? Structural Biology Center, National Institute of Genetics and School of
Genetics, Faculty of Life Sciences, Graduate University for Advanced Studies,
Mishima, Shizuoka 411-8540, Japan
* Present address: Laboratory for Cell Migration, RIKEN Center for Developmental
Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 605-0074, Japan
Author for correspondence (e-mail:
ikatsura{at}lab.nig.ac.jp)
Accepted 9 April 2003
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SUMMARY |
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Key words: C. elegans, Protein tyrosine phosphatase, Cholesterol, Dauer larva
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INTRODUCTION |
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The dauer larva of the nematode Caenorhabditis elegans serves as a
good model system for these issues. Under favorable conditions, C.
elegans develops successively through four larval stages (L1-L4) to the
reproductive adult in 3 days. However, under unfavorable conditions, it forms
an alternative third-stage larva called dauer larva and arrest development.
The dauer larva can survive for periods of several weeks or months without
aging, while the adult animal can live only for about 2 weeks. When favorable
conditions are encountered, the dauer larva begins to feed and resumes
development to the adult stage (Riddle and
Albert, 1997). It is known that the decision to enter the dauer
stage is regulated by three environmental cues: food, temperature and a
dauer-inducing pheromone that reflects population density
(Golden and Riddle, 1982
;
Golden and Riddle, 1984a
;
Golden and Riddle, 1984b
). At
least two of them, food and dauer pheromone, are thought to be sensed by
chemosensory neurons with ciliated endings directly exposed to the external
environment (Albert et al.,
1981
; Perkins et al.,
1986
; Bargmann and Horvitz,
1991
; Vowels and Thomas,
1992
; Thomas et al.,
1993
; Schackwitz et al.,
1996
). The sensory neurons that participate in dauer formation
have been identified by laser microsurgery. When sensory neurons ADF, ASG, ASI
and ASJ are killed at the L1 stage, wild-type animals become dauer larvae and
arrest development regardless of environmental conditions
(Bargmann and Horvitz,
1991
).
To identify genes involved in dauer signaling, many mutants that show
abnormality in the dauer decision have been isolated
(Riddle et al., 1981;
Albert et al., 1981
;
Swanson and Riddle, 1981
;
Malone and Thomas, 1994
;
Inoue and Thomas, 2000
). Such
mutants, named daf (dauer formation abnormal), consist of two groups:
dauer formation-constitutive (Daf-c) mutants, which enter the dauer stage even
under conditions appropriate for reproductive growth, and dauer
formation-defective (Daf-d) mutants, which develop as non-dauers even under
harsh conditions. By epistasis analyses, these daf genes have been
ordered into three pathways that act in parallel to regulate dauer formation
(Vowels and Thomas, 1992
;
Thomas et al., 1993
;
Gottlieb and Ruvkun, 1994
).
Molecular analyses revealed that these pathways correspond to cell-signaling
pathways conserved among many animals, namely, cGMP
(Coburn et al., 1998
;
Birnby et al., 2000
),
TGFß (Georgi et al.,
1990
; Estevez et al.,
1993
; Schackwitz et al.,
1996
; Ren et al.,
1996
; Patterson et al.,
1997
; Inoue and Thomas,
2000
) and insulin/IGF-I
(Morris et al., 1996
;
Kimura et al., 1997
;
Ogg et al., 1997
;
Lin et al., 1997
;
Ogg and Ruvkun, 1998
)
pathways. The signals through the three pathways are integrated by a nuclear
hormone receptor DAF-12, suggesting that the developmental decision is
regulated by a lipophilic hormone(s)
(Antebi et al., 2000
;
Snow and Larsen, 2000
).
Recently it was reported that one of the Daf-c genes, daf-9, encodes
a cytochrome P450 and acts upstream of daf-12. The results indicate
that DAF-9 is probably involved in the biosynthetic pathway for a ligand(s) of
DAF-12 (Gerisch et al., 2001
;
Jia et al., 2002
).
daf-9 mutants as well as special daf-12 mutants that show a
Daf-c phenotype form dauer-like larvae, which differ from normal dauer larvae
in that their pharynx pumps and is not radially constricted. Furthermore, the
Daf-c phenotypes resulting from weak alleles of these genes are greatly
enhanced by cholesterol deprivation, consistent with the involvement of
steroids in the function of daf-12 and daf-9. C. elegans
cannot synthesize sterols, and steroid hormones in C. elegans are
considered to be made from cholesterol provided by culture media
(Chitwood, 1999
).
Conventional Daf-c genes contain mutations that cause highly penetrant
Daf-c phenotypes. In addition, there are many other genes that, when mutated,
result in only low-penetrance Daf-c, synthetic Daf-c (syn-Daf) and/or Hid
(high temperature-induced dauer formation) phenotypes. Syn-Daf mutants and Hid
mutants form dauer larvae only in a certain mutant background and at 27°C
(a temperature too high for the reproduction of C. elegans),
respectively (Bargmann et al.,
1990; Avery, 1993
;
Katsura et al., 1994
;
Iwasaki et al., 1997
;
Prasad et al., 1998
;
Take-Uchi et al., 1998
;
Ailion et al., 1999
;
Sze et al., 2000
;
Sym et al., 2000
;
Daniels et al., 2000
;
Ailion and Thomas, 2000
;
Lanjuin and Sengupta, 2002
).
The presence of such genes suggests that the daf pathways may be
bifurcated further or modified by other pathways. Analysis of these genes will
help to elucidate the complicated regulatory network of dauer decision and
provide a new insight into the environmental regulation of development.
