Research article
2595
FGF signaling functions in the hypodermis to regulate fluid balance
in C. elegans
Peng Huang and Michael J. Stern*
Yale University School of Medicine, Department of Genetics, I-354 SHM, PO Box 208005, New Haven, CT 06520-8005, USA
*Author for correspondence (e-mail: michael.stern@yale.edu)
Accepted 17 February 2004
Development 131, 2595-2604
Published by The Company of Biologists 2004
doi:10.1242/dev.01135
Summary
Signaling by the Caenorhabditis elegans fibroblast growth
factor receptor EGL-15 is activated by LET-756, a
fibroblast growth factor, and attenuated by CLR-1, a
receptor tyrosine phosphatase. Hyperactive EGL-15
signaling results in a dramatic Clr phenotype characterized
by the accumulation of clear fluid within the
pseudocoelomic space, suggesting that regulated EGL-15
signaling is essential for fluid homeostasis in C. elegans. To
determine the cellular focus of EGL-15 signaling, we
identified an enhancer element (e15) within the egl-15
promoter, which is both necessary for the promoter activity
and sufficient when duplicated to drive either egl-15 or clr1 rescue activity. This enhancer drives GFP expression
in hypodermal cells. Consistent with this finding,
immunofluorescence studies of EGL-15 indicate that EGL15 is expressed in hypodermal cells, and hypodermal
promoters can drive full clr-1 and egl-15 rescue activity.
Moreover, a mosaic analysis of mpk-1, which acts
downstream of egl-15, suggests that its suppression of Clr
(Soc) function is required in the hypodermis. These results
suggest that EGL-15 and CLR-1 act in the hypodermis to
regulate fluid homeostasis in worms.
Introduction
EGL-15 signaling leads to a spectrum of phenotypic
consequences. Mutations that compromise the activity of CLR1 result in hyperactive EGL-15 signaling, leading to a dramatic
Clear (Clr) phenotype characterized by the accumulation of
clear fluid within the pseudocoelomic space (Kokel et al.,
1998). A similar Clr phenotype can be observed in transgenic
animals bearing arrays expressing a constitutively activated
form of EGL-15, called EGL-15(neu*) (Kokel et al., 1998).
Conversely, animals that completely lack egl-15 activity arrest
early in larval development (Let), while severe hypomorphic
egl-15 alleles result in a Scrawny (Scr) phenotype (DeVore et
al., 1995). Genetic screens for suppressors of clr-1, or soc
mutants, have identified multiple components in the EGL-15
signaling pathway, including EGL-15, SOC-1, SOC-2/SUR-8,
SEM-5/GRB2 and LET-341/SOS (Borland et al., 2001;
DeVore et al., 1995). In addition, a candidate gene approach
has demonstrated the involvement of LET-60/RAS and
members of the MAPK cascade in mediating the essential
function of EGL-15 signaling (Borland et al., 2001; Schutzman
et al., 2001). The Clr and Soc phenotypes suggest that EGL15 signaling is crucial to regulating fluid homeostasis in
worms. Increased EGL-15 signaling leads to fluid
accumulation and confers a Clr phenotype, while decreased
EGL-15 signaling results in a Soc, Scr or Let phenotype.
Although the Clr and Soc phenotypes have provided a
powerful genetic tool for assembling the components of an
EGL-15 signaling pathway, the biological basis of these
phenotypes remains elusive. The accumulation of fluid within
the pseudocoelom in Clr animals could be due to an imbalance
between fluid intake and fluid excretion. Identifying the
Fibroblast growth factor (FGF) receptors are a class of receptor
tyrosine kinases that play pivotal roles in transducing
extracellular cues to elicit diverse biological responses such as
mitogenesis, angiogenesis, cell proliferation, differentiation
and cell migration (Ullrich and Schlessinger, 1990). Signaling
by FGF receptors is initiated by FGF ligand binding, which
induces receptor dimerization, stimulation of its tyrosine
kinase activity and the activation of the RAS/MAPK cascade.
FGF signaling plays many important roles during normal
development, and alterations in the level of FGF signaling have
been implicated in human skeletal disorders (Webster and
Donoghue, 1997) and tumorigenesis (Basilico and Moscatelli,
1992).
In the nematode Caenorhabditis elegans, egl-15 encodes the
FGF receptor and plays a crucial role in multiple aspects
of development (Borland et al., 2001). Two major events
mediated by EGL-15 are the migrations of the hermaphrodite
sex myoblasts (SMs) and an early essential function (DeVore
et al., 1995). There are two known FGFs in C. elegans, encoded
by the genes egl-17 and let-756. EGL-17 is the chemoattractant
that guides the migrations of SMs to their precise final
positions (Burdine et al., 1998). LET-756 appears to be
required for the essential function of EGL-15, as animals that
lack LET-756 activity arrest at an early larval stage (Roubin et
al., 1999), similar to the phenotype caused by lack of the EGL15 receptor (DeVore et al., 1995).
The essential function of EGL-15 is attenuated by the action
of a receptor tyrosine phosphatase (RTP) known as CLR-1
(Kokel et al., 1998). Genetic perturbation of the strength of
Supplemental data available online
Key words: FGF receptor, EGL-15, Hypodermis, Fluid balance
2596 Development 131 (11)
cellular focus of EGL-15 activity would lead to a better
understanding of the mechanism by which EGL-15 signaling
regulates fluid balance in worms. Here we present evidence
indicating that CLR-1 and EGL-15 function in the same cells,
and that the EGL-15 signaling pathway acts in the hypodermis
to regulate fluid homeostasis.
Materials and methods
Strains and genetics
The wild-type C. elegans strain used was Bristol, N2. Standard
methods were used for maintenance and manipulation of strains
(Brenner, 1974). The following genes, mutations and chromosomal
rearrangements were used: LGI: unc-74(e883), szT1(I;X)[lon2(e678)]; LGII: clr-1(e1745ts), clr-1(e2530); LGIII: dpy-17(e164),
mpk-1(oz140), let-756(s2887), ncl-1(e1865), unc-36(e251), unc32(e189), sDp3(III;f); LGIV: soc-2(n1774), dpy-20(e1282ts); LGX:
egl-15(n1456), egl-15(n1783). Three integrated arrays were obtained
during this work: ayIs25[egl-15(+) (20 ng/µl); Pmyo-3::GFP (5
ng/µl)], ayIs26[Phsp16-2::let-756 (50 ng/µl); Pmyo-2::GFP (5 ng/µl)],
and ayIs29[egl-15(+) (5 ng/µl); Pmyo-2::GFP (5 ng/µl); pGEM5Z (70
ng/µl)]. ayIs25 and ayIs26 were generated by Psoralen-UV
mutagenesis (S. Clark, personal communication); ayIs29 was a
spontaneous integrant. ayIs26 and ayIs29 were mapped to LGIII and
LGIV, respectively.
