pKK62 and pKK63, were used as the basic
egl-18/elt-6::GFP reporters. Each contains the 792 bp fragment
surrounding intron 2 of the egl-18 gene (positions 622-1413 relative
to the egl-18 ATG) and 200 bp of the egl-18 basal promoter
immediately upstream of the ATG. pKK62 contains the gfp-coding region
(derived from pPD95.67; all pPD vectors are gifts of A. Fire) fused in frame
shortly after the egl-18 ATG, and pKK63 contains the gfp and
ß-galactosidase coding regions (derived from pPD96.04) fused at the same
site. Transgenic animals carrying either construct showed GFP expression in
the VPCs, their descendants and VC neurons. Some lines also showed GFP
expression in the intestinal cells. pKK63 was used to characterize the normal
egl-18/elt-6 expression pattern, and pKK62 for generating mutant
versions. Several pKK62 derivatives were made as follows (mutated bases are in
lower case).
Site-directed mutagenesis for pKK70, pKK73 and pKK74 was performed using the QuickChange kit (Stratagene) according to the manufacturer's protocol. Constructs were sequenced to confirm the targeted mutations.
Each construct ( 100 µg/ml) was co-injected with
unc-119(+) (pDP#MM016B,
100 µg/ml) into
unc-119(ed4) hermaphrodites. We observed qualitatively similar,
albeit weak, expression at lower levels (
50 µg/ml) of injected DNA.
Some lines showed weak, widespread neuronal expression, apparently an artifact
attributable to the unc-119(+)maker. To confirm expression
patterns, some constructs were also co-injected into N2 worms with
ceh-22::GFP and gcy-5::GFP constructs (each at
50
µg/ml, gifts of P. Okkema and D. Garbers, respectively) as co-injection
markers.
Effects of reducing lin-39 activity on
egl-18/elt-6::GFP
To examine egl-18/elt-6::GFP expression in animals with reduced
lin-39 activity, we performed two experiments. First, we used
n709, a temperature-sensitive allele of lin-39, to construct
a strain, JR2195, containing the n709 mutation as well as an
integrated array with egl-18/elt-6::GFP (wIs129) and an
integrated array with ajm-1::GFP (jcIs1).
ajm-1::GFP was used to identify unfused P5.p-P7.p cells during the
late L2 through mid L3 stages, and the percentage of cells expressing
egl-18/elt-6::GFP in JR2195 animals was compared with the percentage
in a lin-39(+) strain (JR2193) carrying wIs129 and
jcIs1. Both lin-39(n709) and wIs129 were
temperature-sensitive: at higher temperature, more VPCs fused in n709
animals and more VPCs expressed GFP in wIs129 animals. The experiment
was performed at 20°C, which allowed some VPCs to remain unfused in
n709 animals and GFP expression was detectable in the majority of
P5.p-P7.p cells in wIs129 animals.
Second, we compared JR2193 worms (containing egl-18/elt-6::GFP and
ajm-1::GFP) soaked in lin-39 dsRNA with worms soaked in
either H2O or control dsRNA. L1 larvae were soaked in concentrated
(2 mg/ml) dsRNA or H2O in the presence of food for
16
hours. The larvae were transferred to plates and allowed to continue to
develop before examination by fluorescence microscopy. Soaking in
lin-39 dsRNA caused the majority of VPCs to fuse, but some remained
unfused, and only the unfused P5.p-P7.p cells during the late L2 to mid-L3
stages were scored. Data from animals soaked in H2O were combined
with those from animals soaked in control dsRNA, as they were comparable. The
worms were grown at 25°C throughout the experiment.
Overexpression of EGL-18 in lin-39(RNAi) animals
Two egl-18 heat-shock constructs, pKK8 and pKK9, were made by
cloning the entire egl-18-coding region into vectors pPD49.78
(hsp-16.2) and pPD49.83 (hsp-16.41). Both constructs were
co-injected with ceh-22::GFP and gcy-5::GFP markers into N2
hermaphrodites to obtain JR2268. lin-39 RNAi by feeding was carried
out as described (Gleason et al.,
2002). The timing of this RNAi protocol does not interfere with
the early function of lin-39 (VPC generation), but does affect the
later function of lin-39 (VPC fate specification). Control (N2) and
experimental (JR2268) animals were given five heat shocks (37°C for 15
minutes) during the L2 and L3 stages. The first heat shock was administered at
the midpoint of the L2 stage, followed by a heat shock 1 hour later, and then
three more heat shocks every 2.25 hours. These animals were allowed to
continue development at 20°C and were then scored by Nomarski microscopy
for the number of VPCs adopting induced vulval fates as described above.
