Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
* Author for correspondence (e-mail: ruvkun{at}molbio.mgh.harvard.edu)
Accepted 12 January 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: daf-9, daf-12, Dauer, Gonadal migration, Cytochrome P450, Insulin, C. elegans, TGFß
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genetic mosaic analysis showed that the DAF-2 insulin/IGF-I like receptor
and the DAF-4 type II TGFß receptor control reproductive development in a
cell nonautonomous manner (Apfeld and
Kenyon, 1998; Inoue and
Thomas, 2000
; Wolkow et al.,
2000
). A secondary signal is thought to be responsible for
communication between the peptide hormone responsive tissues, such as the
nervous system, and the rest of the body. Genetic analysis suggests that
daf-9 functions downstream of or in parallel to daf-2 and
daf-7 and upstream of daf-12
(Gerisch et al., 2001
;
Jia et al., 2002
).
daf-9 encodes a cytochrome P450 enzyme, whereas daf-12
encodes a nuclear receptor (Antebi et al.,
2000
; Gerisch et al.,
2001
; Jia et al.,
2002
). As cytochrome P450 enzymes mediate steroid hormone
synthesis in mammals and Drosophila
(Miller, 1988
;
Warren et al., 2002
), and
because daf-9 acts upstream of the nuclear receptor gene
daf-12 (Gerisch et al.,
2001
; Jia et al.,
2002
), DAF-9 may mediate the production of a lipophilic hormone
that regulates DAF-12 activity. Based on its action downstream of
daf-2 and daf-7 in the genetic epistasis analysis,
daf-9 expression or activity may in turn be regulated by the upstream
daf-2 and daf-7 signaling pathways.
Two classes of daf-9 mutant alleles have been described. Animals
carrying strong loss-of-function alleles arrest as dauers unconditionally and
seldom recover (Gerisch et al.,
2001; Jia et al.,
2002
). Ultrastructural studies revealed that daf-9 dauers
display intermediate dauer morphology in selected tissues
(Albert and Riddle, 1988
). This
suggests that parallel pathways may operate in conjunction with daf-9
to complete the global remodelling in dauer animals. A second class of
daf-9 alleles confers weak loss-of-function phenotypes: reversible
dauer arrest and a gonadal migration defect
(Antebi et al., 1998
;
Gerisch et al., 2001
;
Jia et al., 2002
). Allele and
temperature specific extension of adult life span has also been reported for
daf-9 mutant animals (Gerisch et
al., 2001
; Jia et al.,
2002
). Furthermore, there are complex interactions between
daf-9, germline and daf-2 signaling pathways in the control
of adult life span, reminiscent of those reported for daf-12
(Larsen et al., 1995
;
Gems et al., 1998
;
Hsin and Kenyon, 1999
).
The DAF-9 protein sequence is most similar to the mammalian CYP2 family of
P450 enzymes, which are responsible for degradation of steroidal and
xenobiotic compounds (Nebert and Russell,
2002). Nevertheless, DAF-9 is unlikely to be a functional
homologue of the mammalian CYP2 enzymes. This is because daf-9
expression is not induced by a range of xenobiotic compounds
(Menzel et al., 2001
), a
feature of the mammalian CYP2 enzymes
(Waxman, 1999
). A biosynthetic
role for DAF-9 was supported by the observations that cholesterol deprivation
causes a gonadal migration defect, similar to that of hypomorphic
daf-9 mutant animals (Gerisch et
al., 2001
). Furthermore, cholesterol withdrawal inhibits recovery
from dauer arrest by daf-9 hypomorphs
(Jia et al., 2002
). This
argues that daf-9 may participate in the modification of cholesterol
in the biosynthetic pathways to steroid hormones.