Promoter analysis of egl-15
A series of truncated egl-15 promoters was generated by PCR
amplification. The size and the 5′ position of each promoter/5′UTR
fragment (the number of each referring to its position relative to the
initiating ATG) is as follows: 2.0 kb, –1993; 1.7 kb, –1694; 1.5 kb,
–1530; 1.3 kb, –1332; 1.1 kb, –1168; 0.8 kb, –815. These promoters
were inserted upstream of the clr-1 genomic coding sequence
(NH#268) to replace the endogenous clr-1 promoter/5′UTR fragment.
The resulting constructs were assayed for clr-1-rescuing activity as
described (see below).
To generate Pe15*2::GFP and Pe15*2::lacZ, e15 (from –1530 to
–1296 in the egl-15 promoter) was inserted duplicated upstream of the
minimal pes-10 promoter of pPD97.78 and pPD95.21 (a gift from A.
Fire), respectively between the HindIII and StuI sites. The resulting
constructs (NH#1100 and NH#1090, respectively) were injected at 20
ng/µl into dpy-20 animals with pMH86 [dpy-20(+)] as the cotransformation marker at 50 ng/µl.
Tissue-specific expression of egl-15 and clr-1
The promoters used in tissue-specific expression of egl-15 and clr1 included: Pdpy-7, Prol-6 (hypodermal expression); Punc-54 (body wall
muscle expression); Paex-3, Punc-14, Psnb-1 (neuronal expression)
(Gilleard et al., 1997; Iwasaki et al., 1997; Mello et al., 1991; Nonet
et al., 1998; Ogura et al., 1997; Okkema et al., 1993). These
promoters were obtained by PCR amplification to yield fragments
that span the following regulatory regions (the numbers indicate the
base pair relative to the initiating ATG): Pdpy-7, from –609 to –1;
Prol-6, from –976 to –1; Punc-54, from –1428 to –1; Paex-3, from –1464
to –1; Punc-14, from –1738 to –1; Psnb-1, from –3330 to –1. For the
egl-15 and clr-1 promoter swapping experiments, Pegl-15 is from
–1993 to –1, and Pclr-1 is from –2422 to –1. P∆pes-10, Pe15*1 and
Pe15*2, which contain zero, one and two copies of e15 fused to the
minimal pes-10 promoter, respectively, were generated by PCR
amplification using NH#1100 as a template (see above). To generate
heterologous egl-15, clr-1, and egl-15(neu*) constructs, the
promoter fragments were digested with appropriate restriction
enzymes, and inserted upstream of the egl-15 genomic coding region
(NH#150), the clr-1 genomic coding region (NH#268) and the egl15(neu*) construct (NH#526), respectively, to replace the
endogenous promoters.
Research article
Transgenic rescue assays
clr-1(Clr) rescue assay
clr-1-rescuing activity was assayed in a clr-1(e1745ts) background.
Animals were injected with tester DNA at 50 ng/µl and the cotransformation marker rol-6(su1006) in plasmid pRF4 at 100 ng/µl.
Transgenic lines were obtained at the permissive temperature (15°C)
based on the Roller phenotype. A minimum of 12 Rol animals from
each line were tested for rescue of the Clr phenotype by shifting
animals at the L3-L4 stage to the non-permissive temperature (25°C).
clr-1-rescued animals are non-Clr at 25°C; non-rescued animals are
Clr at 25°C. clr-1-rescuing activity is classified into four categories
based on the percentage of rescued animals: strong (++), >75%
rescued; intermediate (+), 25-75% rescued; weak (+/–), <25%
rescued; or no rescue (–), 0% rescued.
egl-15(Soc) rescue assay
egl-15(Soc)-rescuing activity was assayed in a clr-1(e1745ts); egl15(n1783) background. Animals were injected with tester DNA at 20
ng/µl and the co-transformation marker rol-6(su1006) in plasmid
pRF4 at 100 ng/µl. Transgenic lines were obtained at the permissive
temperature (15°C) based on the Roller phenotype, and scored as
described above for the clr-1(Clr) assay. egl-15-rescued animals are
Clr (non-Soc) at 25°C; non-rescued animals are non-Clr (Soc) at
25°C. egl-15-rescuing activity is classified into four categories similar
to the clr-1-rescuing assay (see above): strong (++), intermediate (+),
weak (+/–) and no rescue (–).
egl-15(Let) rescue assay
egl-15(Let)-rescuing activity was assayed in a unc-74/szT1;
egl-15(n1456)/szT1 background. Animals were injected with
tester DNA at 20 ng/µl and the co-transformation marker
pJKL449.1[Pmyo-2::GFP] at 5 ng/µl. Transgenic lines were obtained
based on GFP expression in the pharynx. The balanced strain
normally does not segregate any non-arrested Unc progeny; rescue is
scored by the presence of viable Unc progeny derived from the nonUnc parental strain.
egl-15(neu*) assay
egl-15(neu*) constructs were injected into wild-type animals at 20
ng/µl with the co-transformation marker pJKL449.1[Pmyo-2::GFP] at
5 ng/µl. The phenotype of GFP-positive F1 transformants was
examined.
Immunofluorescent staining
Mixed-stage populations of each strain were fixed and stained
according to the protocol of Finney and Ruvkun (Finney and Ruvkin,
1990). Affinity-purified rabbit anti-EGL-15 antibodies (Pop) were
used at a concentration of 1:10; the secondary antibody was Alexa
Fluor 546-conjugated mouse anti-rabbit antibody (Molecular Probes)
diluted at 1:250.
Mosaic analysis
Mosaic analysis was carried out as previously described in strains
bearing sDp3, a free chromosomal duplication (Hedgecock and
Herman, 1995). ncl-1 was used as a cell-autonomous marker
(Hedgecock and Herman, 1995). Cells displaying an Ncl phenotype
were assumed to have lost sDp3. The point of sDp3 loss within each
mosaic animal was inferred by assuming the minimum loss of sDp3
consistent with the Ncl phenotype pattern of each animal. The
following representative cells from each lineage were scored for the
Ncl phenotype: CANL/R, RID, ALA, ASKL, ADLL (from AB.al);
MI, I5, ALML/R, BDUL/R (from AB.ar); ASIL, excretory pore,
HSNL, PHBL, QL, V5L (from AB.pla); excretory duct, excretory cell,
K, repD, PHAL, hyp8/9, repVL, U, F, imL, hyp10 (from AB.plp);
ASKR, ADLR, ASIR, QR, V5R, HSNR, PHBR (from AB.pra);
PHAR, hyp8/9, repVR, B, body muscle, hyp10 (from AB.prp);
vccL/R, dccL/R, dtcA/P, anchor cell, M4, imR, body muscles (from
EGL-15 signaling in the hypodermis 2597
MS); hyp11, body muscles (from C); body muscles (from D).