LIN-39 and CEH-20 protein purification
6His-tagged versions of LIN-39 and CEH-20 were produced using the pRSET
vectors pJKL430 and pRL434, respectively (gifts from J. Liu and A. Fire.)
BL21-Codon Plus cells (Stratagene) carrying these vectors were grown to an O.D
of 0.6-0.8 and induced with 1 mM IPTG for 3-4 hours. Cells were lysed in
buffer A [8 M urea/10 mM Tris HCl (pH 8.0)/100 mM
NaH2PO4/20 mM BME/30 mM imidazole]. Proteins were
purified on a Superflow Ni2+-NTA column (Qiagen) via FPLC (BioRad
BioLogic HR Workstation). Proteins were renatured on the column using a linear
gradient of buffer B (500 mM NaCl/20 mM Tris HCl pH 8.0/20% glycerol) and
eluted with buffer E [250 mM imidazole/300 mM NaCl/50mM
NaH2PO4 (pH 8.0) plus protease inhibitors]. Fractions (1
ml) were collected and those with LIN-39 or CEH-20 were pooled and glycerol
was added to 50% for storage. Proteins are greater than 80% pure based on
Coomassie staining.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays were performed as described
(Chang et al., 1995) with
modifications. DNA binding was carried out at 4°C in a volume of 15 µl
with 250 ng of LIN-39 and/or 2.5 µg of CEH-20 and final buffer conditions
of 2 µg poly dI-dC/75 mM NaCl/1 mM EDTA/1 mM DTT/10 mM Tris HCl (pH 8.0)/2
µg BSA/25% glycerol. 32P labeled oligonucleotide (5000 cpm) was
added per reaction. After 30 minutes, samples were loaded onto a 6%
polyacrylamide gel run in 0.5xTBE buffer at 100 V. To make labeled
oligonucleotides, 5 pmole of one oligonucleotide was incubated with 10 µCi
of 32P (300 Ci/mmol, NEN) and T4 polynucleotide kinase (New England
Biolabs) at 37°C for 10 minutes and then at 80°C for 5 minutes.
Complementary strand oligonucleotide (10 pmole) was then added, incubated an
additional 5 minutes at 80°C and then slow cooled to room temperature. The
labeled, double-stranded oligonucleotide was purified over a Centri-Spin-20
column (Princeton Separation). The oligonucleotides used (one strand only) are
as follows (lower case indicates mutated bases).
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RESULTS |
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egl-18 chromosomal mutants are lumpy and uncoordinated at hatching
and show partially penetrant embryonic or larval lethality (see
Table 1), defects in alae
(seam-specific cuticular structures) and the vulva, and an occasional roller
(Rol) phenotype (Fig. 1)
(Eisenmann and Kim, 2000). We
found that these egl-18 phenotypes can be rescued with a transgenic
elt-5 gene (see Materials and Methods). Moreover, we identified
molecular legions in the elt-5-coding region in all four previously
identified egl-18 alleles (Fig.
2). Three alleles, n475, ga97 and n162,
contained nonsense mutations; the fourth, n474, carried a deletion of
a single base pair, causing a frame-shift and introduction of a premature stop
codon. All four alleles are predicted to encode polypeptides that are
truncated before the DNA-binding domain. In addition, we found that
ok290 mutants, recently isolated by the C. elegans Genome
Knockout Consortium, show similar phenotypes to the previously described
egl-18 mutants. This latter mutation is a deletion of an 816 bp
fragment of elt-5 that removes the zinc-finger region. We conclude
that egl-18 encodes the ELT-5 GATA factor.
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egl-18 is functionally redundant with elt-6
Whereas 100% of the progeny of adults injected with high doses of
egl-18 dsRNA arrest by the early L1 larva stage
(Koh and Rothman, 2001),
egl-18 chromosomal mutants that are likely null alleles based on
their molecular lesions (e.g. the n475 mutation truncates over 90% of
the protein), show only partially penetrant lethality
(Table 1). The survival of
egl-18 mutants may be attributable to intact elt-6 activity.