Here we present genetic and molecular analyses of a new syn-Daf gene, sdf-9. It encodes a protein tyrosine phosphatase-like molecule, which seems to increase the activity of DAF-9 or help the execution of the DAF-9 function. The cells expressing sdf-9 are the same as those expressing daf-9 in the head and have been identified as XXXL/R cells in this study. These cells seem to play a key role in the metabolism of the steroid hormone(s) for DAF-12 that regulates dauer formation.
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MATERIALS AND METHODS |
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Dauer formation assays
Four to six adult hermaphrodites were allowed to lay eggs at 20°C for
4-7 hours on 6-cm NGM plates on which E. coli had been grown. Parent
animals were then removed and the plates were incubated at the assay
temperature. Dauer and non-dauer animals were counted after 3-3.5 days at
20°C, 2.5-3 days at 25°C, and 2-2.5 days at 27°C.
Molecular cloning of sdf-9
The sdf-9 mutations were previously mapped to the right arm of
chromosome V (N. Suzuki, T. Ishihara and I. Katsura, unpublished). More
precise mapping was performed as follows. From the genotype
sdf-9(ut187)/unc-51(e369)rol-9(sc148), 12/12 Rol non-Unc recombinants
were non-syn-Daf, and thus sdf-9(ut187) mapped near or on the right
side of rol-9. Using SNPs (Wicks et al., 2001), sdf-9(ut187)
was placed to the right of cosmid ZC15 (data not shown). Cosmids and PCR
fragments from this region of the genome were introduced into
unc-31(e169); sdf-9(ut187) animals, and the resulting transgenic
lines were tested for the rescue of the Daf-c phenotype. A 14.8 kb PCR
fragment containing the predicted ORFs Y44A6D.4 and Y44A6D.5
(C. elegans Sequencing
Consortium, 1998) rescued the Daf-c phenotype. This fragment was
digested with SpeI and NaeI, and the resulting 8.6 kb
fragment containing Y44A6D.4 was subcloned into the
SpeI-EcoRV site of pBluescript KS(+) (Stratagene Co.) to
generate the plasmid pKO1. pKO1 was digested with EcoRV and
HindIII, and the 5.7 kb fragment was cloned into the
HincII-HindIII site of pBluescript KS(+) to generate the
plasmid pKO2, which also rescued the Daf-c phenotype (data not shown).
Allele sequencing
Genomic DNA was isolated from the sdf-9 mutant animals, and the
sdf-9 region was PCR-amplified. Mutation sites were determined by
sequencing the mixed products of two independent PCR reactions.
cDNA cloning
The exon-intron structure of sdf-9 gene was deduced by sequencing
RT-PCR products amplified with gene-specific primers. The 5' end of the
gene was determined by sequencing PCR products obtained with SL1 leader
sequence and gene-specific primers, while the 3' end was determined by
sequencing 3' RACE products with gene-specific and artificial adapter
primers.
Germline transformation
Germline transformation was carried out as described previously
(Mello et al., 1991), at a
concentration of 1-60 ng/µl test DNA with 5-15 ng/µl coinjection marker
(myo-3::GFP, gcy-10::GFP, or pRF4(rol-6)) and 40-95
ng/µl carrier DNA (pBluescript KS(+)).
Reporter constructs
In this work, we used the modified Fire vectors pKOGzero and pKORzero as
GFP and RFP (dsRed) vectors, respectively, for studying gene expression.
pKOGzero was made by ligating fragments of three Fire vectors: a 2.9 kb
XhoI-ApaI fragment of pPD95.67, a 98 base
NcoI-XhoI fragment of pPD104.53 and a 1.6 kb
XhoI-ApaI fragment of pPD104.91. pKORzero was made by
substituting RFP cDNA for the GFP cDNA of pKOGzero. An 8.3-kb C.
elegans genomic DNA containing the sdf-9 gene was amplified with
primers 5'-ATACGGAGCGCAAGGCTGTG-3' and
5'-GGCTTTGGGATCCACCACGGGCGGC-3' and digested with HincII
and BamHI. The resulting 6.2 kb fragment was subcloned into the
HincII-BamHI site of pKOGzero to make pKOG6. pKOG6 was
digested with KpnI to remove the nuclear localization signal, and
self-ligated to generate pKOG7 (SDF-9::GFP; a 3.5 kb promoter and the entire
coding region fused to GFP cDNA). pKOG6 was digested with HindIII and
PvuII and the 3.5 kb fragment was cloned into the
HindIII-HincII site of pKOGzero and pKORzero to generate
pKOG8 (sdf-9p::GFP1; a 3.5 kb promoter region fused to GFP cDNA) and
pKOR1 (sdf-9p::RFP; a 3.5 kb promoter region fused to RFP cDNA).
pKOG8 was digested with KpnI to remove the nuclear localization
signal and self-ligated to generate pKOG9 (sdf-9p::GFP2). The coding
region (ApaI fragment) of the sdf-9 gene from pKO1 was
cloned into pBluescript KS(+) to generate pKO20 (sdf-9 promoterless
gene). The daf-9 promoter region was amplified by PCR as described
previously (Jia et al., 2002),
and inserted into the PstI site of pKO20 to generate pKO20-d9
(daf-9p::sdf-9(promoterless)). For DAF-9::GFP, a 7.7 kb PCR
product from the daf-9 genomic DNA containing a 5.4 kb 5'
upstream sequence and a 2.3 kb coding sequence was cloned into pKOGzero.
Mosaic analysis for the identification of sdf-9-expressing
cells using cell-specific GFP markers
The following cell-specific GFP markers were used: egl-4.a::GFP
for IL1VL/R (Fujiwara et al.,
2002), gcy-8::GFP for AFDL/R
(Yu et al., 1997
),
gpa-4::GFP for ASIL/R (Jansen et
al., 1999
), T23G5.5::GFP for CEPDL/R
(Jayanthi et al., 1998
) (T.