Intestinal cells do not display a Ncl phenotype. Animals that have lost
sDp3 in the MS-lineage could possibly have lost sDp3 also in the E
lineage.
mpk-1 mosaics
We screened approximately 1500 Unc or weakly Unc progeny of clr1(e1745ts); mpk-1 ncl-1 unc-36; sDp3 at the L3-L4 stage for Ncl
mosaics using Nomarski optics at the permissive temperature (15°C).
Eighteen mosaic animals were identified; 17 of these animals had
sDp3 loss in AB descendants, and one animal was P1(–). These
animals were recovered and shifted to the non-permissive temperature
(25°C). The phenotype was subsequently determined after at least 10
hours at 25°C. To identify P1 mosaics, we shifted approximately
50,000 synchronized progeny of clr-1(e1745ts); mpk-1 ncl-1 unc-36;
sDp3 at the L3-L4 stage to the non-permissive temperature (25°C),
and screened for animals that were Soc non-Unc or semi-Clr non-Unc
using a dissecting microscope after 10 hours at 25°C and then for Ncl
mosaics using Nomarski optics. We identified two semi-Clr non-Unc
and one Soc non-Unc animals with losses in P1. P1 losses are
expected in approximately one out of every 400 random animals
(Hedgecock and Herman, 1995). Thus, we expected to identify
approximately 125 P1(–) mosaics in our screen. These mosaic animals
should all be phenotypically Soc non-Unc if loss of mpk-1 activity in
the P1 lineage were sufficient to suppress the Clr phenotype.
Therefore, it is likely that the vast majority of P1(–) animals had
already turned Clr by the time we screened them after 10 hours at
25°C. To test the possibility that loss in P1 might merely delay a
strong Clr phenotype to mosaic animals, we screened for P1 mosaic
animals by looking for an intermediate phenotype at an earlier time
after transfer to the restrictive temperature. We screened an additional
82,000 synchronized animals after 4-5 hours at 25°C using a
dissecting microscope, looking for animals that were semi-Clr nonUnc, and then for Ncl mosaics using Nomarski optics. Eight P1(–)
animals were identified this way; they were recovered at 25°C, and
their terminal phenotype was analyzed after at least 10 hours at 25°C.
In addition to mosaic animals, we identified a number of animals in
which the duplication had broken down. In the first screen we
identified six Soc non-Unc animals, and in the modified screen we
identified 23 Soc non-Unc animals that probably represent breakdown
products of sDp3. Their classification as duplication breakdown
events was based on one of the three following criteria: (1) the Ncl
phenotype pattern of the animal was not consistent with its Soc nonUnc phenotype; (2) the Ncl phenotype pattern and the Soc non-Unc
phenotype of the animal was contradictory to our previous
observations; or (3) the animal had a complicated pattern of sDp3 loss.
Since all of these animals were sterile, progeny testing was not
possible to verify their classification.
let-756 mosaics
We screened approximately 10,000 progeny of dpy-17 let-756 ncl-1
unc-36; sDp3 hermaphrodites for animals that were Unc non-Dpy or
Dpy non-Unc using a dissecting microscope. These animals were then
screened for Ncl mosaics using Nomarski optics. We identified 80
Unc non-Dpy animals, all of which had losses in the AB lineage, as
expected (see Fig. 5 and Table S1 at http://dev.biologists.org/
supplemental/). Some of them were recovered, and the phenotypes of
their progeny were checked to verify that the parents were mosaic
animals rather than the result of duplication breakdown events. We
identified only one Dpy Unc, two Dpy non-Unc, and two semi-Dpy
non-Unc animals from the screen. The Ncl phenotype of these animals
was determined using Nomarski optics. These animals were
recovered, the phenotypes of their progeny determined, and the
genotype of the let-756 locus of the mosaic animals and their progeny
analyzed by single-worm PCR. Because the genotypes of these
animals and their progeny did not correlate with their phenotypes,
these Dpy animals were classified as the result of duplication
breakdown events. We further screened approximately 750 randomly
selected L3-L4 animals for mosaics directly based on their Ncl
phenotype. We identified 31 mosaic animals using this approach, and
the Ncl phenotype patterns are represented in Fig. S1
(http://dev.biologists.org/supplemental/).
Results
Genetic interactions between let-756, egl-15 and clr-1
Since EGL-15 is the sole FGF receptor in C. elegans, it is
reasonable to hypothesize that the LET-756 FGF acts as one of
its ligands. Nonetheless, support for this hypothesis is based
solely on the similarity of their lethal null phenotypes and the
scrawny phenotypes of severe hypomorphs (Roubin et al.,
1999). While these data are consistent with the hypothesis that
these genes act in the same pathway, the general and nonspecific nature of these phenotypes does not offer strong
evidence that LET-756 functions as a ligand for EGL-15. To
test this hypothesis more rigorously, we sought additional
genetic evidence that would support the hypothesis that these
components act as a ligand–receptor pair.
If LET-756 acts as a ligand for EGL-15, then constitutive
activation of EGL-15 might bypass the requirement for LET756, and overexpression of LET-756 might stimulate the
more specific Clr phenotype characteristic of EGL-15
hyperactivation. Consistent with the first prediction, the Let
phenotype of let-756(s2887) could be suppressed by the
overexpression of wild-type EGL-15 (Fig. 1D) or by the lowlevel expression of hyperactive EGL-15(neu*) (data not
shown). Furthermore, overexpression of LET-756 can cause a
Clr phenotype that is dependent on EGL-15 signaling. let-756
was overexpressed using the heat shock promoter Phsp16-2
(Stringham et al., 1992) to drive expression of LET-756. Heat
shock of animals bearing a transgenic array containing
Phsp16-2::let-756 in a background overexpressing egl-15
resulted in a dramatic Clr phenotype (Fig. 1E). This phenotype
was completely suppressed when the same experiment was
performed in a soc-2 mutant background (Fig. 1F). Moreover,
heat shock of the same array in the absence of overexpressed
egl-15 results in animals that do not display any obvious
phenotypes (data not shown). These experiments show that
overexpression of LET-756 can confer a Clr phenotype that
requires EGL-15 and some of the same components that
mediate EGL-15 signaling. These results show that LET-756
can regulate the same processes as EGL-15, supporting the
hypothesis that LET-756 is the FGF ligand for this function of
EGL-15.
To establish the relationship between LET-756, an activating
ligand for EGL-15, and CLR-1, a negative regulator of EGL15 signaling, we tested genetic interactions between these two
components. Our results show that progressive reduction of
clr-1 function promotes increasing suppression of the let756 phenotype by increasing net EGL-15 signaling. The
temperature-sensitive allele clr-1(e1745ts) suppressed the
lethality of let-756(s2887) homozygotes at the permissive
temperature (15°C), but the animals appeared scrawny (Fig.