Indeed, interfering with elt-6 function by treatment with
elt-6 dsRNA in egl-18 chromosomal mutants caused nearly
fully penetrant late-embryonic/early-larval lethality
(Table 1). These results
indicate that egl-18 and elt-6 are functionally redundant
during embryonic development and imply that egl-18 mutations affect
egl-18 activity alone, elt-6 dsRNA affects elt-6
activity alone, and egl-18 dsRNA affects both egl-18 and
elt-6 activity. This is consistent with previous observations
suggesting that egl-18 and elt-6 are both monocistronically
and dicistronically transcribed and that egl-18 dsRNA interferes with
expression of an elt-6 reporter gene containing the
egl-18-coding region (Koh and
Rothman, 2001
).
egl-18 and elt-6 appear to function redundantly in vulval
development as well as viability. Whereas approx. two-thirds of
egl-18(RNAi) animals rescued for lethality by the extrachromosomal
array wEx1070 are Vulvaless (i.e., all VPCs adopted either the F or
tertiary fate, Table 2 and
Fig. 3), such strong vulval
defects were rarely observed in egl-18 chromosomal mutants
(Eisenmann and Kim, 2000)
(Fig. 1). This difference is
unlikely to result from non-specific interference of genes other than
egl-18 and elt-6, as dsRNA made from two non-overlapping
regions of egl-18 yielded essentially the same results (not shown).
Rather, these results imply that egl-18(RNAi) affects both
egl-18 and elt-6 activity, which function redundantly. For
simplicity, we will refer to animals subjected to egl-18 dsRNA as
egl-18(RNAi) mutants, though it is probable that both egl-18
and elt-6 activity is compromised in such animals.
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Vulval defects in the absence of egl-18 and elt-6
function
To further characterize the vulval defects in egl-18(RNAi);
wEx1070 animals (in which lethality is rescued but egl-18/elt-6
are not expressed in the vulval primordium) we followed the cell lineages of
the VPCs in ten animals (Table
3). Almost all the VPCs followed adopted inappropriate cell fates.
Many VPCs inappropriately adopted the F fate, and the P5.p-P7.p cells often
stopped dividing after one or two cell divisions (`SS' or `NNNN'). Previous
lineage analysis of egl-18 chromosomal mutants
(Eisenmann and Kim, 2000)
revealed similar, but milder, defects in VPC fusion and number of divisions.
These results indicate that EGL-18 and ELT-6 are key regulators of vulval
development.
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To determine when VPCs fuse in egl-18(RNAi); wEx1070 animals, we
examined expression of ajm-1::GFP (a marker of epithelial adherens
junctions) (Koppen et al.,
2001; Mohler et al.,
1998
) at several times from late L1 through L3 stages. In
wild-type animals, six Pn.p cells (P3.p-P8.p) remain unfused during the late
L1 and early L2 stages. During the ensuing L2 and L3 stages, only P3.p fuses,
and does so only 50% of the time; thus, there are five or six Pn.p cells
surrounded by the ajm-1::GFP signal at this stage. In all
egl-18(RNAi); wEx1070 animals examined, six Pn.p cells expressed
ajm-1::GFP through the early L2 stage. However, beginning around the
mid-L2 stage, egl-18(RNAi); wEx1070 animals often contained fewer
than five Pn.p cells demarcated by ajm-1::GFP, indicating that some
VPCs had inappropriately fused (Fig.
3 and data not shown). Fusion occurred between the mid L2 and L3
stages, which correspond to the time at which the V3.p cells fuse in wild-type
animals. These results implicate egl-18 and elt-6 in
maintaining VPCs in an unfused state during later stages of vulval
development. However, it remains possible that these genes also function in
the generation of the VPCs (see Discussion).
The vulval defects observed in egl-18/elt-6 mutants resemble those
in animals with reduced lin-39 function
(Clandinin et al., 1997;
Clark et al., 1993
;
Maloof and Kenyon, 1998
),
suggesting a close relationship between lin-39 and
egl-18/elt-6.
egl-18/elt-6::GFP is expressed in the VPC lineages and VC
neurons
To further investigate the role egl-18 and elt-6 play in
vulval development, we examined their larval expression. Previous work
demonstrated that these genes are expressed in many cell types, apparently
under the control of separable enhancer elements for different cell types
(Koh and Rothman, 2001). Using
a series of partial promoter reporter constructs
(Koh and Rothman, 2001
) (data
not shown), we identified an
800 bp region surrounding intron 2 of
egl-18 that includes a vulval enhancer. We found that a reporter
construct (pKK63) containing this
800 bp element and an
200 bp basal
promoter fragment of egl-18 is sufficient to drive GFP expression in
the VPCs and their descendants as well as in the six VC motoneurons that
innervate vulval muscles (Fig.
4A-C), which are likely to be co-regulated with vulval cells.