Ishihara, unpublished) and ttx-3::GFP for AIYL/R
(Hobert et al., 1997
).
egl-4.a::GFP, gcy-8::GFP, a mixture of gpa-4::GFP
and ttx-3::GFP, and a mixture of gpa-4::GFP and
T23G5.5::GFP, respectively, were injected into wild-type animals
together with sdf-9p::RFP (pKOR1) to make animals that carry an
extrachromosomal array containing one or two GFP markers and
sdf-9p::RFP. The transgenic animals were grown on normal NGM plates
to generate spontaneous mosaic animals, which were observed under a microscope
at the L3 to adult stage. Mosaic animals were identified by the presence of
sdf-9p::RFP in only one of the two sdf-9-expressing cells.
Such animals were found at a frequency of several percent among animals
showing RFP fluorescence, and examined for the presence or absence of each GFP
marker, using both Nomarski and fluorescence optics.
Cell ablation
Laser microsurgery was carried out essentially as described previously
(Bargmann and Avery, 1995).
pKOG8 (sdf-9p::GFP1) was injected into unc-31(e169) animals
together with gcy-10::GFP and pBluescript KS(+), and the established
array was introduced into N2, daf-3(e1376), daf-16(m27), and
daf-12(m20) animals. The cells expressing sdf-9p::GFP
(XXXL/R cells, according to our assignment) were ablated in L1 larvae, which
were then cultivated on NGM plates at 25°C. After 2 days, the numbers
dauer larvae, dauer-like larvae and non-dauers were scored. The success of
cell ablation was judged by the loss of sdf-9p::GFP fluorescence, and
animals showing the fluorescence were ignored. Control animals, that were
treated in the same way as the operated animals except that no cells were
killed, did not form dauer or dauer-like larvae.
Expression of sdf-9 under the control of the daf-9
promoter
pKO20 (sdf-9 promoterless gene) and pKO20-d9
(daf-9p::sdf-9(promoterless)), were injected into
unc-31(e169); sdf-9(ut187) animals together with gcy-10::GFP
and pBluescript KS(+), and the transgenic lines were tested for dauer
formation.
Suppression of the Daf-c phenotype of various mutations by the
wild-type daf-9 transgene
A 9.3-kb PCR product from the daf-9 genomic region containing
5.4-kb 5' upstream sequence, 2.3-kb coding sequence, and 1.6-kb 3'
sequence was injected with gcy-10::GFP and pBluescript KS(+) into
unc-31(e169), unc-31(e169);sdf-9(ut187), daf-7(e1372) and
daf-2(e1370) animals, respectively. Multiple independent transgenic
lines were tested for dauer formation. For the experiments with the
sdf-9(ut163) mutant, the arrays were established in the
unc-31(e169) background and introduced into sdf-9(ut163)
animals by crossing. sdf-9(ut187); Ex[daf-9(+), gcy-10::GFP]
animals were made from unc-31(e169); sdf-9(ut187); Ex[daf-9(+),
gcy-10::GFP].
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RESULTS |
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Position of sdf-9 gene in the dauer pathway
To determine the position of the sdf-9 gene in the dauer pathway,
we carried out genetic epistasis experiments. Under reproductive growth
conditions, the sdf-9(ut163) single mutant formed dauer-like larvae
at 25°C. The daf-16(m27) and daf-12(m20) mutations
suppressed this phenotype, while daf-3(e1376) enhanced it
(Table 3). The presence of the
daf-5(e1386) mutation had only a very small effect on the Daf-c
phenotype of sdf-9: N2, daf-5, ut163 and
daf-5;ut163 mutants produced 0% (n=236), 0%
(n=416), 80% (n=256), and 52% (n=256) dauer and
dauer-like larvae, respectively, at 25.5°C on NGM minus cholesterol. We
also tested suppression of the Daf-c phenotype of unc-31(e169); sdf-9
at 20°C, using three sdf-9 alleles, ut163, ut174 and
ut187. The results were essentially the same as the suppression of
sdf-9(ut163), i.e., daf-16 and daf-12 mutations
suppressed the unc-31; sdf-9 Daf-c phenotypes, while daf-3
enhanced it (data not shown). The results indicate that sdf-9 acts
upstream of daf-16 and daf-12 but downstream of or in
parallel with daf-3 and daf-5. Unidentified interaction may
exist between daf-3 and sdf-9, because the
daf-3(e1376) mutation slightly enhanced the Daf-c phenotype of
sdf-9(ut163), as in the case of daf-3(mgDf90) and
daf-12(rh273daf-c) (Gerisch et
al., 2001).
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This nonsense mutation would result in a truncated protein lacking the PTP active site and hence these alleles are likely to be null.
Expression pattern of sdf-9
We investigated the expression pattern of sdf-9 using GFP and RFP
fusion genes. pKOG7 (SDF-9::GFP) rescued the Daf-c phenotype of
unc-31(e169); sdf-9(ut187) (data not shown), indicating that the GFP
fusion protein is functional and that the place and the time of this
expression are sufficient for normal dauer regulation. The expression of
SDF-9::GFP was observed from late embryos to adults, and primarily in two
cells anterior to the nerve ring. The SDF-9::GFP fusion protein was localized
in the cytoplasm. The fluorescence intensity was essentially constant from the
threefold stage of embryos to L4 larvae, but slightly weaker at the adult
stage. To analyze the cell shape, a promoter-fusion GFP construct,
sdf-9p::GFP2, was introduced into wild-type animals. Like SDF-9::GFP,
sdf-9p::GFP2 was expressed strongly in two ventral cells anterior to
the nerve ring (Fig. 5A), but
additional faint signals were occasionally observed in several neurons
posterior to the nerve ring. The cells expressing sdf-9p::GFP2 and
SDF-9::GFP had two (or sometimes three) short processes, which had a complex
shape and looked different from those of sensory neurons. In some transgenic
animals, one of the processes occasionally extended to the anterior tip of the
head (Fig. 5B). In dauer
larvae, one of the processes was long and extended posteriorly, but did not
seem to enter the nerve ring (Fig.