In this paper, we addressed the following questions. Where is the site of action of daf-9 gene function? How does daf-9 interact with the daf-2 and daf-7 signaling pathways? Is daf-9 expression regulated at a transcriptional level? We addressed these questions by expressing a functional GFP-tagged DAF-9 protein under the control of the endogenous daf-9 promoter and other well-established tissue-specific promoters. We find that daf-9 directs larval development and gonadal migration in a cell nonautonomous manner and its action is intricately linked to daf-2, daf-7 and daf-12 activities.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mgEx661-662: Ex[daf-9p::daf-9 genomic::GFP], mgEx663-664: Ex[dpy-7p::daf-9 cDNA::GFP; mec-7::GFP], mgEx669-670: Ex[sdf-9p::daf-9 cDNA::GFP; mec-7::GFP], mgEx665-666: Ex[che-2p::daf-9 cDNA::GFP; mec-7::GFP] and mgEx667-668: Ex[col-12p::daf-9 cDNA::GFP; mec 7::GFP].
In all tables, line 1 refers to the odd-numbered extrachromosomal array and line 2 refers to the even-numbered extrachromosomal array.
Generation of daf-9p::daf-9::GFP transgenic lines
The genomic region spanning all exons of daf-9 plus 7 kb of
non-coding sequence 5' to the initiator codon of the daf-9b
isoform was amplified by PCR and subcloned into the
SalI/BglII sites of pPD95.69 (kindly provided by A. Fire) in
two steps. The nuclear localisation signal of pPD95.69 was removed as a
result. The genomic region spanning the first exon, the first intron and seven
residues of the second exon together with 3 kb of non-coding sequence 5'
to the initiator codon of the daf-9b isoform was amplified by PCR and
subcloned into the BglII site of pPD95.75 (kindly provided by A.
Fire). The ligation junctions of the above constructs were sequenced to ensure
that the daf-9-coding sequence was in-frame with the GFP coding
sequence. The genomic region encompassing all exons of the daf-9 plus
3 kb of non-coding sequence 5' to the initiator codon of daf-9b
isoform was amplified by PCR and subcloned into the
BglII/AgeI sites of pPD95.75. Intron 1 of the
daf-9b isoform was removed from the last construct by recombinant PCR
and the product subcloned into the BglII/AgeI sites of
pPD95.75. The nuclear receptor consensus half site in intron 1 of the
daf-9b isoform was mutated into a LexA binding site by recombinant
PCR and the product subcloned into the BglII/AgeI sites of
pPD95.75. For the last three constructs, all exons and introns of
daf-9 were fully sequenced.
The above constructs were injected into N2 wild-type animals at 10 to 30 ng/µl. pBluescript was used to normalise the total concentration of injection mix to 100 ng/µl. Extrachromosomal arrays which gave robust daf-9::GFP expression were introduced into + / szT1 [lon-2(e678)]; daf-9(e1406) dpy-7(sc27) / szT1 animals by genetic crosses.
Generation of tissue specific daf-9::GFP transgenic lines
Tissue specific daf-9::GFP transgenic constructs were generated by
assembling three PCR products by a modified recombinant PCR method
(Hobert, 2002). The following
tissue specific promoters were amplified from N2 genomic DNA: sdf-9
(3.7 kb), che-2 (2.5 kb), dpy-7 (0.4 kb) and col-12
(1 kb) (Johnstone and Barry,
1996
; Fujiwara et al.,
1999
; Ohkura et al.,
2003
). The primer sequences defining the 5' end of the
promoter in these transgenes are as follows:
Each promoter fragment encompasses sequence immediately 5' to the start codon of the respective gene. GFP-coding sequence and unc-54 3' UTR sequence were amplified by PCR using pPD95.75 as template. The coding sequence of the daf-9b isoform was amplified by PCR using a daf-9 cDNA clone as template (kindly provided by Y. Kohara). Purified PCR products were injected into N2 wild-type animals at 5 ng/µl in the presence of mec-7::GFP plasmid at 30 ng/µl. pBluescript was used to normalise the total concentration of injection mix to 100 ng/µl. Extrachromosomal arrays which gave robust daf-9::GFP expression were introduced into + / szT1 [lon-2(e678)]; daf-9(e1406) dpy-7(sc27) / szT1 animals by genetic crosses.