Similar vulval expression was observed when GFP was fused to the start codon
of either egl-18 or elt-6 in a reporter containing
8 kb
of contiguous genomic sequence that includes this
800 bp region
(Fig. 4D and not shown),
suggesting that the
800 bp region is likely to be a vulval enhancer for
both genes. As the expression levels and patterns of pKK63 showed substantial
variability, even among chromosomal integrants of the transgene, our
characterization of the spatial and temporal pattern of
egl-18/elt-6::GFP expression is based on the composite pattern that
emerged from examination of many animals.
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When expression of egl-18/elt-6::GFP is first detected in VPCs
around the mid-L2 stage, all six VPCs are equally likely to express GFP
(Fig. 4A). However, beginning
at around the late-L2/early-L3 stage, until the VPCs divide in the mid-L3
stage, the expression in P5.p-P7.p is generally higher than in the other VPCs,
and P6.p often shows the strongest expression
(Fig. 4B). Expression persists
in the descendants of P5.p-P7.p (Fig.
4C) through the L4 stage, and P6.p descendants typically show
stronger expression than descendants of P5.p and P7.p. This pattern is similar
to that of lin-39 expression in the developing vulva
(Maloof and Kenyon, 1998), and
suggests that, like lin-39, egl-18/elt-6 may be upregulated by
Rasmediated vulval induction.
egl-18/elt-6 act downstream of lin-39 activity in
the VPCs
We tested the relationship between lin-39 and
egl-18/elt-6, by analyzing the effect of reducing lin-39
activity on expression of the latter. We found that lin-39(RNAi)
animals show virtually undetectable expression of egl-18/elt-6::GFP
(not shown). However, almost all VPCs adopt the fused fate in
lin-39(RNAi) animals; the lack of egl-18/elt-6 expression
might simply be a consequence of fusion per se. We therefore examined the
effects of partial reduction of lin-39 function on
egl-18/elt-6::GFP expression, conditions in which at least some VPCs
remained unfused (see Materials and Methods). We found that while 76%
(n=160) of unfused P5.p-P7.p cells in lin-39(+)
worms expressed GFP, only 42% (n=85) did so in animals carrying
n709, a lin-39 reduction-of-function allele; this is a
highly significant difference (2(1)=26.7,
P<0.0001). Furthermore, we found that soaking of L1 larvae in
lin-39 dsRNA significantly reduced the fraction of unfused P5.p-P7.p
cells that express egl-18/elt-6::GFP from 92% (n=110) for
the control soak to 55% (n=128) (
2(1)=39.0,
P<0.0001). In both experiments, reduction of lin-39
activity resulted in lowered egl-18/elt-6::GFP expression.
Consistent with the model in which egl-18 and elt-6 act
downstream of LIN-39 Hox during vulval development, we found that
overexpression of egl-18 from heat-shock promoters is sufficient to
partially rescue vulval defects in lin-39(RNAi) animals. Among
lin-39(RNAi) control animals subjected to heat-shock (five 15 minute
pulses at 37°C), only 36% (n=92) showed wild-type vulval
invaginations at the L4 stage. By contrast, significantly more (76%;
n=98; 2(1)=30.3; P<0.0001)
lin-39(RNAi) animals carrying hs-egl-18 had normal
invaginations following the same heat-shock regimen. In the absence of
heat-shock, lin-39(RNAi) had approximately equal effects on both
wild-type (17% with normal invaginations; n=93) and
hs-egl-18-bearing (16% with normal invaginations; n=93)
animals.
A Hox/PBC-binding site is essential for vulval expression of
egl-18/elt-6::GFP reporters
Hox proteins appear to require co-factors to achieve DNA-binding
specificity (for reviews, see Mann and
Affolter, 1998; Mann and Chan,
1996
). The most extensively studied of the Hox co-factor genes are
the Drosophila extradenticle (exd) and mammalian pre-B
cell homeobox 1 genes, collectively referred to as PBC genes. Hox and PBC
proteins form heterodimers that bind DNA in vitro. C. elegans
contains one known Exd homolog, CEH-20, which appears to act as a Hox
co-factor (Liu and Fire,
2000
). Consistent with the possibility that egl-18 and
elt-6 are direct targets of LIN-39 Hox, we found several consensus
Hox/PBC-binding sites (TGATNNAT) in the egl-18 and elt-6
genomic region (Fig. 5). Two of
these [site 1 (TGATATAT) and site 2 (TGATTGAT)] are present in intron 2 of
egl-18, which is included in the
800 bp promoter element that
directs GFP expression in the VPC lineages and VC neurons. Several lines of
evidence indicate that site 1, but not site 2, is important for
vulval-specific expression of egl-18/elt-6. First, alteration of 6 bp
in site 1 eliminated expression in the VPC lineages and VC neurons, whereas a
similar mutation that alters 4 bp of site 2 had no obvious effect on reporter
expression (Fig. 5; Table 4). Second, a reporter in
which 544 base pairs surrounding only Site 1 is present showed expression in
the vulva and VC neurons (Table
4), albeit at an attenuated level compared with the reporter
containing both sites. Mutation of Site 1 from this construct eliminated
vulval and VC expression (Table
4). Finally, comparison of the egl-18 sequence of C.