5C). The positions of the two cells were variable, with the
right-hand cell occasionally found anterior to the metacorpus (19%,
n=508).
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Identification of sdf-9-expressing cells
We first chose the candidates of the sdf-9-expressing cells
(Fig. 7) by their positions,
using figure 2 of White et al.
(White et al., 1986). Then,
ciliated sensory neurons (hatched in Fig.
7) were excluded from the candidates, because we found that the
cells expressing sdf-9p::RFP did not express che-2p::GFP
(data not shown), which is known to be expressed in most ciliated sensory
neurons including those shown in Fig.
7 (Fujiwara et al.,
1999
).
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There is additional evidence supporting the proposal that the sdf-9::RFP-expressing cells are XXXL/R. If we assume that sdf-9L/R are XXXL/R, as many as 94% of the mosaic animals could be explained by a single loss of the extrachromosomal array, and only 6% of the mosaic animals require two losses and none require three losses, for their formation (see supplemental data at http:/dev.biologists.org/supplemental/). In contrast, if sdf-9L/R were any of the other cells shown in Fig. 6B, much higher percentages of mosaic animals would have suffered multiple losses (see Table S3 at http:/dev.biologists.org/supplemental/). Since multiple losses of the extrachromosomal array in one animal do not occur so frequently, the results support the above conclusion.
Killing of XXXL/R cells induces the formation of dauer-like
larvae
To determine whether the sdf-9-expressing cells, which we
identified as XXXL/R cells, are involved in dauer formation, we killed them in
L1 larvae by laser microbeam surgery. At 25°C, about 30% of the operated
wild-type animals became dauer-like larvae resembling those of sdf-9
and having a non-constricted, pumping pharynx
(Table 3). Furthermore, about
60% of the operated unc-31(e169) animals formed Unc but otherwise
normal dauer larvae that resemble unc-31; sdf-9 dauer larvae
(Table 3). We also killed these
cells in some Daf-d mutants: daf-3(e1376), daf-16(m27) and
daf-12(m20). When the XXXL/R cells were killed, daf-3
animals formed dauer-like larvae, whereas daf-16 and daf-12
animals formed neither dauer nor dauer-like larvae
(Table 3). These results
suggest that XXXL/R cells are important in preventing formation of dauer-like
larvae and that the position of their function in the dauer pathway is the
same as that of sdf-9.
Expression of sdf-9 gene under the control of the
daf-9 or npc-1 promoter rescues the Daf-c phenotype of
unc-31; sdf-9
Because sdf-9 is expressed in the daf-9-expressing cells
in the head, we expressed the sdf-9 gene under the control of the
daf-9 promoter and tested whether this is sufficient for the
wild-type phenotype of sdf-9 gene. The Daf-c phenotype of the
unc-31(e169); sdf-9(ut187) double mutant was suppressed by the
sdf-9 gene under the control of a daf-9 promoter (see Table
S4 at
http:/dev.biologists.org/supplemental/),
but not by the promoterless sdf-9 gene (data not shown). The result
indicates that the expression driven by the daf-9 promoter is
sufficient for the function of the sdf-9 gene in normal dauer
regulation. By using GFP and RFP fusion genes, we already showed that the
cells in which sdf-9 is strongly expressed are a subset of cells in
which daf-9 is strongly expressed. The result in this section
confirms this conclusion through the function of sdf-9.
We also expressed sdf-9 under the control of other promoters.
npc-1 and npc-2 are homologs of the Niemann-Pick type C
disease gene, which plays a role in intracellular sterol transport
(Hoekstra and van IJzendoorn,
2000; Ioannou,
2001
). In C. elegans, it is known that the double mutant
npc-2; npc-1 shows a Daf-c phenotype
(Sym et al., 2000
). We found
that npc-1p::GFP was expressed in the sdf-9-expressing
cells. Furthermore, the expression of sdf-9 under the control of the
npc-1 promoter rescued the Daf-c phenotype of unc-31(e169);
sdf-9(ut187) (data not shown). These results suggest that the
sdf-9-expressing cells, which we identified as XXXL/R, may be
important in the metabolism of steroids or steroid hormone signaling.
Cholesterol deprivation enhances the weak Daf-c phenotype of
sdf-9 mutants
It was reported that cholesterol deprivation enhances the weak Daf-c
phenotype of daf-9(rh50) and daf-12(rh284) mutants
(Gerisch et al., 2001). Since
sdf-9 resembles daf-9 in dauer-like larvae and expression
pattern, we investigated the dauer formation of sdf-9 mutants on NGM
plates without a supplement of cholesterol (NGM minus cholesterol). The
sdf-9 Daf-c phenotype was enhanced by cholesterol deprivation
(Table 2). We confirmed that a
functional SDF-9::GFP (pKOG7) rescued this Daf-c phenotype of the
sdf-9(ut163) mutant (see Table S5 at
http:/dev.biologists.org/supplemental/).
We also found that increased concentrations of cholesterol tend to suppress
the Daf-c phenotype of sdf-9 mutants
(Table 2). These results show
that cholesterol or its metabolite regulates dauer formation in cooperation
with the regulatory pathway including the sdf-9 gene.
