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{at}yale.edu)
Accepted 17 February 2004
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SUMMARY |
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Key words: FGF receptor, EGL-15, Hypodermis, Fluid balance
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Introduction |
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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 EGL-15 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 EGL-15 signaling leads to a spectrum of
phenotypic consequences. Mutations that compromise the activity of CLR-1
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 EGL-15 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 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.
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Materials and methods |
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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 co-transformation 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
clr-1 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
egl-15(neu*) construct (NH#526), respectively, to replace the
endogenous promoters.
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
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. 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);
egl-15(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 non-Unc 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 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
clr-1(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 non-Unc, 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 non-Unc 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/).
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Results |
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If LET-756 acts as a ligand for EGL-15, then constitutive activation of
EGL-15 might bypass the requirement for LET-756, 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
low-level 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.
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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 egl-15 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::egl-15), 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 egl-15(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 egl-15(neu*) as well as the weaker rescue of
egl-15(Soc) by Pclr-1::egl-15 compared with
Pegl-15::egl-15 (Table
1A).
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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.
|
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 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
EGL-15 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 ncl-1
(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 EGL-15 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.
|
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.
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Discussion |
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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 LET-23(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 EGL-15
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 aex-3 and the unc-14 promoters
drove strong expression of EGL-15 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 egl-15(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 let-756 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.
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 bloodbrain 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.
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ACKNOWLEDGMENTS |
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Footnotes |
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