Assay for dauer arrest
Adults were allowed to lay eggs on nematode-growth plates for 3 hours at
room temperature, and progeny were incubated at 20°C for 68 and 92 hours
or 25°C for 55 hours. Dauers were distinguished by a radially constricted
body, dauer alae and a constricted pharynx. Dauer assays for each strain were
repeated at least three times.
Lifespan assay
Adult lifespan of various strains was determined at 25°C, with agar
plates containing 0.1 g/ml FUDR to prevent growth of progeny. Synchronous
populations of worms from 3 hour egglays on nematode-growth medium plates were
allowed to develop at 15°C until young adult stage, before being
transferred to FUDR plates and shifted to 25°C. Worms were monitored every
2-4 days and were scored as dead when they no longer responded to gentle
prodding with a platinum wire. Lifespan is defined as the time elapsed from
the day when worms were put on FUDR plates (day 0) to when they were scored as
dead. Worms that crawled off the plates were excluded from calculations.
Lifespan assays were repeated at least twice.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We used the collagen gene dpy-7 promoter to direct hypodermal
expression of daf-9 during larval stages
(Gilleard et al., 1997). A GFP
tag was appended to the DAF-9 C terminus so that tissue specific DAF-9
expression could be confirmed by visualization of the GFP signal. Restoration
of daf-9 activity in the hypodermis alone was sufficient to prevent
dauer arrest of daf-9(e1406) mutant animals
(Table 1); these transgenic
animals developed into reproductive adults with the same growth rate as
animals carrying a daf-9 transgene driven by its own promoter. Next,
we expressed daf-9 exclusively in XXXL/R cells under the control of
the sdf-9 gene promoter (Ohkura
et al., 2003
). At 20°C, daf-9 expression in the two
XXXL/R cells was sufficient to prevent dauer arrest of daf-9(e1406)
mutant animals (Table 1).
Nevertheless, 5-20% of daf-9(e1406) animals that expressed
daf-9 in the XXXL/R cells arrested as dauers at 25°C
(mgEx667, mgEx668; n=898). This was not due to
downregulation of the sdf-9p::daf-9::GFP transgene at
25°C as no dramatic diminution of GFP fluorescence was observed in the
XXXL/R cells. By contrast, dpy-7 promoter-driven, daf-9
expression in the hypodermis led to complete suppression of dauer arrest of
daf-9(e1406) animals at 25°C (mgEx663, mgEx664;
n=300). Therefore, hypodermal daf-9 expression or a general
increase in daf-9 expression level may be crucial in promoting
reproductive development at elevated, dauer-inducing temperatures. Taken
together, daf-9 activity in the hypodermis is sufficient to
orchestrate reproductive development in an otherwise daf-9 deficient
animal. Hence, our observations support the hypothesis that daf-9
inhibits dauer arrest in a cell nonautonomous manner.
daf-9 controls gonadal migration cell-nonautonomously
In contrast to the strong loss-of-function allele daf-9(e1406),
daf-9 weak loss of function alleles confer a gonadal migration defect,
where the distal tip cells fail to migrate dorsally at the third larval stage
(Gerisch et al., 2001;
Jia et al., 2002
). This
indicates that daf-9 also plays a role in gonadal migration during
reproductive development. Transgenic expression of daf-9 under the
control of its endogenous promoter fully rescued daf-9(e1406) mutant
animals and no gonadal migration defect was observed at the L4 or adult stage
(Table 2). Similarly, normal
gonadal migration was observed in daf-9(e1406) mutant animals when
daf-9 was expressed exclusively in the hypodermis or the XXXL/R cells
under the control of dpy-7 or sdf-9 promoters, respectively
(Fig. 1A and
Table 2). Taken together, our
results suggest that daf-9 acts in a cell nonautonomous manner to
direct the movement of the distal tip cells that drive proper gonadal
migration, and that expression either from the broadly distributed hypodermal
cells or the anterior XXXL/R cells supplies sufficient signal to do so.