The Daf-c phenotype of sdf-9 single and unc-31;
sdf-9 double mutants is suppressed by the overexpression of the wild-type
daf-9 gene
We examined the expression of DAF-9::GFP in the wild type and the following
mutant backgrounds: unc-31(e169), sdf-9(ut174), sdf-9(ut187),
unc-31(e169); sdf-9(ut174), and unc-31(e169); sdf-9(ut187). The
expression did not change in these mutant backgrounds (data not shown), but
the transgene weakly suppressed the Daf-c phenotype of unc-31; sdf-9
mutants, although it weakly enhanced the dauer formation of the
unc-31 mutant (data not shown). We therefore examined whether the
wild-type daf-9 gene suppresses the Daf-c phenotypes of
sdf-9 single mutants on NGM minus cholesterol and unc-31;
sdf-9 double mutants on NGM. As shown in
Table 5, the extrachromosomal
array of the wild-type daf-9 gene strongly suppressed the Daf-c
phenotypes of sdf-9(ut163), sdf-9(ut187) and unc-31(e169);
sdf-9(ut187), but not those of daf-7(e1372) and
daf-2(e1370), in which daf-9p::GFP was also expressed (data
not shown). These results suggest that SDF-9 enhances the function but not the
expression of DAF-9.
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DISCUSSION |
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SDF-9 enhances the DAF-9 function
The results of this study showed that the function of sdf-9 is
closely related to that of daf-9. First, like daf-9 mutants,
sdf-9 mutants form dauer-like larvae under reproductive growth
conditions, and normal dauer larvae under dauer-inducing conditions. Moreover,
like daf-9, the formation of dauer-like larvae by sdf-9
mutants is enhanced by cholesterol deprivation. Second, sdf-9 is
expressed in the two head cells in which daf-9 is expressed. Third,
like the Daf-c phenotype of daf-9, the Daf-c and syn-Daf phenotypes
of sdf-9 are suppressed by daf-12 but not by
daf-3.
In some aspects, however, sdf-9 mutants show weaker phenotypes
than daf-9 mutants, or the sdf-9 gene has only a part of the
properties of daf-9 gene. First, even a putative null mutant of
sdf-9 shows only a weak, temperature-sensitive Daf-c phenotype, while
many daf-9 mutants show strong, nonconditional Daf-c phenotypes.
Second, the formation of dauer-like larvae is suppressed by daf-16 in
the case of sdf-9 mutants but not in daf-9 mutants. Third,
sdf-9 is expressed essentially only in two cells in the head, while
daf-9 is expressed in hypodermis, vulval blast cells and spermatheca
at some stages of development, besides the two head cells. Fourth,
sdf-9 mutants seem to show normal longevity, at least at 25°C
(K.O., T.I. and I.K., unpublished), while daf-9 mutants show
increased longevity under certain conditions
(Gerisch et al., 2001;
Jia et al., 2002
). These
results may be explained, if the sdf-9 function is partially
redundant, if sdf-9 controls only part of the multiple functions of
daf-9, or if sdf-9 just enhances the daf-9
function.
The functional relationship between sdf-9 and daf-9 can be further speculated on. This study revealed that the Daf-c and syn-Daf phenotypes of sdf-9 are suppressed by the overproduction of the wild-type daf-9 gene, and that DAF-9::GFP is expressed even in the sdf-9 mutant background. The results show that SDF-9 enhances the function but not the synthesis of DAF-9. SDF-9 may increase the activity of the DAF-9 P450 molecule or may help the execution of the DAF-9 function, for instance, through the metabolism or transport of the substrate or product of DAF-9.
Position of sdf-9 in the dauer regulatory pathway
Although we have discussed that sdf-9 is closely related to
daf-9 in its function, the position of the sdf-9 gene in the
dauer regulatory pathway is yet another issue to be discussed. The Daf-c
phenotypes of sdf-9 as well as daf-9 were suppressed by
daf-12 but not by daf-3 or daf-5. These results
indicate that the sdf-9 gene, like daf-9, acts upstream of
daf-12, but downstream of or in parallel with the TGFß pathway
genes daf-3 and daf-5. Since the sdf-9 Daf-c
phenotype is not enhanced by daf-5, its enhancement by daf-3
is probably related to the Daf-c phenotype of daf-3 (but not
daf-5) at 27°C (Ailion and
Thomas, 2000).
Unlike daf-9, the Daf-c phenotypes of sdf-9 mutations were suppressed by daf-16. This result can be interpreted in the light of the laser microsurgery experiments. The dauer larva formation induced by the ablation of XXXL/R showed the same suppression pattern as that of sdf-9 mutants. Furthermore, like sdf-9 mutations, the operated larvae produced mainly dauer-like larvae in the wild-type background, and normal dauer larvae in the unc-31 background. Those results suggest that the function of XXXL/R is located upstream of daf-16 in the dauer regulatory pathway and that the function of daf-9 in other cells is located downstream of or in parallel with daf-16 in the pathway.
The XXXL/R-specific suppression by daf-16 suggests that the DAF-2
insulin receptor signal transduction may play a tissue-specific role in these
cells, which involves SDF-9 function and whose defect leads to dauer-like
larvae formation. Experiments on daf-2 mosaic animals
(Apfeld and Kenyon, 1998)
revealed that animals that are daf-2() in AB, ABa, ABar, ABp,
or ABplaa cell lineages form dauer-like larvae (`class II dauers'). Since XXXL
and XXXR cells form in the ABplaa and ABarpa lineages, respectively, the
result is in agreement with the idea that loss of daf-2 gene activity
in XXXL/R results in the formation of dauer-like larvae. It remains to be
examined whether daf-2 is expressed in XXXL/R and whether SDF-9
interacts with the DAF-2 tyrosine kinase, especially its
tyrosine-phosphorylated form.