elegans and C. briggsae revealed a highly conserved 27 bp
element surrounding Site 1 (Fig.
5B) but no conservation of site 2. Thus, the site 1 Hox/PBC site
is apparently necessary and sufficient for vulva-specific expression of
egl-18/elt-6::GFP.
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LIN-39/CEH-20 dimers bind Hox/PBC sites in the egl-18/elt-6
vulval enhancer
We performed electrophoretic mobility shift assays to test the hypothesis
that egl-18 and elt-6 are direct targets of LIN-39/CEH-20
heterodimers in the vulva. Indeed, we found that LIN-39 and CEH-20
heterodimers bind in vitro to 30 bp oligonucleotides centered on either the
Hox/PBC site 1 or site 2 (Fig.
6). Whereas binding of LIN-39/CEH-20 to site 1 oligos could be
competed away with excess unlabeled site 1 or 2 oligos, unlabeled site 1
oligos could not compete with site 2 oligos (not shown), implying that site 2
has a higher in vitro affinity for LIN-39/CEH-20 than does site 1. Our results
indicate that LIN-39/CEH-20 heterodimers can bind cooperatively to site 1,
which is essential for expression of the egl-18/elt-6 reporter in the
vulva. Based on these results and the phenotypes of egl-18/elt-6
mutants, it seems likely that LIN-39 regulates vulval development by directly
activating EGL-18 and ELT-6, which in turn repress epidermal fusion and
activate vulval differentiation.
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DISCUSSION |
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All five alleles of egl-18 eliminate the zinc-finger DNA-binding domain of the protein and may represent null or strong loss-of-function alleles. However, they exhibit phenotypes considerably milder than those seen in the egl-18(RNAi); wEx1070 strain. This can be explained by proposing that, while egl-18 chromosomal mutations eliminate only egl-18 activity, egl-18(RNAi) abolishes the activity of both egl-18 and elt-6 because of their dicistronic transcription. The vulval phenotypes observed in egl-18(RNAi); wEx1070 animals are somewhat variable; it is possible that the strongest phenotype seen (i.e., all VPCs adopting the F fate) represents the null phenotype of egl-18 and elt-6 double mutants.
We did not obtain evidence that EGL-18 and ELT-6 control one important
aspect of vulval development: the generation of the VPCs during the L1 stage
(i.e. by preventing fusion of the midbody Pn.p cells). Although inappropriate
fusion of the P3.p-P8.p cells occurred during the late L2 and L3 stages in
egl-18(RNAi); wEx1070 animals, we never observed fusion of P3-8.p
cells in late L1 larvae, the stage at which they fuse in lin-39 null
mutants and when other Pn.p cells fuse in wild-type animals
(Clark et al., 1993;
Wang et al., 1993
). One
possible interpretation of this result is that egl-18 and
elt-6 might regulate Pn.p cell fusion specifically during the L2 and
L3 stages. Several other genes, e.g. bar-1 and sem-4,
regulate Pn.p cell fusion only during the L2 and L3 stages
(Eisenmann et al., 1998
;
Gleason et al., 2002
;
Grant et al., 2000
).
Alternatively, egl-18 and elt-6 may regulate early Pn.p cell
fusion, but such a role was not apparent in our experiments because the
partial promoter used to rescue egl-18(RNAi) lethality drives
detectable levels of ELT-6 expression in P cells of embryos as a component of
the widespread AB lineage expression (Koh
and Rothman, 2001
). Thus, residual levels of ELT-6 in Pn.p cells
of late L1 larvae may be sufficient to repress P3.p-P8.p fusion.