|
|
Even though expression of daf-9 in the ciliated neurons using the
che-2 promoter rescued daf-9(e1406) dauer arrest, more than
70% of the reproductive adults displayed a gonadal migration defect
(Fig. 1B and
Table 2). Such defect is also
observed in animals bearing daf-9 weak loss-of-function alleles. One
possibility is that the daf-9-expressing cells and the target distal
tip cells are too distant from each other. Notably, of the 56 neurons where
the che-2 promoter is active, 49 of them are located in the head,
five in the tail and none in the vicinity of the distal tip cells
(Fujiwara et al., 1999).
However, this is ruled out by the complete suppression of gonadal migration
phenotype of daf-9 mutant animals when daf-9 activity was
restored only in the two XXXL/R cells in the head
(Table 2). We therefore
attribute the gonadal migration defect to a suboptimal level of lipophilic
hormone that is produced by DAF-9 in ciliated neurons. Alternatively, the
complement of enzymes in ciliated sensory neurons may only be able to
synthesize a hormone that promote non-dauer fate but not proper gonadal
migration, unlike the ones in the native XXXL/R cells or hypodermis.
Effect of constitutive daf-9 expression on dauer arrest in daf-7() and daf-2() mutant animals
Genetic analysis suggests that daf-9 functions either downstream
of or in parallel to daf-16 and daf-3 in the dauer pathway
(Gerisch et al., 2001;
Jia et al., 2002
). One
attractive model is that daf-9 expression is regulated at a
transcriptional level by DAF-16 and DAF-3 in response to the daf-2
insulin-like and daf-7 TGFß like signaling pathways,
respectively. Modulation of daf-9 gene expression would in turn alter
the level of a probable lipophilic secondary hormonal signal that promotes
reproductive development. If this is the case, constitutive expression of
DAF-9 may substitute for a loss of daf-7 or daf-2 signaling
and direct reproductive development in daf-7() and
daf-2() mutant animals, which normally arrest as dauers.
To uncouple daf-9 expression from any potential transcriptional control originating from the daf-7 and daf-2 pathways, we introduced transgenes that direct daf-9 expression in the hypodermis or the XXXL/R cells under the control of dpy-7 or sdf-9 promoters, respectively, into daf-7(e1372), daf-1(m40) and daf-2(e1370) mutant animals. The same transgenes were fully functional in rescuing the dauer phenotype and gonadal migration defect of daf-9(e1406) mutant animals.
The daf-7 and daf-1 genes encode a TGFß like ligand
and a type I TGFß receptor, respectively
(Georgi et al., 1990;
Ren et al., 1996
).
Constitutive hypodermal expression of daf-9 suppressed the dauer
arrest phenotype of daf-7(e1372) mutant animals
(Table 3). At 25°C, none of
the transgenic animals arrested at the dauer stage, and the majority of them
became gravid adults while all non-transgenic daf-7(e1372) mutant
animals arrested as dauers. Similar results were obtained when the same
transgenes were introduced into daf-1(m40) animals. By contrast,
daf-9 expression in the XXXL/R cells, verified by GFP fluorescence of
the DAF-9::GFP fusion protein, was unable to prevent dauer arrest of
daf-7(e1372) mutant animals at 25°C
(Table 3), although a fraction
of the dauers did recover spontaneously upon prolonged incubation. Our results
demonstrate that constitutive daf-9 hypodermal, but not XXXL/R,
expression can substitute for the loss of daf-7 neuroendocrine signal
and bypass the block in TGFß signaling in target tissues. This argues a
major role for daf-9 in transducing the daf-7 signal,
perhaps through production of a lipophilic hormone in the hypodermis.