XXXL/R cells are involved in dauer regulation, probably through
steroid hormonal signaling and/or steroid metabolism
We identified the sdf-9- and daf-9-expressing cells in
the head as XXXL/R by mosaic experiments. These cells are known as embryonic
hypodermal cells whose function at later stages is unknown
(Sulston et al., 1983). White
et al. (White et al., 1986
)
included these cells in the list of neurons and their associated cells in the
head (figure. 2 of White et
al.). This study revealed a function of XXXL/R, namely, regulation of dauer
formation. The most direct evidence for this function is that killing of these
cells induces formation of dauer-like larvae in the wild-type background and
that of dauer larvae in the unc-31 background.
Gerisch et al. (Gerisch et al.,
2001) and Jia et al. (Jia et
al., 2002
) also carried out laser microsurgery experiments on
these cells. Although their results are partly different from ours, we think
there is no major contradiction. Gerisch et al.
(Gerisch et al., 2001
)
reported that ablation of daf-9-expressing head cells in 41
daf-9(dh6); Is[daf-9::GFP] animals resulted in
dauer-like larva formation in only four (10%) of them. The difference may be
due to overexpression of daf-9 by Is[daf-9::GFP] as
well as other differences in experimental conditions. Jia et al.
(Jia et al., 2002
) found that
none of the six N2; Ex[daf-9p::GFP] animals formed
dauer-like larvae after killing of the daf-9-expressing head cells,
but they argued that the timing of the surgery might be too late. They also
killed these cells in daf-9(m540);
Ex[daf-9p::daf-9 cDNA::GFP] animals, and all the
four animals in which the surgery was successful formed dauer-like larvae.
XXXL/R cells seem to regulate dauer formation through the functions of
daf-9 and sdf-9, which are expressed in these cells and
whose mutants form dauer-like larvae. The following results show that these
functions are probably related to steroid hormonal signaling and/or steroid
metabolism. First, daf-9 encodes a cytochrome P450 of the CYP2
family, whose members are involved in steroid metabolism
(Gerisch et al., 2001;
Jia et al., 2002
). Second, the
daf-12 gene, which acts downstream of daf-9, encodes a
steroid hormone receptor (Antebi et al.,
2000
). Third, the Daf-c phenotypes of daf-9, sdf-9 and
daf-12(daf-c) mutations are enhanced by the deprivation of
cholesterol (Gerisch et al.,
2001
; Jia et al.,
2002
; this study). Fourth, the expression of SDF-9 under the
control of the npc-1 promoter rescues the Daf-c phenotype of
unc-31(e169); sdf-9(ut187), where npc-1 is a homolog of the
Niemann-Pick type C disease gene, which plays a role in intracellular sterol
transport (Hoekstra and van IJzendoorn,
2000
; Ioannou,
2001
).
In conclusion, we showed that SDF-9, a protein tyrosine phosphatase-like molecule, is involved in the regulation of dauer formation. Its function is closely related to that of DAF-9, a P450 molecule: it probably enhances the activity of DAF-9 or helps the execution of the DAF-9 function. Furthermore, SDF-9 is expressed in two head cells in which DAF-9 is expressed. We identified these cells as XXXL/R cells, which are known as embryonic hypodermal cells but whose function at later stages remains to be studied. Since this study on SDF-9 and former studies on DAF-9 suggested that the functions of these proteins are related to steroid metabolism or steroid hormonal signaling, XXXL/R cells seem to play a key role in the metabolism or function of a steroid hormone(s) that acts in dauer regulation.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ailion, M., Inoue, T., Weaver, C. I., Holdcraft, R. W. and
Thomas, J. H. (1999). Neurosecretory control of aging in
Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA
96,7394
-7397.
Ailion, M. and Thomas, J. H. (2000). Dauer
formation induced by high temperatures in Caenorhabditis elegans.Genetics 156,1047
-1067.
Albert, P. S., Brown, S. J. and Riddle, D. L. (1981). Sensory control of dauer larva formation in Caenorhabditis elegans. J. Comp. Neurol. 198,435 -451.[Medline]
Albert, P. S. and Riddle, D. L. (1988). Mutants of Caenorhabditis elegans that form dauer-like larvae. Dev. Biol. 126,270 -293.[Medline]
Antebi, A., Culotti, J. G. and Hedgecock, E. M.
(1998). daf-12 regulates developmental age and the dauer
alternative in C. elegans. Development
125,1191
-1205.
Antebi, A., Yeh, W-H., Tait, D., Hedgecock, E. and Riddle, D.
L. (2000). daf-12 encodes a nuclear receptor that
regulates the dauer diapause and developmental age in C. elegans.Genes Dev. 14,1512
-1527.
Apfeld, J. and Kenyon, C. (1998). Cell nonautonomy of C. elgans DAF-2 function in the regulation of diapause and life span. Cell 95,199 -210.[Medline]
Avery, L. (1993). The genetics of feeding in
Caenorhabditis elegans. Genetics
133,897
-917.
Bargmann, C. I., Thomas, J. H. and Horvitz, H. R. (1990). Chemosensory cell function in the behavior and development of Caenorhabditis elegans. Cold Spring Harbor Symp. Quant. Biol. 55,529 -538.[Medline]
Bargmann, C. I. and Horvitz, H. R. (1991). Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans. Neuron 7, 729-742.[Medline]
Bargmann, C. and Avery, L. (1995). Laser killing of cells in Caenorhabditis elegans. In Methods in Cell Biology. Vol. 48 Caenorhabditis elegans: Modern Biological Analysis of an Organism (ed. H. F. Epstein and D. C. Shakes), pp. 225-250. San Diego: Academic Press.[Medline]
Birnby, D. A., Malone, E., Vowels, J. J., Tian, H., Colacurcio,
P. L. and Thomas, J. H. (2000). A transmembrane guanylyl
cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of chemosensory
behaviors in Caenorhabditis elegans. Genetics
155,85
-104.