The possibility that EGL-18/ELT-6 might repress cell fusion in the early
Pn.p cells would not be unexpected given the role of these genes in regulating
fusion in other epidermal cells (Koh and
Rothman, 2001) earlier in development. In fact, it is conceivable
that EGL-18/ELT-6 might function broadly to repress fusion in non-syncytial
epidermal cells.
egl-18 and elt-6 are likely to be direct targets of
the LIN-39 Hox protein in the vulva
The 800 bp vulval enhancer surrounding intron 2 of egl-18 is
sufficient to drive egl-18/elt-6::GFP expression in the VPC lineages
and the VC neurons. This function requires an intact Hox/PBC consensus site,
which binds LIN-39/CEH-20 heterodimers in vitro. The effect of lin-39
activity on expression of egl-18/elt-6::GFP, and the observation that
overexpression of EGL-18 rescues vulval defects in animals with reduced
lin-39 activity, further suggest that egl-18 and
elt-6 are direct targets of lin-39 in the vulva and VC
neurons and may mediate the positional regulatory information provided by this
Hox gene.
Although site 1 appears to be necessary for vulval expression of reporter constructs, other results indicate that site 1 is not strictly necessary for egl-18/elt-6 expression in vulval development. In ok290 deletion mutants both sites 1 and 2 are removed, but they show relatively mild vulval phenotypes (64% wild-type vulval invagination at the L4 stage) compared with egl-18(RNAi); wEx1070 animals (8% wild-type; see Table 2). This observation implies that site 1 is not the only regulatory site responsible for egl-18/elt-6 expression in the vulva. Other sites, either the potential Hox/PBC binding sites found throughout the egl-18 and elt-6 genomic region (Fig. 5B), or sites controlled by other regulatory factors, may contribute to egl-18/elt-6 expression during vulval development.
It is interesting to note that while site 2 shows higher affinity in vitro,
site 1 appears to be more critical than site 2 for in vivo reporter expression
and is the only one of the two that is conserved in C. briggsae.
These observations are consistent with previous findings suggesting that in
vivo specificity may be more important than affinity
(Mann and Affolter, 1998). As
the conservation of sequences between C. elegans site 1 and the
corresponding site in C. briggsae extends beyond the Hox/PBC octamer
consensus binding site (23/27 base pairs are identical)
(Fig. 5), there are likely to
be other, as yet unidentified factors that bind the element and that are
important in vivo for specificity of lin-39 activity. Such additional
factors may be required to restrict egl-18/etl-6 expression to a
subset of lin-39-expressing cells. Although the expression patterns
of lin-39 and egl-18/elt-6 overlap, they are not identical:
for example, lin-39 is expressed in all ventral cord neurons in the
mid-body region, whereas egl-18/elt-6::GFP is expressed only in VC
neurons. In addition, widespread expression of LIN-39 by a heat-shock promoter
does not cause ectopic expression of egl-18/elt-6::GFP (not shown).
These results suggest that LIN-39 is insufficient to activate
egl-18/elt-6 expression and that other VPC- and VC-specific
transcription factors may be required for their expression. The 27-mer
conserved enhancer element we have identified may prove useful in discovering
such tissue-specific factors as well as additional binding partners of the
LIN-39 Hox protein.
Despite systematic attempts at discovering targets of Hox genes in the fly
(e.g. Gould et al., 1990;
Mastick et al., 1995
),
homeotic response elements of only a few target genes have been characterized
in detail (e.g. Capovilla et al.,
1994
; McCormick et al.,
1995
; Pederson et al.,
2000
; Regulski et al.,
1991
). In C. elegans, only one Hox-responsive element,
that of hlh-8, which functions in postembryonic mesoderm development,
has been characterized (Liu and Fire,
2000
). Comparison of multiple Hox-responsive elements, including
the lin-39-responsive element we have identified, may be helpful in
understanding how Hox genes regulate their target genes.
Our finding that the GATA factors regulate fusion in the development of two
different cell types (seam and vulva) suggests the possibility that they are
key regulators of fusion more generally, acting on the same set of target
genes in multiple cell types. For example, they may be intermediaries that
integrate developmental cues and repress genes that promote cell fusion, such
as the recently identified eff-1 gene, which is required for all cell
fusions in the C. elegans epidermis
(Mohler et al., 2002;
Witze and Rothman, 2002
).
However, as they promote distinct differentiated cell fates depending on the
context (e.g., seam fate versus vulval fate), they must also have distinct
sets of target genes. Discovery of such common and distinct targets of
egl-18 and elt-6 may help to elucidate how multiple
signaling pathways and Hox genes achieve diverse developmental tasks.
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ACKNOWLEDGMENTS |
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