1G), a phenotype indicative of compromised EGL-15 signaling
(Borland et al., 2001; DeVore et al., 1995). Further reduction
of clr-1 activity, achieved by placing clr-1(e1745ts) in trans to
the null allele clr-1(e2530), resulted in essentially wild-typelooking animals at the permissive temperature (15°C) (Fig.
2598 Development 131 (11)
Research article
Fig. 1. Genetic interactions between let-756, egl-15 and clr-1. (A) Wild type. (B) let-756 unc-32 arrested L1 larva. (C) clr-1(e2530) Clr
hermaphrodite. (D) let-756 unc-32; ayIs25[egl-15(+)] Scr hermaphrodite. (E) ayIs26[Phsp16-2::let-756]; ayIs25[egl-15(+)] Clr hermaphrodite.
(F) ayIs26[Phsp16-2::let-756]; soc-2; ayIs25[egl-15(+)] Soc hermaphrodite. (G) clr-1(e1745ts); let-756 unc-32 Scr hermaphrodite, at 15°C.
(H) clr-1(e1745ts)/clr-1(e2530); let-756 unc-32 non-Clr non-Scr hermaphrodite, at 15°C. (I) clr-1(e2530); let-756 unc-32 viable semi-Clr
hermaphrodite. All animals were photographed as adults. (E-F) Animals were heat shocked for 30 minutes at 37°C, and allowed to recover at
20°C for 5.5 hours. The Clr phenotype in (C) and (E) can be discerned from the appearance of the intestine floating within the fluid-filled
pseudocoelomic cavity. Abbreviated genotypes: 0, null allele; ts, temperature-sensitive allele; ++, transgenic overexpression. Scale bar:
200 µm.
1H). Finally, the double-null strain clr-1(e2530); let756(s2887) is viable and partially Clr (Fig. 1I), demonstrating
that the loss of the negative regulator not only can bypass the
requirement for LET-756 but also allow hyperactivity of basal
levels of EGL-15. These results are consistent with a model in
which there is a balance established by the actions of LET-756
and CLR-1 on EGL-15 activity, with the basal, unregulated
activity of EGL-15 resulting in a slight gain-of-function
phenotype that is independent of the LET-756 ligand.
Rescue analysis indicates EGL-15 functions in the
hypodermis
Interchangeability of the egl-15 promoter and the clr-1
promoter
Both EGL-15 and CLR-1 are membrane-associated receptors
(DeVore et al., 1995; Kokel et al., 1998). Since the intracellular
phosphatase activity of CLR-1 is absolutely required for its
negative regulation of EGL-15 signaling (Kokel et al., 1998),
it is likely that both CLR-1 and EGL-15 act in the same cells
to mediate their downstream effects. We tested this hypothesis
using promoter swapping experiments to see whether the egl15 promoter and the clr-1 promoter could interchangeably
provide the functions of the endogenous promoters. When the
clr-1 promoter was used to drive egl-15 expression (Pclr-1::egl15), the resulting temporal and spatial expression of egl-15 was
able to rescue both strong and weak egl-15 loss-of-function
phenotypes. This construct could rescue the Soc phenotype
caused by egl-15(n1783) as well as the Let phenotype caused
by the null allele egl-15(n1456) (Table 1A). Conversely, when
the egl-15 promoter was used to drive clr-1 expression
(Pegl-15::clr-1), the resulting temporal and spatial expression of
clr-1 was able to rescue the Clr phenotype of clr-1(e1745ts)
(Table 2A). In addition, a construct in which the clr-1 promoter
drives egl-15(neu*) expression (Pclr-1::egl-15(neu*)) could
confer a dominant Clr phenotype similar to that seen when egl15(neu*) was expressed from the egl-15 promoter (Table 1A).
The interchangeability of these two promoters suggests that
CLR-1 and EGL-15 probably function in the same cells to
carry out their normal functions. It is interesting to note that
the clr-1 promoter appears to be weaker than the egl-15
promoter. This is reflected both in the lower penetrance of the
Clr phenotype when Pclr-1 is used to drive expression of egl15(neu*) as well as the weaker rescue of egl-15(Soc) by
Pclr-1::egl-15 compared with Pegl-15::egl-15 (Table 1A).
Identification of the egl-15 enhancer element e15
We have taken several approaches to address the cellular focus
of the essential function of EGL-15. The first of these
approaches was to identify an enhancer element in the egl-15
promoter that is necessary and sufficient for rescue of both egl15 and clr-1. To look for important regulatory elements within
the egl-15 promoter, we fused truncated egl-15 promoters to
the clr-1 coding sequence, and tested their ability to rescue the
clr-1 mutant phenotype (Fig. 2). The degree of clr-1 rescue
activity is a sensitive readout for expression in the appropriate
subset of cells. Full-scale clr-1 rescue activity was found using
promoter fragments as small as 1.5 kb (Fig. 2). Smaller
promoters, such as the 1.3 kb or the 1.1 kb egl-15 promoters,
EGL-15 signaling in the hypodermis 2599
Table 1. Tissue-specific rescue of egl-15
Extent of rescue of
egl-15(Soc) lines†
n
Rescued
egl-15(Let)
lines‡
% Clr phenotype in
egl-15(neu*) F1s§
(n)
3
5
5/5
2/2
84.2% (329)
25.3% (645)
6
3
0
6
5
7
ND¶
ND
4/4
ND
ND
84.3% (637)
0
3
15
0
12
4
12
15
1
12
2/2
2/2
2/2††
1/1
0/2
99.6% (479)
94.4% (621)
0.4% (275)
66.8% (277)
0% (369)
Transgene
promoter‡‡
++
+
+/–
--
(A) Interchangeability of the
egl-15 and clr-1 promoters
Pegl-15
Pclr-1
3
0
0
3
0
2
0
0
(B) Expression of egl-15 driven
by the e15 element
P∆pes-10
Pe15*1
Pe15*2
0
0
7
0
1
0
0
1
0
(C) Tissue-specific expression
of egl-15
Pdpy-7 (hypodermis)
Prol-6 (hypodermis)
Paex-3 (neurons)
Punc-14 (neurons)
Punc-54 (body wall muscles)
4**
4
0
1
0
0
4
0
0
0
0
1
0
0
0
‡‡Major site of expression of the tissue-specific promoter is indicated in parentheses.
†egl-15(Soc) rescue assays were performed in a clr-1(e1745ts); egl-15(n1783) background.
At the non-permissive temperature (25ºC), egl-15-rescued animals
are Clr, whereas non-rescued animals retain the Soc phenotype. Rescuing activity is classified as strong (++), intermediate (+), weak (+/–), or no rescue (–). n, the
number of transgenic lines scored.
‡Transgenic lines were isolated in an unc-74/szT1; egl-15(n1456)/szT1 background; lines were scored as rescued if the parental line could segregate a viable
unc-74; egl-15(n1456) strain bearing the extrachromosomal array.