Bowers, W. S., Ohta, T., Cleere, J. S. and Marsella, P. A. (1976). Discovery of insect anti-juvenile hormones in plants. Science 193,542 -547.[Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
The C. elegans Sequencing Consortium
(1998). Genome sequence of the nematode C. elegans: a
platform for investigating biology. Science
282,2012
-2018.
Chitwood, D. J. (1999). Biochemistry and
function of nematode steroids. Crit. Rev. Biochem. Mol.
Biol. 34,273
-284.
Coburn, C. M., Mori, I., Ohshima, Y. and Bargmann, C. I.
(1998). A cyclic nucleotide-gated channel inhibits sensory axon
outgrowth in larval and adult Caenorhabditis elegans: a distinct
pathway for maintenance of sensory axon structure.
Development 125,249
-258.
Conover, D. O. and Kynard, B. E. (1981). Environmental sex determination: Interaction of temperature and genotype in a fish. Science 213,577 -579.
Daniels, S. A., Ailion, M., Thomas, J. H. and Sengupta, P.
(2000). egl-4 acts through a transforming growth
factor-beta/SMAD pathway in Caenorhabditis elegans to regulate
multiple neuronal circuits in response to sensory cues.
Genetics 156,123
-141.
Estevez, M., Attisano, L., Wrana, J. L., Albert, P. S., Massague, J. and Riddle, D. L. (1993). The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development. Nature 365,644 -649.[CrossRef][Medline]
Fujiwara, M., Ishihara, T. and Katsura, I.
(1999). A novel WD40 protein, CHE-2, acts cell-autonomously in
the formation of C. elegans sensory cilia.
Development 126,4839
-4848.
Fujiwara, M., Sengupta, P. and McIntire, S. L. (2002). Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase. Neuron 36,1091 -1102.[Medline]
Georgi, L. L., Albert, P. S. and Riddle, D. L. (1990). daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase. Cell 61,635 -645.[Medline]
Gerisch, B., Weitzel, C., Kober-Eisermann, C., Rothers, V. and Antebi, A. (2001). A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span. Dev. Cell 1,841 -851.[Medline]
Golden, J. W. and Riddle, D. L. (1982). A pheromone influences larval development in the nematode Caenorhabditis elegans. Science 218,578 -580.[Medline]
Golden, J. W. and Riddle, D. L. (1984a). The Caenorhabditis elegans dauer larva: Developmental effects of pheromone, food and temperature. Dev. Biol. 102,368 -378.[Medline]
Golden, J. W. and Riddle, D. L. (1984b). A pheromone-induced developmental switch in C. elegans: temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proc. Natl. Acad. Sci. USA 81,819 -823.[Abstract]
Gottlieb, S. and Ruvkun, G. (1994). daf-2,
daf-16 and daf-23: genetically interacting genes controlling
dauer formation in Caenorhabditis elegans. Genetics
137,107
-120.
Guan, K. L. and Dixon, J. E. (1990). Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science 249,553 -556.[Medline]
Guan, K. L. and Dixon, J. E. (1991). Evidence
for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate
intermediate. J. Biol. Chem.
266,17026
-17030.
Hobert, O., Mori, I., Yamashita, Y., Honda, H., Ohshima, Y., Liu, Y. and Ruvkun, G. (1997). Regulation of interneuron function in the C. elegans thermoregulatory pathway by the ttx-3 LIM homeobox gene. Neuron 19,345 -357.[Medline]
Hoekstra, D. and van IJzendoorn, S. C. (2000). Lipid trafficking and sorting: how cholesterol is filling gaps. Curr. Opin. Cell Biol. 12,496 -502.[CrossRef][Medline]
Inoue, T. and Thomas, J. H. (2000). Targets of TGF-ß signaling in C. elegans dauer formation. Dev. Biol. 217,192 -204.[CrossRef][Medline]
Ioannou, Y. A. (2001). Multidrug permeases and subcellular cholesterol transport. Nat. Rev. Mol. Cell. Biol. 2,657 -668.[CrossRef][Medline]
Iwasaki, K., Staunton, J., Saifee, O., Nonet, M. and Thomas, J. H. (1997). aex-3 encodes a novel regulator of presynaptic activity in C. elegans. Neuron 18,613 -622.[Medline]
Jansen, G., Thijssen, K. L., Werner, P., van der Horst, M., Hazendonk, E. and Plasterk, R. H. (1999). The complete family of genes encoding G proteins of Caenorhabditis elegans. Nat. Genet. 21,414 -419.[CrossRef][Medline]
Jayanthi, L. D., Apparsundaram, S., Malone, M. D., Ward, E.,
Miller, D. M., Eppler, M. and Blakely, R. D. (1998). The
Caenorhabditis elegans gene T23G5.5 encodes an antidepressant- and
cocaine-sensitive dopamine transporter. Mol.
Pharmacol. 54,601
-609.
Jia, Z., Barford, D., Flint, A. J. and Tonks, N. K. (1995). Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B. Science 268,1754 -1758.[Medline]
Jia, K., Albert, P. S. and Riddle, D. L.
(2002). DAF-9, a cytochrome P450 regulating C. elegans
larval development and adult longevity. Development
129,221
-231.
Katsura, I., Kondo, K., Amano, T., Ishihara, T. and Kawakami,
M. (1994). Isolation, characterization and epistasis of
fluoride-resistant mutants of Caenorhabditis elegans.Genetics 136,145
-154.
Kimura, K. D., Tissenbaum, H. A., Liu, Y. and Ruvkun, G.
(1997). daf-2, an insulin receptor-like gene that
regulates longevity and diapause in Caenorhabditis elegans.Science 277,942
-946.
Lanjuin, A. and Sengupta, P. (2002). Regulation of chemosensory receptor expression and sensory signaling by the KIN-29 Ser/Thr kinase. Neuron 33,369 -381.[Medline]
Lin, K., Dorman, J. B., Rodan, A. and Kenyon, C.