Alternatively, daf-9 may act in parallel of daf-7 in
promoting reproductive development.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
daf-9 acts upstream of daf-12 and antagonizes its
activity in the dauer pathway (Gerisch et
al., 2001; Jia et al.,
2002
). Two simple models consistent with the genetic analysis are
that daf-9 participates in the synthesis of a daf-12
antagonist, or that daf-9 degrades a daf-12 agonist. We
favour the former model. First, expression of daf-9 in single tissues
is sufficient to direct reproductive development of other tissues, suggesting
that daf-9 mediates the synthesis of an endocrine signal. Second,
daf-9 expression is restricted to three tissues, while a ubiquitous
expression pattern was reported for daf-12
(Antebi et al., 2000
;
Gerisch et al., 2001
;
Jia et al., 2002
). It is
conceivable that once the daf-12 ligand is generated in XXXL/R,
hypodermis or spermatheca by DAF-9, it would diffuse to target tissues via the
pseudocoelom. At this stage, we cannot exclude the possibility that the DAF-12
ligand may be degraded by DAF-9. However, this necessitates transport of the
DAF-12 ligand throughout the body to the three daf-9-expressing
tissues for destruction. Another model involves co-expression of
daf-9, and other P450 enzyme(s) that are genuinely involved in
daf-12 ligand synthesis, in the same tissue. The level of agonist
produced, and DAF-12 transcriptional potential, would then depend on the
relative activity of the competing P450 enzymes. This is similar to cases in
Drosophila and mammals, where hormone availability is modulated
locally by enzymes, such as cytochrome P450, in target tissues
(Luu-The, 2001
;
Gilbert et al., 2002
). The
validity of this model awaits identification of additional enzymes that
participate in daf-12 ligand metabolism.
The daf-9-expressing head cells have been identified as XXXL/R
where the che-2 promoter is inactive
(Ohkura et al., 2003). This is
intriguing because expression of daf-9 in ciliated sensory neurons
using the che-2 promoter was clearly able to suppress dauer arrest of
daf-9() animals and hence substitute for the loss of
daf-9 activity in XXXL/R and other tissues. We obtained similar
results when daf-9 expression in ciliated sensory neurons was driven
by the osm-6 promoter (H.Y.M. and G.R., unpublished). Nevertheless,
ectopic daf-9 expression in mechanosensory neurons using the
mec-7 promoter did not suppress the dauer arrest phenotype of
daf-9() animals (Gerisch
and Antebi, 2004
). To reconcile the different observations, we
propose the following models. As XXXL/R cells are adjacent to the
che-2-expressing, but not the mec-7-expressing, neurons
(e.g. IL1s), one can imagine intercellular shuttling of lipophilic
intermediates of hormone synthesis over a short distance. Alternatively,
cytochrome P450 enzymes that normally act upstream and downstream of DAF-9 may
be present in the ciliated neurons and given the cholesterol derived substrate
is available, daf-9 expression in these neurons may be sufficient to
produce the bona fide hormone.
daf-9(+) activity in ciliated sensory neurons was sufficient to
rescue the dauer arrest phenotype of daf-9-deficient animals, even
though these animals display a gonadal migration defect. This phenocopies
animals bearing daf-9 weak loss-of-function alleles
(Gerisch et al., 2001;
Jia et al., 2002
). Perhaps
DAF-9 in ciliated sensory neurons can only produce a suboptimal dose of its
cognate lipophilic hormone that is nevertheless sufficient to prevent dauer
arrest. However, it is known that particular head chemosensory neurons emit
key signals to control dauer arrest
(Bargmann and Horvitz, 1991
;
Ren et al., 1996
;
Schackwitz et al., 1996
), and
they may be the target cells that respond to a paracrine signal generated by
DAF-9. It is conceivable that daf-9 may be involved in the synthesis
of an endocrine signal for gonadal migration and a second paracrine signal for
reproductive development. Accordingly, DAF-9 may be proximal in a hormone
synthesis pathway in which the product of DAF-9 can be further modified by
multiple downstream P450 enzymes to yield different lipophilic signaling
molecules. This model predicts that the ciliated sensory neurons are unable to
produce the endocrine signal that direct proper gonadal migration despite
ectopic expression of daf-9, because of a lack of its downstream
partners.