§Promoter::egl-15(neu*) constructs were injected into wild-type animals. F1 transformants were identified by expression of the P
myo-2::GFP co-transformation
marker, and the percentage of transformants with the Clr phenotype was determined. n, the number of F1 transformants.
¶ND, not determined.
**clr-1(e1745ts); egl-15(n1783); ayEx[Pdpy-7::egl-15] animals were Clr even at the permissive temperature (15°C).
††Rescued animals from both lines were Scrawny.
Table 2. Tissue-specific rescue of clr-1
(A) Interchangeability of the egl-15
and clr-1 promoters
(B) Expression of clr-1 driven
by the e15 element
(C) Tissue-specific
expression of clr-1
Extent of rescue of clr-1 lines†
Transgene
promoter§
++
+
+/–
--
n
Pclr-1
Pegl-15
7
6
0
0
0
0
0
0
7
6
P∆pes-10
Pe15*1
Pe15*2
0
0
14
0
4
0
2
3
0
8
1
0
10
8
14
Pdpy-7 ‡ (hypodermis)
Prol-6 (hypodermis)
Paex-3 (neurons)
Punc-14 (neurons)
Psnb-1 (neurons)
Punc-54 (body wall muscles)
4
14
1
2
3
0
4
0
3
0
0
0
1
0
7
0
0
4
3
0
4
0
0
2
12
14
15
2
3
6
§Major site of expression of the tissue-specific promoter is indicated in parentheses.
†clr-1 rescue assays were performed in a clr-1(e1745ts) background. clr-1 rescuing activity
is classified as strong (++), intermediate (+), weak (+/–), and no
rescue (- -). n, the number of transgenic lines scored.
‡P
dpy-7::clr-1 was injected at 5 ng/µl; all other constructs were injected at 50 ng/µl.
show decreased rescue activity (Fig. 2). These results suggest
that the 234 bp DNA segment that is the difference between
the 1.5 kb and 1.3 kb promoters (e15, from –1530 to –1296)
is necessary for maximal egl-15 promoter activity (Fig. 2).
To test whether e15 is also sufficient for egl-15 and clr-1
rescue, we fused one or two copies of e15 to the pes-10
minimal promoter (P∆pes-10). The resulting artificial promoters
(named Pe15*1 and Pe15*2, respectively) were used to drive
either egl-15 or clr-1 expression. Strikingly, the duplicated e15
enhancer, Pe15*2, was able to drive full-scale rescue activity of
egl-15, similar to the endogenous egl-15 promoter (Table 1B).
One copy of the e15 enhancer, Pe15*1, showed a reduced extent
of rescue activity, while the minimal promoter, P∆pes-10,
showed only background levels of rescue activity (Table 1B).
Since a single e15 element is not sufficient to drive EGL-15
expression to confer full rescue activity, additional elements
might be present between the 1.1 kb and 0.8 kb promoters to
help drive normal expression of EGL-15. The significant
rescue activity found for the 1.1 kb promoter fragment is
consistent with this hypothesis (Fig. 2). Similar results were
obtained using the clr-1 rescue assay (Table 2B), consistent
with the hypothesis that CLR-1 and EGl-15 act in the same
cells. Additionally, the egl-15(neu*) transgene under the
control of Pe15*2 conferred a Clr phenotype to transgenic
animals with a penetrance comparable to that of egl-15(neu*)
driven by the egl-15 promoter (Table 1B). This analysis thus
identified an enhancer element that is necessary for the egl-15
promoter. Furthermore, when present in two copies, this
2600 Development 131 (11)
Research article
Pegl-15 ::clr-1 constructs
Fig. 2. Identification of the e15 enhancer
element within the egl-15 promoter. egl-15
promoter fragments, from 2.0 kb to 0.8 kb,
were used to drive expression of the clr-1
genomic coding region. Rescue assays were
performed in a clr-1(e1745ts) background.
Rescue activity is classified as strong (++),
intermediate (+), weak (+/–), or no rescue
(– –). n, the total number of transgenic lines
scored. The location of the egl-15 enhancer
(e15, from –1530 to –1296) lies between the
dashed lines.
Pegl-15 Promoter Size
Extent of Rescue of clr-1 lines
––
++
+
+/–
n
clr-1
2.0 kb
6
0
0
0
6
clr-1
1.7 kb
6
0
0
0
6
clr-1
1.5 kb
4
0
1
0
5
clr-1
1.3 kb
1
1
1
2
5
clr-1
1.1 kb
2
0
3
2
7
clr-1
0.8 kb
0
0
4
7
11
e15
element can drive functionally robust expression in cells
relevant to the essential function of egl-15.
To determine the expression pattern of the e15 enhancer, we
constructed GFP and lacZ reporters under the control of Pe15*2.
The Pe15*2::GFP reporter is expressed in the major hypodermis
(Fig. 3A), persisting throughout larval development and into
the adult stage. Pe15*2::GFP was not seen in the lateral
hypodermal blast cells (the seam cells) (Fig. 3A). An identical
expression pattern was observed using the Pe15*2::lacZ reporter
(data not shown). The strong hypodermal expression driven by
the e15 enhancer suggests that an important site of EGL-15 and
CLR-1 function is in hypodermal cells.
Tissue-specific expression of EGL-15 and CLR-1
To test the hypothesis that EGL-15 and CLR-1 function in the
hypodermis, we used tissue-specific promoters to drive either
EGL-15 or CLR-1 expression in mutant rescue assays. When
we expressed EGL-15 from either of two hypodermal
promoters, Pdpy-7 (Gilleard et al., 1997) and Prol-6 (Mello et al.,
1991), we observed robust rescue of egl-15 mutant phenotypes
(Table 1C). By contrast, expression of EGL-15 in all body wall
muscles from the unc-54 promoter (Okkema et al., 1993) failed
to show any rescue activity (Table 1C). These data further
support the hypothesis that EGL-15 functions in the
hypodermis. In addition to the rescue of the Soc and Let
phenotypes, hypodermal expression of EGL-15(neu*) is also
able to confer a highly penetrant Clr phenotype (Table 1C).
These results further suggest that the Clr, Soc and Let
phenotypes are caused by perturbation of the same process in
the same tissue. Interestingly, pan-neuronal expression of
EGL-15 could also show strong rescue activity. Expression of
EGL-15 by the unc-14 promoter (Ogura et al., 1997) can rescue
egl-15 phenotypes, and transgenes containing Punc-14::egl15(neu*) can confer the Clr phenotype (Table 1C). By contrast,
expression of EGL-15 from another pan-neuronal promoter
derived from the aex-3 gene (Iwasaki et al., 1997) failed to
rescue the egl-15(Soc) phenotype, and Paex-3::egl-15(neu*)
transgenes also failed to confer the dominant Clr phenotype
(Table 1C). Although Paex-3::egl-15 was found to rescue the
Let phenotype of egl-15(n1456), the rescued animals were
scrawny, a phenotype indicative of compromised egl-15
signaling (Table 1C). Immunofluorescence studies using antiEGL-15 antibodies have shown that neuronal EGL–15
expression in animals with Punc-14::egl-15 and Paex-3::egl-15
Fig. 3. EGL-15 is expressed in the hypodermis. (A) GFP expression
pattern of Pe15*2::GFP animals. GFP is seen in the hyp7
syncytium, and excluded from the lateral seam cells (*).