(1997). daf-16: an HNF-3/Forkhead family member that can
function to double the life-span of Caenorhabditis elegans.Science 278,1319
-1322.
Malone, E. A. and Thomas, J. H. (1994). A
screen for nonconditional dauer-constitutive mutations in Caenorhabditis
elegans. Genetics 136,879
-886.
Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10,3959 -3970.[Abstract]
Miller, L. M., Waring, D. A. and Kim, S. K.
(1996). Mosaic analysis using a ncl-1(+)
extrachromosomal array reveals that lin-31 acts in the Pn.p cells
during Caenorhabtitis elegans vulval development.
Genetics 143,1181
-1191.
Morris, J. Z., Tissenbaum, H. A. and Ruvkun, G. (1996). A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans.Nature 382,536 -539.[CrossRef][Medline]
Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A. and Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389,994 -999.[CrossRef][Medline]
Ogg, S. and Ruvkun, G. (1998). The C. elegans PTEN homolog, DAF-18, acts in the insulin receptor-like metabolic signaling pathway. Mol. Cell 2,887 -893.[Medline]
Patterson, G. I., Koweek, A., Wong, A., Liu, Y. and Ruvkun,
G. (1997). The DAF-3 Smad protein antagonizes
TGF-ß-related receptor signaling in the Caenorhabditis elegans
dauer pathway. Genes Dev.
11,2679
-2690.
Perkins, L. A., Hedgecock, E. M., Thomson, J. N. and Culotti, J. G. (1986). Mutant sensory cilia in the nematode C. elegans. Dev. Biol. 117,456 -487.[Medline]
Prasad, B. C., Ye, B., Zackhary, R., Schrader, K., Seydoux, G.
and Reed, R. R. (1998). unc-3, a gene required for
axonal guidance in Caenorhabditis elegans, encodes a member of the
O/E family of transcription factors. Development
125,1561
-1568.
Ren, P.-f., Lim, C.-S., Johnsen, R., Albert, P. S., Pilgrim, D.
and Riddle, D. L. (1996). Control of C. elegans
larval development by neuronal expression of a TGF-ß homolog.
Science 274,1389
-1391.
Riddle, D. L., Swanson, M. M. and Albert, P. S. (1981). Interacting genes in nematode dauer larva formation. Nature 290,668 -671.[Medline]
Riddle, D. L. and Albert, P. S. (1997). Genetic and environmental regulation of dauer larva development. In C. elegans II (ed. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess), pp. 739-768. NY: Cold Spring Harbor Laboratory Press.
Schackwitz, W. S., Inoue, T. and Thomas, J. H. (1996). Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans. Neuron 17,719 -728.[Medline]
Snow, M. I. and Larsen, P. L. (2000). Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans. Biochim Biophys Acta 1494,104 -116.[Medline]
Streuli, M., Krueger, N. X., Tsai, A. Y. and Saito, H. (1989). A family of receptor-linked protein tyrosine phosphatases in humans and Drosophila. Proc. Natl. Acad. Sci. USA 86,8698 -8702.[Abstract]
Streuli, M., Krueger, N. X., Thai, T., Tang, M. and Saito, H. (1990). Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR. EMBO J. 9,2399 -2407.[Abstract]
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100,64 -119.[Medline]
Sulston, J. and Hodgkin, J. (1988). Methods. In The Nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 587-606. New York: Cold Spring Harbor Laboratory Press.
Swanson, M. M. and Riddle, D. L. (1981). Critical periods in the development of the Caenorhabditis elegans dauer larva. Dev. Biol. 84, 27-40.[Medline]
Sym, M., Basson, M. and Johnson, C. (2000). A model for Niemann-Pick type C disease in the nematode Caenorhabditis elegans. Curr. Biol. 10,527 -530.[CrossRef][Medline]
Sze, J. Y., Victor, M., Loer, C., Shi, Y. and Ruvkun, G. (2000). Food and metabolic signaling defects in a C. elegans serotonin-synthesis mutant. Nature 403,560 -564.[CrossRef][Medline]
Take-Uchi, M., Kawakami, M., Ishihara, T., Amano, T., Kondo, K.
and Katsura, I. (1998). An ion channel of the
degenerin/epithelial sodium channel superfamily controls the defecation rhythm
in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA
95,11775
-11780.
Thomas, J. H., Birnby, D. A. and Vowels, J. J.
(1993). Evidence for parallel processing of sensory information
controlling dauer formation in Caenorhabditis elegans.Genetics 134,1105
-1117.
Tonks, N. K. and Neel, B. G. (2001). Combinatorial control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 13,182 -195.[CrossRef][Medline]
Vowels, J. J. and Thomas, J. H. (1992). Genetic
analysis of chemosensory control of dauer formation in Caenorhabditis
elegans. Genetics 130,105
-123.
White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. (1986). The structure of the nervous system of the nematode C. elegans. Phil. Trans. R. Soc. Lond. B314,1 -340.
Wishart, M. J., Denu, J. M., Williams, J. A. and Dixon, J.
E. (1995). A single mutation converts a novel phosphotyrosine
binding domain into a dual-specificity phosphatase. J. Biol.
Chem. 270,26782
-26785.
Wishart, M. J. and Dixon, J. E. (1998). Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem. Sci. 23,301 -306.[CrossRef][Medline]
Yu, S., Avery, L., Baude, E. and Garbers, D. L.
(1997). Guanylyl cyclase expression in specific sensory neurons:
a new family of chemosensory receptors. Proc. Natl. Acad. Sci.
USA 94,3384
-3387.
Zhou, G., Denu, J. M., Wu, L. and Dixon, J. E.
(1994). The catalytic role of Cys124 in the dual specificity
phosphatase VHR. J. Biol. Chem.
269,28084
-28090.
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