daf-9 may act in the pathway for the synthesis of a secondary
signal that mediates the cell nonautonomous action of the daf-2
insulin/IGF-I receptor and daf-4 TGFß type II receptor
(Apfeld and Kenyon, 1998;
Inoue and Thomas, 2000
;
Wolkow et al., 2000
;
Gerisch et al., 2001
;
Jia et al., 2002
). In support
of this hypothesis, we found that constitutive expression of daf-9 in
the hypodermis, under the control of a heterologous promoter, was sufficient
to completely suppress the dauer arrest phenotype of daf-7()
and daf-1() animals. The ability to bypass
daf-7/TGFß signaling deficiency by a gain-of-function
daf-9 transgene strongly suggests that daf-9 is a major
transducer of the daf-7 reproductive signal.
Constitutive expression of daf-9 in the hypodermis allowed partial
suppression of the dauer arrest phenotype of daf-2() animals.
It appeared that the dauer program was never initiated in a subset of tissues,
while others were resistant to the excess hormonal signal generated by DAF-9.
We postulate that daf-9 may indeed mediate daf-2 signaling
in the hypodermis, pharynx and vulva. By contrast, it is unlikely to mediate
daf-2(+) activity in the germline and intestine. One possibility is
that daf-2 may exert cell-autonomous action on the latter set of
tissues and does not normally employ daf-9 to relay its activity.
Notably, mosaic analysis suggested that reproductive development of the
germline may require additional daf-2(+) activity that acts in a
cell-autonomous manner (Apfeld and Kenyon,
1998). The partial dauer arrest phenotype displayed by
daf-2(e1370) mutant animals overexpressing daf-9 is similar
to those observed in daf-2(e1370); daf-12(m20) and
daf-2(e1370); daf-12(m583) animals
(Larsen et al., 1995
). The
similarity in the effects of daf-9 overexpression and daf-12
severe loss of function, with respect to the highly tissue-specific phenotype
of partial daf-2 dauers, again highlights the close functional link
between daf-9 and daf-12. Nevertheless, the observation that
daf-2(e1370); daf-12(m20) animals lives twice as long as
daf-2(e1370) animals provides an exception to this notion
(Larsen et al., 1995
), as the
life span of daf-2(e1370) animals could not be altered by
constitutive expression of daf-9 in the hypodermis. It has been
reported that the daf-2 pathway acts in adult animals (up to 4 days
old) to specify life span (Dillin et al.,
2002
); however, the activity of our hypodermal daf-9
transgene ceases in 2-day-old adults. It may also be possible that
daf-2 and daf-9 function independently to control adult life
span.
We initially attempted to suppress dauer arrest of daf-2() and daf-7() animals by expressing daf-9 under the control of its endogenous promoter. Unlike the hypodermis-specific dpy-7 promoter, daf-9 expressed from its own promoter failed to rescue daf-2() or daf-7() animals at the restrictive temperature. We note that the daf-9 promoter is unable to support daf-9 hypodermal expression in daf-2(), daf-7() or natural dauers derived from starvation, even though daf-9 expression persists in XXXL/R. This correlates well with our observations that sdf-9 driven daf-9 expression in XXXL/R alone is not sufficient to prevent dauer arrest of daf-2() and daf-7() animals. Taken together, we propose that favorable growth conditions, transduced in part by the daf-2 and daf-7 signaling pathways may trigger daf-9 hypodermal expression at mid-L2 stage. Given the mass and coverage of the hypodermis across the entire animal, daf-9 expression in this tissue may induce a dramatic increase in lipophilic hormone production that may be crucial in the initiation or reinforcement of the reproductive program. This is demonstrated by our results that constitutive expression of daf-9 in the hypodermis is sufficient to promote reproductive development in dauer constitutive (Daf-c) mutant animals.