(B) Immunofluorescence staining of EGL-15 in the strain ayIs29[egl15(+)] using anti-EGL-15 antibodies. Staining is observed in the
hyp7 syncytium, but absent in the seam cells. The punctate staining
is highly variable, and has not been assigned to any defined
anatomical structure. Animals shown were photographed at the L1
(A) and L2 (B) stage. Anterior is to the left. Scale bar: 50 µm.
transgenes is comparable (data not shown). These data suggest
that EGL-15 expression in the neurons may not be the relevant
site of expression that confers rescue activity, but rather that
low level expression elsewhere might be responsible for the
rescue results.
We performed similar tissue-specific transgene rescue
experiments using these same promoters to drive expression of
clr-1. The same subset of promoters that restored egl-15
function could also restore clr-1 function (Tables 1C and 2C),
further suggesting that EGL-15 and CLR-1 act in the same
tissues. Since hypodermal expression of CLR-1 also shows
robust rescue activity (Table 2C), the hypodermis is also likely
to be the site where CLR-1 normally functions to regulate fluid
balance. However, based on these heterologous rescue
experiments, it remains possible that the nervous system might
EGL-15 signaling in the hypodermis 2601
zygote (P0)
Clr
semi-Soc
Soc
AB
P1
A
P
L
A
R
P
A
EMS
L
P
A
R
P
A
MS
P
A
P2
E
P
C
A
P3
P
D
P4
Fig. 4. Summary of mpk-1 mosaics. The mosaic analysis was carried out in a strain of genotype clr-1(e1745ts); mpk-1 ncl-1 unc-36; sDp3.
Patterns of duplication loss are represented by different colors: red, loss in a single lineage; green, losses in a single lineage and some
independent cell(s); blue, loss in multiple independent lineages. Terminal phenotypes were determined at the non-permissive temperature
(25°C) and represented by different shapes: 䊏, Clr; 䉱, semi-Soc; 䊉, Soc.
be a relevant site for the functions of EGL-15 and CLR-1, since
neuronal promoters such as the unc-14 (Ogura et al., 1997) and
snb-1 promoters (Nonet et al., 1998) can rescue the clr-1
mutant phenotype (Table 2C).
Expression analysis of egl-15
To determine where EGL-15 is normally expressed, we carried
out immunofluorescence studies using anti-EGL-15 antibodies.
We were unable to detect endogenous EGL-15 in wild-type
animals, probably due to the low-level expression of the
endogenous receptor. Therefore, we generated a strain
overexpressing a chromosomally integrated egl-15(+) array
(ayIs29), and performed immunofluorescence staining on this
strain using affinity-purified anti-EGL-15 antibodies. EGL-15
expression was observed in hypodermal cells as well as the sex
myoblasts, the type I vulval muscles and some unidentified
neurons in the head (Fig. 3B and data not shown; a more
detailed analysis will be reported elsewhere). The staining is
specific to EGL-15, since no signal is observed with secondary
antibody alone (data not shown). Hypodermal expression is
obvious throughout all four larval stages, with stronger
expression in the early larval stages. A similar pattern of EGL15 expression has been observed for many other arrays
containing transgenic egl-15 constructs (data not shown). The
expression in the hypodermis is very similar to that observed
in Pe15*2::GFP animals, in which expression was observed in
the major hypodermis but was excluded from the seam cells
(Fig. 3A). A similar expression pattern can be observed in
rescued egl-15(null) animals expressing an egl-15 transgene
driven by a hypodermal promoter (Prol-6 and Pdpy-7) (data not
shown). Hypodermal expression of EGL-15 is also reported in
animals expressing Pegl-15::lacZ (Hope Lab Expression Pattern
Database: http://129.11.204.86:591/default.htm), consistent
with the antibody staining that we have observed.
Mosaic analysis of the Soc function of mpk-1
The data described above support the hypothesis that the Let,
Clr and Soc phenotypes of egl-15 and clr-1 mutants are due to
various perturbations of a single process that occurs in the same
place for both EGL-15 and CLR-1. The cellular focus of the
Soc function can thus provide additional insight into the site
of action of this pathway. Multiple downstream components of
the EGL-15 signaling pathway have been identified based on
the Soc phenotype (DeVore et al., 1995; Schutzman et al.,
2001). To investigate the cellular focus of the Soc function, we
carried out a mosaic analysis of one of the soc genes, mpk-1,
encoding MAP kinase. mpk-1 had already been shown to be
amenable to mosaic analysis using the well-characterized free
duplication sDp3 and the cell-autonomous lineage marker ncl1 (Church et al., 1995). To analyze its Soc phenotype, we
constructed the strain clr-1(e1745ts); mpk-1 ncl-1 unc-36;
sDp3 for mosaic analysis. The sDp3 free duplication carries a
wild-type copy of each of the mpk-1, ncl-1 and unc-36 genes.
At the restrictive temperature (25°C), clr-1(e1745ts) mutants
are Clr, but animals homozygous for the additional mutation
mpk-1(oz140) are Soc with complete penetrance. Therefore,
non-mosaic animals that carry the duplication [P0(+)] are Clr
at the restrictive temperature (25°C), since the wild-type copy
of the mpk-1 gene on the duplication allows hyperactive EGL15 signaling to cause fluid accumulation. By contrast, lack of
MPK-1 in animals that lose the duplication [P0(–)] prevents
the transduction of hyperactive EGL-15 signaling, resulting in
a Soc phenotype. unc-36 is known to function in the AB.p
lineage (Kenyon, 1986; Yuan and Horvitz, 1990). It was
included to identify animals that had not inherited the
duplication [P0(–)] by their Unc, Soc phenotype, and to
facilitate the identification of animals with mosaic losses in
descendants of the AB blastomere.
We identified 17 animals with sDp3 losses in AB
descendants at the permissive temperature (15°C) (see
Materials and methods), including two AB(–), two AB.a(–)
and three AB.p(–) animals, as well as animals with losses
within the AB.p sublineage (Fig. 4). Interestingly, all these
animals displayed the Clr phenotype when shifted to the
restrictive temperature (25°C). These results suggest that loss
of mpk-1 activity in the AB lineage is not sufficient to suppress
the Clr phenotype. Therefore, mpk-1(+) activity within P1
descendants can function to prevent the Soc phenotype.
2602 Development 131 (11)
Research article
zygote (P0)
AB
P1
A
P
L
A
R
P
A
EMS
L
P
A
R
P
A
MS
P
A
P2
E
P
C
A
P3
P
D
P4
Fig. 5. Summary of let-756 mosaics in the AB lineage. The mosaic analysis was carried out in the background of dpy-17 let-756 ncl-1 unc-36;
sDp3. Mosaic animals were identified based on their Unc non-Dpy phenotypes. Patterns of duplication loss are represented by different colors:
red, loss in a single lineage; green, losses in a single lineage and some independent cell(s); blue, losses in multiple independent lineages.
If P1 losses would confer a Soc phenotype, then P1(–)
mosaic animals would be easily identified phenotypically as
Soc non-Unc. However, Soc non-Unc mosaic animals were
extremely rare (see Materials and methods). Strikingly, nine
additional P1 mosaic animals that were identified by alternative
approaches (see Materials and methods) displayed a Clr
phenotype at the restrictive temperature (25°C) (Fig. 4). We
conclude that loss of mpk-1 activity in the P1 lineage is also
not sufficient to suppress the Clr phenotype. Therefore, the
presence of mpk-1(+) activity within either the AB lineage or
the P1 lineage can prevent the Soc phenotype, and, conversely,
loss of mpk-1 activity in both the AB and P1 lineage is required
for the suppression of the Clr phenotype. Since the hypodermis
has significant contributions from both the AB and the P1
lineage, the results of our mpk-1 mosaic analysis are consistent
with MPK-1 acting in the hypodermis to carry out its Soc
function.
Mosaic analysis of let-756
We also performed a mosaic analysis of let-756 to identify the
site where its expression is required for its essential function.
Loss of LET-756 at this site would result in lethality, thereby
prohibiting the identification of this class of mosaics. Since
LET-756 is predicted to be a ligand, such an analysis could
determine its site of expression rather than its site of action.
Similar to the mpk-1 mosaics, we took advantage of the free
chromosomal duplication sDp3, which also carries a wild-type
copy of let-756. Mutations in unc-36 and dpy-17 were included
to help facilitate the identification of specific classes of
potential mosaic animals. dpy-17 is required in the P1 lineage
for a portion of its activity (Kenyon, 1986; Yuan and Horvitz,
1990). Therefore, AB(–) or AB.p(–) animals are Unc non-Dpy,
while P1(–) animals are semi-Dpy non-Unc.
We first screened dpy-17 let-756 ncl-1 unc-36; sDp3 animals
for either Unc non-Dpy or Dpy non-Unc mosaic animals. Unc
non-Dpy animals were identified as expected, and the vast
majority of these animals had losses in either AB, AB.p, or
descendants in the AB.p lineage (Fig. 5 and Table S1 at
http://dev.biologists.org/supplemental/). Besides being Unc,
these mosaic animals were completely wild type, suggesting
that lack of let-756(+) expression in the AB lineage is not
essential for viability. By contrast, Dpy non-Unc animals were
extremely rare, and the few Dpy non-Unc animals identified
turned out to result from the breakdown of the free duplication
(see Materials and methods). This striking finding suggests that
the cellular focus of let-756 is not easily separable from that
of dpy-17. DPY-17 is thought to be required in the P1 lineage
(Kenyon, 1986; Yuan and Horvitz, 1990). Based on the lack of
P1 losses, we conclude that LET-756 expression, like DPY-17,
is likely required in the P1 lineage. Consistent with our mosaic
analysis, a Plet-756::GFP reporter shows robust expression in
the body wall muscles (Bülow et al., 2004), all but one of which
is P1-derived.
We also screened randomly for mosaic animals using the Ncl
lineage marker. As expected, losses within the AB lineage were
identified, while no P1(–) mosaic animals were identified (see
Fig. S1 at http://dev.biologists.org/supplemental/). These data
are consistent with a requirement for LET-756 expression in
cells derived from the P1 lineage. Interestingly, mosaic animals
with losses in the entire MS lineage and part of the C lineage
can also be identified (see Fig. S1 at http://dev.biologists.org/
supplemental/). Since both the MS and the C lineages
contribute a significant portion of the body wall muscles in the
animal (35% and 40%, respectively), this result suggests that
LET-756 expression is not required in all the body wall
muscles.
Discussion
The Clr phenotype of hyperactive EGL-15 signaling in C.
elegans has been powerfully exploited to identify components
of an FGF signaling pathway. Here we present evidence
supporting the hypothesis that EGL-15 and CLR-1 act together
in the same cells to mediate their downstream effects. These
effects appear to play a physiological role in fluid homeostasis,
as opposed to a developmental role. Such a physiological role
for EGL-15 is supported by the rapid phenotypic response of
clr-1(e1745ts) mutants when shifted to the non-permissive
temperature. Further support derives from the fact that this
response can occur at any time during larval development.
EGL-15 signaling in the hypodermis 2603
To understand the biological function of the CLR-1/EGL-15
pathway, we analyzed the site of action of EGL-15 signaling
components involved in this function of EGL-15. Three lines of
evidence indicate that EGL-15 signaling acts in the hypodermis
to regulate fluid balance. First, EGL-15 is expressed in the
hypodermis, as shown by immunofluorescence as well as an
analysis of the e15 enhancer. Second, hypodermal expression
of either EGL-15 or CLR-1 by tissue-specific promoters can
restore their normal functions. Finally, our results of a mosaic
analysis of mpk-1, which acts downstream of egl-15, are
consistent with MPK-1 acting in the hypodermis to carry out its
Soc function.
Fluid homeostasis results from maintaining the balance
between fluid inflow and outflow rates. To date, laser ablation
experiments and mutant analysis have implicated the excretory
system and the canal-associated (CAN) neurons as important
regulators of fluid balance in C. elegans. The excretory system
consists of four cells: the excretory cell, the excretory duct cell,
the excretory pore cell and the binucleate gland cell (Nelson et
al., 1983). Laser ablation of the excretory cell, the duct cell or
the pore cell results in a Clr phenotype (Nelson and Riddle,
1984). A mosaic analysis of let-60 ras lent further support for
the role of the excretory system in fluid regulation. The rod-like
dead larvae resulting from severely compromised LET23(EGFR)/LET-60(RAS)/MAP kinase signaling display a
dramatic Clr phenotype that appears to be due to the failure to
specify the fate of the excretory duct cell (Yochem et al., 1997).
Although more severe in its lethal phenotype, the fluid
accumulation in the pseudocoelom is very similar to that seen in
clr-1 mutants. Ablation of the CAN neurons also results in a Clr
phenotype (Forrester et al., 1998). This is further supported by
the analysis of ceh-10 null mutants that display both a Clr
phenotype and CAN neurons that fail to migrate and differentiate
properly (Forrester et al., 1998). The tight association of the
CAN neurons with the canals of the excretory cell suggests that
these cells work together to regulate fluid efflux. Here we present
evidence indicating that EGL-15 signaling controls fluid
homeostasis by acting in the hypodermis. Interestingly, the
excretory canals form extensive gap junctions to the hypodermis
(Nelson et al., 1983), suggesting a mechanism that might couple
their influences on maintaining fluid balance.