The execution of the dauer program is crucially dependent on
daf-12 (Antebi et al.,
2000). Therefore, it is not surprising that its activity should be
tightly regulated to prevent entry into diapause under favorable growth
conditions. This is unlikely to be achieved through modulation of
daf-12 expression as it is expressed at high levels at the L2 stage
in the hypodermis (Antebi et al.,
2000
). Instead, we propose that daf-12 activity is
regulated by the synthesis of a DAF-12 antagonist by DAF-9. According to this
model, high level of daf-12 expression and transcriptional activity
will be counteracted by an increase in hypodermal daf-9 expression
that is daf-12 dependent. However, attenuation or prevention of
daf-9 hypodermal expression may be a prerequisite for the dauer
program, as seen in daf-2() and daf-7()
animals. In this case, DAF-12 may disengage from the daf-9 promoter.
Alternatively, DAF-12 may repress daf-9 hypodermal expression through
recruitment of co-repressor complexes in response to dauer inducing
signals.
A DAF-12 response element is located within intron 1 of the daf-9b isoform. This 181 bp sequence also appears to specify daf-9 spermathecal expression independent of DAF-12. A nuclear receptor consensus half site (TGTTCT) is found within this sequence, which is also evident in an analogous location in the C. briggsae daf-9 gene. Surprisingly, mutation of the nuclear receptor consensus binding site eliminated daf-9 spermathecal expression without affecting the hypodermal expression. We propose that an unidentified nuclear receptor may bind to the TGTTCT element and regulate daf-9 expression in the spermatheca. As the consensus binding site for DAF-12 is not known at present, we cannot exclude the possibility that DAF-12 may bind directly to other parts of the 181 bp intron 1. Alternatively, DAF-12 may be part of a transcriptional cascade and indirectly control daf-9 hypodermal expression through other transcription factors.
Our results show that daf-9 signals at a pivotal position in the dauer pathway to integrate daf-2 insulin-like and daf-7 TGFß-like signaling pathways. The next challenge will be to identify the DAF-9 substrate that should shed light on the nature of the hormonal signal.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Caenorhabditis elegans. Development
125,1191
-1205.
Antebi, A., Yeh, W. H., Tait, D., Hedgecock, E. M. 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. elegans daf-2 function in the regulation of diapause and life span. Cell 95,199 -210.[Medline]
Bargmann, C. I. and Horvitz, H. R. (1991). Control of larval development by chemosensory neurons in Caenorhabditis elegans. Science 251,1243 -1246.[Medline]
Birnby, D. A., Link, E. M., 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.
Chawla, A., Repa, J. J., Evans, R. M. and Mangelsdorf, D. J.
(2001). Nuclear receptors and lipid physiology: opening the
X-files. Science 294,1866
-1870.
Dillin, A., Crawford, D. K. and Kenyon, C.
(2002). Timing requirements for insulin/IGF-1 signaling in C.
elegans. Science 298,830
-834.
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408,325 -330.[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.
Gems, D., Sutton, A. J., Sundermeyer, M. L., Albert, P. S.,
King, K. V., Edgley, M. L., Larsen, P. L. and Riddle, D. L.
(1998). Two pleiotropic classes of daf-2 mutation affect larval
arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans.
Genetics 150,129
-155.
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. and Antebi, A. (2004). Hormonal
signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer
diapause in response to environmental cues.
Development 131,1765
-1776.