Mosaic analysis of mpk-1 not only suggests action of EGL15 signaling in the hypodermis, but also strongly discounts the
importance of EGL-15 activity in the excretory system. Our
analysis suggests that MPK-1 acts in cells derived from both
the AB and P1 lineages to carry out its Soc function. Therefore,
the cellular focus of mpk-1 could either be in multiple tissues
or in a tissue that derives from both the AB and P1 lineages.
The hypodermis has major contributions from both the AB and
P1 lineages (Sulston et al., 1983) and similar mosaic results
have been interpreted as supporting function in the hypodermis
(Herman and Hedgecock, 1990). Consistent with this
hypothesis, tissue-specific expression of EGL-15 and CLR-1
shows that hypodermal expression is sufficient to restore their
normal functions. Importantly, all four cells in the excretory
system are derived from the AB.p sublineage. Since lack of
mpk-1 activity in either AB or AB.p is not sufficient to suppress
the Clr phenotype caused by a clr-1 mutation, EGL-15 signaling
does not appear to act in the excretory system exclusively.
Tissue-specific transgene expression also strongly suggests
hypodermal action, despite some indications that neuronal
expression might be important. Supporting a hypodermal site
of action, two different hypodermal promoters (Pdpy-7 and
Prol-6) can drive strong rescue activity for all related EGL-15
functions as well as CLR-1 function. We have also shown by
immunofluorescence that a major site of EGL-15 expression is
in the hypodermis. Furthermore, an essential enhancer element
from the egl-15 promoter, e15, which is sufficient for robust
transgenic rescue of EGL-15 functions when duplicated, drives
expression in the hypodermis. Combined with the mosaic
results, we believe that the most likely site of action for
the essential function of EGL-15 is in the hypodermis.
Interestingly, however, we also observed strong transgenic
rescue for a number of pan-neural promoters that have been
reported to be neuronal-specific. Two out of three pan-neural
promoters (Punc-14 and Psnb-1) showed strong transgenic rescue,
while the third, Paex-3, showed no rescue activity. Both the aex3 and the unc-14 promoters drove strong expression of EGL15 in neurons, as assayed using immunohistochemistry. Thus,
it appears that it is not EGL-15 expression in the neurons that
accounts for the observed transgenic rescue, but rather that low
level expression elsewhere might confer rescue activity. The
mpk-1 mosaic analysis further refutes a pan-neural site of
action of EGL-15. Although 97% of all neurons, including the
CAN neurons, are derived from the AB lineage (Sulston et al.,
1983), loss of mpk-1 activity in the AB lineage alone is not
sufficient to confer a Soc phenotype. Specific action in the
CAN neurons is even more strongly refuted by the mosaic data,
since these cells derive exclusively from the AB lineage. This
evidence is further bolstered by our results that expression of
EGL-15 in the CAN neurons, using either the ceh-10 promoter
or the ceh-23 promoter (Forrester et al., 1998; Svendsen and
McGhee, 1995), failed to rescue the Soc phenotype of egl15(n1783) (P.H. and M.J.S., unpublished). The combined data
from all these approaches strongly support EGL-15 signaling
acting in the hypodermis to regulate fluid balance. It is
interesting to note that the same RAS/MAPK cascade is
exploited in two distinct processes in maintaining fluid
homeostasis in worms: a developmental role in the
specification of the excretory duct cell fate regulated by EGF
signaling (Yochem et al., 1997) and a physiological role to
control fluid influx regulated by FGF signaling.
The use of a receptor-mediated response pathway allows
responding to fluid imbalance by regulating the availability of
the appropriate EGL-15 ligand. Our data support the function
of the LET-756 FGF as the ligand for this function of EGL-15.
To understand how LET-756 regulates EGL-15 signaling, we
performed a mosaic analysis of let-756 to determine its cellular
source. Our data indicate that LET-756 expression is not
required in the AB lineage. Since all the cells that have been
previously implicated in fluid homeostasis, such as the
excretory system and the CAN neurons, are derived from the
AB lineage, this result suggests that these cells are unlikely to
be the cellular source of LET-756. Instead, the cellular focus
of let-756 is tightly associated with that of dpy-17, which is
required in P1-derived cells for a portion of its activity
(Kenyon, 1986; Yuan and Horvitz, 1990). Interestingly, a let756 reporter is expressed extensively in body wall muscles
(Bülow et al., 2004), which derive predominantly from P1.
Understanding the regulation of LET-756 in response to
different physiological conditions may lead to new insights
into how C. elegans maintains its fluid balance.
2604 Development 131 (11)
Based on the phenotypic analysis of mutations affecting
EGL-15 signaling, EGL-15 activity in the hypodermis either
functions to promote fluid intake or inhibit fluid excretion. This
could be achieved by regulating the permeability of the
hypodermis. Interestingly, FGF2–/–/FGF5–/– double-mutant
mice show enhanced permeability of the blood–brain barrier,
which is accompanied by reduced levels of intermediate
filaments as well as tight junction proteins (Reuss et al., 2003).
Alternatively, EGL-15 might regulate the activity of ion
channels, which could provide the driving force for water
movement. In mammals, the kidney is the major organ for
maintenance of global fluid balance as well as ion homeostasis.
Interestingly, FGF-23 is implicated in phosphate homeostasis
in the kidney, which may similarly aid in maintaining systemic
fluid balance. Overexpression of FGF-23, or a mutant form of
FGF-23 that is resistant to proteolysis, results in inhibition
of phosphate reabsorption by the type II sodium-dependent
phosphate (Na/Pi) co-transporter in renal epithelial cells,
leading to hypophosphatemia (Kumar, 2002).
Our findings indicate that the EGL-15 FGFR signaling
pathway is probably the master switch in the hypodermis
regulating fluid balance in C. elegans, and provide an important
foundation from which to explore mechanisms by which FGF
signaling functions to regulate fluid balance.
We thank R. Roubin for discussions, for providing preliminary
expression data for let-756 reporters, and for several let-756
constructs. We also thank: O. Hobert for discussions and sharing
unpublished results; D. Baillie for dpy-17 let-756 unc-32; sDp3; E.
Lambie for providing mpk-1 ncl-1 unc-36; sDp3; C. Z. Borland for
making the initial observation that Pegl-15::clr-1 can rescue clr-1; I. E.
Sasson for help in the integration of ayIs25 and observing EGL-15
expression in the hypodermis; T. Lo for generating ayIs29; and M. R.
Koelle, L. Cooley and members of our laboratory for a critical reading
of this manuscript. Some nematode strains used in this work were
provided by the Caenorhabditis Genetics Center, which is funded by
the NIH. This work was supported by NIH grant GM50504 to M.J.S.
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