Gerisch, B., Weitzel, C., Kober-Eisermann, C., Rottiers, V. and Antebi, A. (2001). A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span. Dev. Cell 1,841 -851.[Medline]
Gilbert, L. I., Rybczynski, R. and Warren, J. T. (2002). Control and biochemical nature of the ecdysteroidogenic pathway. Annu. Rev. Entomol. 47,883 -916.[CrossRef][Medline]
Gilleard, J. S., Barry, J. D. and Johnstone, I. L. (1997). cis regulatory requirements for hypodermal cell-specific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7. Mol. Cell Biol. 17,2301 -2311.[Abstract]
Hobert, O. (2002). PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques 32,728 -730.[Medline]
Hsin, H. and Kenyon, C. (1999). Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399,362 -366.[CrossRef][Medline]
Inoue, T. and Thomas, J. H. (2000). Targets of TGF-beta signaling in Caenorhabditis elegans dauer formation. Dev. Biol. 217,192 -204.[CrossRef][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.
Johnstone, I. L. and Barry, J. D. (1996). Temporal reiteration of a precise gene expression pattern during nematode development. EMBO J. 15,3633 -3639.[Abstract]
Kenyon, C., Chang, J., Gensch, E., Rudner, A. and Tabtiang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366,461 -464.[CrossRef][Medline]
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.
Larsen, P. L., Albert, P. S. and Riddle, D. L.
(1995). Genes that regulate both development and longevity in
Caenorhabditis elegans. Genetics
139,1567
-1583.
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.
Luu-The, V. (2001). Analysis and characteristics of multiple types of human 17beta- hydroxysteroid dehydrogenase. J. Steroid Biochem. Mol. Biol. 76,143 -151.[CrossRef][Medline]
Menzel, R., Bogaert, T. and Achazi, R. (2001). A systematic gene expression screen of Caenorhabditis elegans cytochrome P450 genes reveals CYP35 as strongly xenobiotic inducible. Arch. Biochem. Biophys. 395,158 -168.[CrossRef][Medline]
Miller, W. L. (1988). Molecular biology of steroid hormone synthesis. Endocr. Rev. 9, 295-318.[Medline]
Nebert, D. W. and Russell, D. W. (2002). Clinical importance of the cytochromes P450. Lancet 360,1155 -1162.[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]
Ohkura, K., Suzuki, N., Ishihara, T. and Katsura, I.
(2003). SDF-9, a protein tyrosine phosphatase-like molecule,
regulates the L3/dauer developmental decision through hormonal signaling in C.
elegans. Development
130,3237
-3248.
Patterson, G. I., Koweek, A., Wong, A., Liu, Y. and Ruvkun,
G. (1997). The DAF-3 Smad protein antagonizes
TGF-beta-related receptor signaling in the Caenorhabditis elegans dauer
pathway. Genes Dev. 11,2679
-2690.
Pierce, S. B., Costa, M., Wisotzkey, R., Devadhar, S.,
Homburger, S. A., Buchman, A. R., Ferguson, K. C., Heller, J., Platt,
D. M., Pasquinelli, A. A. et al. (2001). Regulation of DAF-2
receptor signaling by human insulin and ins-1, a member of the unusually large
and diverse C. elegans insulin gene family. Genes Dev.
15,672
-686.
Ren, P., 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-beta homolog.
Science 274,1389
-1391.
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: 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]
Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. and Driscoll, M. (2000). Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat. Genet. 24,180 -183.[CrossRef][Medline]
Timmons, L., Court, D. L. and Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263,103 -112.[CrossRef][Medline]
Warren, J. T., Petryk, A., Marques, G., Jarcho, M., Parvy, J.
P., Dauphin-Villemant, C., O'Connor, M. B. and Gilbert, L. I.
(2002). Molecular and biochemical characterization of two P450
enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA
99,11043
-11048.
Waxman, D. J. (1999). P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR. Arch. Biochem. Biophys. 369, 11-23.[CrossRef][Medline]
Wolkow, C. A., Kimura, K. D., Lee, M. S. and Ruvkun, G.
(2000). Regulation of C. elegans life-span by insulinlike
signaling in the nervous system. Science
290,147
-150.
Related articles in Development: