1 Department of Biochemistry and Molecular Biology, Rutgers University,
Piscataway, NJ 08854, USA
2 Cancer Institute of New Jersey, New Brunswick, NJ 08853, USA
* Author for correspondence (e-mail: patterson{at}mbcl.rutgers.edu)
Accepted 30 September 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dauer, Cyclin dependent kinase inhibitor, Dachshund box, Nematode
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A transforming growth factor ß superfamily (TGFß) pathway is
required for a normal dauer decision, and is thought to act as a step in a
neuroendocrine pathway that couples external cues to dauer development
(Riddle and Albert, 1997;
Patterson and Padgett, 2000
).
Food availability and population density are sensed as chemosensory cues.
These inputs, along with temperature, are sensed during early larval stages
(Golden and Riddle, 1984b). High food and low pheromone stimulate
transcription of daf-7, the gene for the ligand in the TGFß
pathway (Ren et al., 1996
;
Schackwitz et al., 1996
).
DAF-7 binds to the receptors DAF-1 and DAF-4, which probably function in
neurons (Inoue and Thomas,
2000
; Gunther et al.,
2000
). DAF-8 and DAF-14 are Smads that appear to be the direct
targets of the receptors in the dauer TGFß pathway (A. O. Z. Estevez, PhD
thesis, University of Missouri, 1997)
(Riddle and Albert, 1997
;
Inoue and Thomas, 2000
). These
Smads antagonize the function of another Smad called DAF-3
(Patterson et al., 1997
). The
daf-5 gene has similar genetic properties to daf-3
(Thomas et al., 1993
), and so
may be acting as a co-factor to DAF-3.
Little is known about events controlled by the receptors and Smads in this
pathway, or the mechanism by which the pathway controls the dauer decision.
Genetic analysis places the TGFß pathway upstream of daf-12 (a
gene encoding a nuclear hormone receptor) and daf-9 (a gene encoding
a putative biosynthetic enzyme for a hormone that regulates DAF-12)
(Thomas et al., 1993;
Antebi et al., 1998
;
Gerisch et al., 2001
;
Jia et al., 2002
). The
daf-9 gene appears to be expressed in the XXX cell, which is little
studied, but has neuronal properties and is located in a head ganglion
(Ohkura et al., 2003
). These
facts, as well as the expression of DAF-7 in neurons and the suggested
function of DAF-4 in neurons (Inoue and
Thomas, 2000
), suggest a model in which DAF-3 and DAF-5 function
in the nervous system. However, some evidence can be interpreted as suggesting
a function for DAF-3 and DAF-5 outside the nervous system. For example, a
small sequence element derived from a pharynx specific promoter binds DAF-3 in
vitro, and mediates daf-3-dependent repression of a reporter gene in
the pharynx (Thatcher et al.,
1999
).
The dauer TGFß pathway is unconventional in that upstream components
of the pathway directly or indirectly antagonize DAF-3, a Smad protein
(Patterson et al., 1997). In
other pathways, Smad transcription factors are activated, not repressed, by
the upstream components of the pathway. Anti-Smads antagonize receptor or Smad
function (Shi and Massague,
2003
), but DAF-3 is antagonized by receptor signaling, and
functions in the absence of receptor signaling to regulate genes that control
dauer formation (Patterson et al.,
1997
).
Genetic analysis is consistent with DAF-5 acting as a cofactor of the DAF-3 Smad. The most extensively studied roles of TGFß superfamily pathways are in the control of cell fate and in control of the cell cycle. The neuroendocrine role of the dauer pathway is very different. The dauer TGFß pathway has evolved a unique mode of signaling, in which the receptors and receptor-activated Smads antagonize another Smad protein, DAF-3. Studies that reveal mechanisms of daf-5 function will help us understand how signaling pathways evolve new functions.
We show that DAF-5 is a diverged homolog of human Sno and Ski, which
antagonize TGFß signaling in cell culture by binding Smads, preventing
their interaction with each other and with co-activators, and by recruiting
co-repressors (Akiyoshi et al.,
1999; Luo et al.,
1999
; Stroschein et al.,
1999
; Sun et al.,
1999
; Xu et al.,
2000
; Frederick and Wang,
2002
). DAF-5, Sno and Ski share a domain we call the SDS box; this
domain in Ski mediates its interaction with Smad4. Yeast two-hybrid
experiments demonstrate that DAF-5 interacts with DAF-3, and deletion studies
are consistent with this interaction being mediated by the same domains as in
the vertebrate Ski/Smad interaction. This functional similarity, combined with
our phylogenetic analysis of DAF-5/Sno/Ski homologs suggests that, despite a
highly divergent sequence, DAF-5 is an ortholog of Sno and Ski. DAF-5 is the
first example of a Sno/Ski with a genetically defined function in a TGFß
pathway. We identified a mutation hotspot in the conserved Dachbox domain.
These mutants are the first direct evidence for a function for the Dachbox in
TGFß signaling. Our analysis of regulation of a pharynx-specific promoter
does not support a previously hypothesized role for daf-3 and
daf-5 in the pharynx. Furthermore, we show that daf-5 is
expressed and functions in the nervous system. Therefore, we propose a model
in which daf-3 and daf-5 have evolved novel functions that
allow them to act in a neurosecretory pathway to control C. elegans
dauer developmental arrest.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mapping and transgenic strains
DNA polymorphism and mapping data were submitted to Wormbase
(http://www.wormbase.org).
Clones were injected as previously described
(Mello et al., 1991) with pRF4
used as transformation marker.
Orthology and paralogy
We use the terms ortholog and paralog as defined in
(Fitch, 1970) and
(Jensen, 2001
). Simply put,
orthologs are duplicates of a character (e.g. a gene sequence) that arise from
speciation events, whereas paralogs arise from a duplication within a single
genome. Orthology and paralogy are typically hypotheses that are created based
on sequence comparisons and other data. These definitions make no suggestion
of conserved function.
Yeast-two-hybrid assays
Yeast two hybrid assays were performed as described
(Walhout and Vidal, 2001).
His/3-AT growth assay was scored on scale 0-3, and ß-gal activity assay
was scored on scale 0-5. All protein-coding sequences of interest were fused
to the activation domain and the DNA-binding domain, and all assays were
performed with both AD/DB combinations. We assigned `+' scores based on the
total of His/3-AT and ß-gal scores from the two combinations: ±,
1-3; +, 4-7; ++, 8 or 9; +++, 10-13; ++++, 14-16.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
This alignment is very informative regarding the relationship of DAF-5 to other members of the family. Sno and Ski are found in humans and all major groups of vertebrates, but in insects have only one ortholog of these two proteins (see Materials and methods for definitions of ortholog and paralog). Sno and Ski are more similar to each other than either is to the single Drosophila or mosquito ortholog; therefore Sno and Ski are probably paralogs that were duplicated after the divergence of the protostome and deuterostome lineages. We have named the insect genes Snowski (Snk) to reflect the orthology to both Sno and Ski.
A new family of proteins closely related to the Snowski group is shown in Fig. 1C,D. Humans have two genes in this group. We have named these genes Skate (for Ski-related gene) and Icy (for Ski sequence family). Drosophila and mosquito each have a gene that is much more similar to human Icy and Skate than to Drosophila Snk. We have named the single Drosophila and mosquito genes iceskate (isk) to reflect their orthology to both Icy and Skate.
We suggest that DAF-5 is an ortholog of Snowski or Iceskate. First, DAF-5 clearly has an SDS box, which is not found in any other protein in C. elegans or C. briggsae. This SDS box is more similar to the Snowski group than to the Iceskate group, including amino acids that are important for the ability of Ski to bind Smad proteins. Second, DAF-5 binds the DAF-3 Smad (see below). This binding is mediated by the SDS box in Ski, and may thus be a conserved function of the SDS box. Third, the rate of divergence in the Snowski/Iceskate family is so high that relatively modest sequence conservation is not surprising. This rapid change can be seen when examining the SDS box of B. malayi and potato cyst nematode Snowski. These two nematode proteins are more different from each other than insect Snowski is from Human Sno and Ski. The DAF-5 gene is even more rapidly diverging in the Caenorhabditis genus. C. briggsae and C. elegans proteins average more than 70% amino acid identity. The DAF-5 sequence is only 40% identical overall between C. briggsae and C. elegans. In fact, in the Dachbox and SDS box, the difference between C. elegans and C. briggsae DAF-5 is greater than the difference between insect and human Snowski.
A mutation cluster in the Dachbox domain
Sequencing of daf-5 mutants identified a mutation hotspot. We
identified mutations in 15 daf-5 alleles
(Fig. 1B; see Table S2 at
http://dev.biologists.org/supplemental).
All five missense mutants were found in a 16 amino acid stretch of the 627
amino acid protein. This hotspot is in the region of the Dachbox where DAF-5
is most similar to Snowski, Iceskate and Dachshund. Three of the mutants
affect two very strongly conserved residues (two of the mutants are identical
but independently isolated). One mutant has an in frame deletion of this
region, and the final mutant is in a glycine that is unique to DAF-5. This
region is critically important for DAF-5 function; the sa310 mutation
(E162K) has a phenotype as severe as putative null alleles
(Table 1). In vitro analysis
showed that an insertion of four amino acids next to residue 168, which is
homologous to the residue that is mutated in daf-5(sa310), eliminates
the transforming and myogenic activity of vSki
(Zheng et al., 1997). Thus,
the Dachbox is required for a gain-of-function phenotype of vSki, but how this
function relates to wild-type function of cSki or to TGFß signaling has
not been experimentally determined. A point mutation in the Dachbox disrupts
the interaction of Ski with NCoR (Ueki and
Hayman, 2003
). However, this mutation affected Vitamin D
receptor-dependent gene expression, but not TGFß-dependent gene
expression. These in vitro experiments suggest possible functions for the
Dachbox, but our daf-5 mutants are the first missense mutants
identified in any Sno/Ski gene, and thus the first in vivo evidence
for the wild-type function of a particular domain of Sno/Ski.
DAF-5 binds to Smad DAF-3
A yeast two-hybrid screen for proteins that interact with DAF-3 identified
the predicted protein W01G7.1 (M. Tewari, P. J. Hu, G. B. Ruvkun and M. Vidal,
personal communication), which we show is DAF-5. We used yeast two-hybrid
assays to identify regions of DAF-3 and DAF-5 required for interaction. We
find that the DAF-3 MH2 domain strongly interacts with a DAF-5 fragment that
is truncated after the SDS domain (Fig.
2, see Table S3 at
http://dev.biologists.org/supplemental).
Thus, as in vertebrates, the region downstream of the SDS box is dispensable
for binding to Smads and the Smad domain that binds Sno/Ski is the MH2 domain
(Akiyoshi et al., 1999;
Luo et al., 1999
;
Stroschein et al., 1999
;
Sun et al., 1999
;
Frederick and Wang, 2002
;
Wu et al., 2002
). These
interaction results suggest that DAF-3 and DAF-5 function as part of a
transcriptional regulatory complex.
|
|
|
|
DAF-5 is required for cell cycle arrest
In vertebrate cells, Smad proteins control the cell cycle by regulating
transcription of cyclin kinase inhibitor genes
(Moustakas and Kardassis,
1998). The division of hypodermal seam cells is arrested in dauers
because CKI-1, a cyclin kinase inhibitor, is transcriptionally upregulated,
and this upregulation is inhibited by wild-type daf-7
(Hong et al., 1998
),
suggesting that daf-5 may directly or indirectly upregulate
cki-1. We tested whether cell cycle arrest in dauers requires
daf-5. The reporter, rnr-1::GFP, drives the expression of
GFP in the seam cells as they enter S phase and through the division at the
beginning of L3. daf-7 (e1372) mutants do not express
rnr-1::GFP at the corresponding time, which reflects seam cell cycle
arrest in dauers (Hong et al.,
1998
). We observed that 47% of N2 show rnr-1::GFP
expression (Fig. 4A,
Table 5), whereas none of the
daf-1(sa184)-induced dauers does. Thus, this daf-1
mutation causes a complete arrest of the cell cycle. Interestingly, in
daf-5(sa310); daf-1(sa184) double mutants, the
percentage of animals with green seam cells is 31%, which is similar to wild
type (Fig. 4B;
Table 5). The reason that we
did not see rnr-1 expression in some N2 or daf-5; daf-7
double mutants even after careful synchronization was probably due to the
transient expression of the rnr-1 gene and variation in the timing of
cell cycle among individual animals. We conclude that a daf-5
mutation suppresses the cell cycle arrest caused by daf-1.
|
|
Regulation of myo-2
Previous work is consistent with DAF-5 and DAF-3 having a function in the
adult pharynx, but we found that expression of DAF-5 solely in the nervous
system can cause dauer arrest. Therefore, we re-examined the role of DAF-3 and
DAF-5 in adult pharynx. DAF-3 was shown to bind to a 5 bp sequence within a 32
base pair regulatory element isolated from the C. elegans myo-2
promoter (the `C subelement'). This regulatory element drives pharynx-specific
gene expression when placed upstream of a minimal promoter
(Thatcher et al., 1999).
Expression from this construct in adults is strongly repressed in mutants of
daf-7 and other Daf-c mutants in the TGFß pathway, but a
mutation of daf-3 or daf-5 relieves this repression.
However, all of this regulation was observed with the C subelement removed
from its normal context and concatamerized upstream of a minimal promoter, and
we wished to see if this regulation occurs within the normal context of the
full myo-2 promoter.
We used a GFP reporter fused to a full-length myo-2 promoter (with
1.6 kb upstream of the translation start) to examine regulation by genes in
the TGFß pathway. If the full-length promoter is regulated similarly to
the C subelement reporter, we expected that expression would be repressed in
daf-7 mutant adults relative to wild-type adults, and that mutations
in daf-5 or daf-3 would alleviate that repression. However,
expression in adults was indistinguishable in wild type and in daf-7
mutants (Table 6). Therefore,
the regulation of the full-length promoter is unlike the regulation of the C
subelement reporter. Similarly, in wild-type L2 stage larvae and
daf-7 larvae in the corresponding L2d stage, expression was
indistinguishable between N2 and daf-7, and expression in the
daf-7;daf-5 and daf-7;daf-3 double mutants was modestly
reduced. Finally, we see that expression in the daf-7 dauer is less
than in wild type or the daf-7;daf-3 and daf-7;daf-5 double
mutants. The C subelement reporter also shows a reduction in expression in
dauers, comparable with what we see. However, unlike in adults, the reduction
of expression of the C subelement reporter in dauers is not dependent on
daf-3 (Thatcher et al.,
1999), and is therefore mediated by some other pathway. In
summary, we see no evidence that the full-length myo-2 promoter is
regulated by daf-3 or daf-5.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of DAF-5 identifies a role for a Snowski family protein in TGFß signaling in vivo
Recent work in cell culture has showed that Sno and Ski function in vitro
as key factors in TGFß regulation of gene expression and cell division
(Akiyoshi et al., 1999;
Luo et al., 1999
;
Stroschein et al., 1999
;
Sun et al., 1999
;
Wang et al., 2000
;
Xu et al., 2000
;
Frederick and Wang, 2002
;
Miyazono et al., 2003
;
Ueki and Hayman, 2003
). These
studies suggest numerous possible functions for Sno and Ski in TGFß
signaling, but understanding the specific contexts in which Sno and Ski
function in vivo will require further work. Some studies of Sno/Ski function
in vivo have used gain-of-function phenotypes in Xenopus development
(Wang et al., 2000
) or in
cancer (Frederick and Wang,
2002
; Miyazono et al.,
2003
). These studies have identified mechanisms of Sno/Ski action
and suggest possible roles for the proteins in vivo, but are accompanied by a
caveat. Overexpression and gain-of-function mutants might cause the genes to
act in an event that is not part of the normal function of the gene. Mouse
Ski and Sno mutants have interesting phenotypes, including
skeletal and muscular developmental defects and cancer susceptibility
(Berk et al., 1997
;
Shinagawa et al., 2000
;
Shinagawa et al., 2001
). These
mutants will be useful in dissecting the wild-type functions of Sno and Ski in
TGFß signaling; however, the phenotypes of these mutants are complex, and
Sno and Ski function in multiple signaling pathways
(Dahl et al., 1998
;
Nomura et al., 1999
), so tying
a specific phenotype to the effect of Sno or Ski on TGFß signaling will
require careful analysis. Whereas Sno and Ski function in many pathways,
daf-5 mutants exclusively affect phenotypes controlled by the dauer
TGFß pathway; this fact makes the assignment of function much more
straightforward in C. elegans.
DAF-5 and the Snowski/Iceskate gene families
From analysis of the Snowski family, we identified several interesting new
genes and relationships among them. First, we show that Sno and
Ski are likely to be paralogs that arose after the divergence of
protostomes and deuterostomes, perhaps as late as the divergence of
urochordates and cephalochordates (because sea squirt has only one ortholog).
Second, we identify the Iceskate family: an uncharacterized, highly
conserved Snowski-related gene family. Genes in the Iceskate
family have a striking difference from Snowski. Iceskate shows
virtually no similarity to a set of amino acids in the SDS box of Ski that
provide critical hydrogen bonds and van der Waals contacts in the Ski/Smad4
structure (Wu et al., 2002).
To our knowledge, Iceskate proteins have not been studied experimentally; the
functional relationship of Iceskate to Sno/Ski is an interesting issue for
future research. Third, we find that Snowski is changing rapidly in nematode
evolution. In Caenorhabditis, we see that daf-5 is changing
rapidly, much more rapidly than the typical C. elegans gene, which
may explain the relatively low primary sequence conservation between DAF-5 and
other Snowski proteins.
Despite the rapid evolution of DAF-5, it binds DAF-3, a Smad. We find that the region downstream of the SDS box is entirely dispensable for DAF-5 binding to Smads, and the MH2 domain of DAF-3 is sufficient for interaction with DAF-5. The region of DAF-5 homologous to the region of Ski that contacts Smad4 is shown in Fig. 1C. Sno, Ski, DAF-5 and all other members of this family have a conserved zinc finger in the SDS domain. This zinc finger is an important structural component of Ski, and presumably all members of this family, because it is absolutely conserved. Only a subset of the residues of Ski that contact Smad4 are conserved in DAF-5. The binding of DAF-5 to DAF-3 may be somewhat different from the binding of Ski to Smad4, because DAF-3 is also greatly diverged from Smad4.
Evolution of the dauer TGFß pathway
The rapid change in daf-5 in the Caenorhabditis genus
prompts the question of to what extent have daf-5 and other members
of the TGFß pathway evolved new functions? The model for Sno/Ski function
in vertebrate TGFß signaling is shown in
Fig. 5A. Briefly, activation of
TGFß receptors causes phosphorylation of Smads. This activation allows
the Smads to bind Sno/Ski proteins and cause their degradation. With Sno/Ski
gone, the Smads are free to regulate gene expression. However, Sno
and Ski transcription is activated by Smads, leading to accumulation
of Sno/Ski protein after receptor activation. Sno and Ski bind to Smads and
recruit a variety of co-repressors, which prevent Smads from activating gene
expression.
|
DAF-5 acts in the nervous system
The question of where the TGFß pathway is acting is important, and
evidence is not conclusive. Many of the genes in the pathway have broad
patterns of expression, including many non-neuronal cells
(Patterson et al., 1997;
Gunther et al., 2000
;
Inoue and Thomas, 2000
). We
found that expression from a neuron-specific promoter efficiently rescued the
daf-5 mutant, and that expression in muscle or hypodermis did not
rescue. This result is consistent with daf-5 acting in neurons to
regulate a hormonal cue for dauer.
Experiments to directly address the site of action of DAF-4 have been
reported (Inoue and Thomas,
2000), and suggest that this gene also has a neuronal focus of
action. In these experiments, a daf-4 cDNA was fused to several
tissue-specific reporters. A promoter expected to give neuronal and intestinal
expression fused to a daf-4 cDNA rescued a daf-4 mutant, but
putative intestine-specific, muscle-specific and other promoters did not.
However, the authors could not monitor the expression of the constructs
directly, and they were appropriately cautious in interpreting their results.
Our results suggest that unexpected expression is not just a formal
possibility. Rather, misexpression of `tissue specific' reporter constructs
may be common. Several of our constructs gave unexpected expression patterns
(Table 4). The unc-14
promoter has been reported to be neuron-specific, but we saw expression
elsewhere. The unc-119, dpy-7, dpy-30 and ges-1 promoters
gave unexpectedly weak or undetectable expression, although in the case of
dpy-7, we were able to correct the problem by isolating transgenic
strains with stronger expression.
Two reports suggest functions for daf-3 and daf-5 outside
the nervous system, which is inconsistent with our conclusion. One report
suggested that daf-3 and daf-5 function in the pharynx to
directly regulate gene expression in adults
(Thatcher et al., 1999). This
work used a reporter with a small element derived from the myo-2
reporter (the `C subelement'). However, our examination of the full-length
myo-2 promoter indicates that daf-3 and daf-5 do
not regulate myo-2 gene expression in adults. In dauers, the
full-length promoter does show regulation similar to that of a reporter
containing only the C subelement, but regulation of the C subelement reporter
in dauers is daf-3-independent. This daf-3-independent
downregulation may be caused by a general reduction in expression of
housekeeping genes that is seen in dauers (T. Liu and G.I.P., unpublished). We
suggest that daf-3 and daf-5 do not actually regulate the
expression of myo-2 in a normal context. daf-3 and
daf-5 may function in the nervous system to regulate a secondary
signal that in turn regulates the reporter with the C subelement.
Cell division arrest in hypodermis and other non-neuronal cells of dauers
is dependent on a cyclin kinase inhibitor, cki-1
(Hong et al., 1998). This gene
is similar to a gene that is directly regulated by Smads in vertebrates
(Moustakas and Kardassis,
1998
). In C. elegans, this gene is repressed in a
daf-7 mutant, and we found that this regulation was daf-5
dependent, which suggests that daf-3 and daf-5 could act
outside the nervous system to directly regulate this gene. However, our
demonstration that daf-5 expression in the nervous system is
sufficient for dauer arrest suggests that regulation of cki-1 is
indirect.
Biological role of TGFß signaling in C. elegans
We observed a phenotype of daf-5 that has not been previously
reported; daf-5 mutants are Egl-c, meaning that compared with wild
type, daf-5 mutants have fewer eggs retained in the uterus and lay
eggs with younger embryos. This phenotype suggests that the daf-5
mutants have hyperactive egg-laying behavior. We were concerned that a low
rate of egg production in the spermatheca might cause this phenotype, but we
found that the rate of egg production was not correlated with the Egl
phenotype. We found that all of the mutant genotypes in our experiment produce
eggs at a rate of 1/2 to 2/3 that of wild type (data not shown), but have
dramatically different Egl phenotypes. For example, the daf-5 single
mutants make eggs at the same rate as the daf-8; daf-5; daf-14 triple
mutants, which are not Egl-c. This new result suggests that daf-5
functions to restrain egg laying even when the TGFß pathway is active.
The role of daf-5 in egg laying is not TGFß pathway independent,
as daf-c; daf-5 double mutants are not significantly Egl-c.
In addition to dauer and egg laying, the dauer TGFß pathway also
regulates social feeding behavior and fat accumulation
(Trent et al., 1983;
Thomas et al., 1993
). In
addition, daf-3 and daf-5, but not the other Smads or
receptors, have a role in chemotaxis to both olfactory and gustatory cues
(Daniels et al., 2000
). All of
these behaviors and developmental events are also affected by chemosensory
input. Thus, the role of the TGFß pathway can be said to be a general
role in coupling chemosensory events to developmental and behavioral output.
How might this pathway be coupled to these events? One possibility is that all
of these events are affected, directly or indirectly, by a hormonal cue or
cues, and that the TGFß pathway regulates the expression of genes needed
for production of the hormone(s). Orientation to a chemical gradient is quick,
occurring within minutes. This is perhaps too brief an interval for the
hormone to be acting during the chemotactic process, but a hormone might cause
structural changes in the nervous system that alter chemotactic behavior.
A second interesting possibility is suggested by recent work that has
identified new functions for TGFß superfamily signaling in the nervous
system. Retrograde signals between Drosophila neurons and their
postsynaptic partner cells can affect the nature of the synaptic connection
(Aberle et al., 2002;
Marques et al., 2002
) as well
as the neurotransmitters produced by the presynaptic cells
(Allan et al., 2003
), and
genetic analysis suggests that these retrograde signals are mediated by Gbb, a
TGFß superfamily ligand, and Wit, a TGFß superfamily receptor. Thus,
the dauer TGFß pathway might act to affect the synaptic connections or
other properties of neurons to alter signaling in the nervous system. This
model is not mutually exclusive with the hormone regulation model above, as
changes in connectivity or neurotransmitter production might affect synaptic
signaling to a neurosecretory cell that makes hormone.
Gene regulation by the Ski superfamily
Study of the Ski and Sno proteins has provided an opportunity to learn how
a co-repressor can play a variety of roles in different regulatory events.
Perhaps the next important task will be to tie the biochemical and cell
biological mechanisms identified in cell culture systems to important
functions of Sno and Ski in vivo. Cancer biology is one field that may provide
this connection, as the role of Ski and Sno in cancerous cells suggests
testable hypotheses about the role of these proteins in the normal context.
Sno and Ski function in multiple pathways, which makes understanding the role
of the proteins in any one pathway difficult. In C. elegans, DAF-5
appears to function predominantly or exclusively in a single TGFß
pathway. This simplicity has allowed us the unique discovery of an unambiguous
connection of a Sno/Ski family member to a specific regulatory event in vivo,
and this discovery provides a genetic system in which to pursue further
understanding of Sno/Ski function.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aamodt, E. J., Chung, M. A. and McGhee, J. D. (1991). Spatial control of gut-specific gene expression during Caenorhabditis elegans development. Science 252,579 -582.[Medline]
Aberle, H., Haghighi, A. P., Fetter, R. D., McCabe, B. D., Magalhaes, T. R. and Goodman, C. S. (2002). Wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33,545 -558.[Medline]
Ailion, M. and Thomas, J. H. (2000). Dauer
formation induced by high temperatures in Caenorhabditis elegans.Genetics 156,1047
-1067.
Akiyoshi, S., Inoue, H., Hanai, J., Kusanagi, K., Nemoto, N.,
Miyazono, K. and Kawabata, M. (1999). c-Ski acts as a
transcriptional co-repressor in transforming growth factor-ß signaling
through interaction with Smads. J. Biol. Chem.
274,35269
-35277.
Allan, D. W., St Pierre, S. E., Miguel-Aliaga, I. and Thor, S. (2003). Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell 113,73 -86.[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.
Bargmann, C. I. and Horvitz, H. R. (1991). Control of larval development by chemosensory neurons in Caenorhabditis elegans. Science 251,1243 -1246.[Medline]
Berk, M., Desai, S. Y., Heyman, H. C. and Colmenares, C.
(1997). Mice lacking the ski proto-oncogene have defects
in neurulation, craniofacial, patterning, and skeletal muscle development.
Genes Dev. 11,2029
-2039.
Dahl, R., Kieslinger, M., Beug, H. and Hayman, M. J.
(1998). Transformation of hematopoietic cells by the Ski
oncoprotein involves repression of retinoic acid receptor signaling.
Proc. Natl. Acad. Sci. USA
95,11187
-11192.
Daniels, S. A., Ailionb, M., Thomas, J. H. and Sengupta, P.
(2000). egl-4 acts through a transforming growth
factor-ß/SMAD pathway in Caenorhabditis elegans to regulate
multiple neuronal circuits in response to sensory cues.
Genetics 156,123
-141.
Fitch, W. (1970). Distinguishing homologous from analogous proteins. Syst. Zool. 19, 99-113.[Medline]
Frederick, J. P. and Wang, X. F. (2002). Smads `freeze' when they ski. Structure 10,1607 -1611.[CrossRef][Medline]
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]
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]
Golden, J. and Riddle, D. (1984a). A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proc. Natl. Acad. Sci. USA 81,819 -823.[Abstract]
Gunther, C., Georgi, L. and Riddle, D. (2000).
A Caenorhabditis elegans type I TGF ß receptor can function in
the absence of type II kinase to promote larval development.
Development 127,3337
-3347.
Hong, Y., Roy, R. and Ambros, V. (1998).
Developmental regulation of a cyclin-dependent kinase inhibitor controls
postembryonic cell cycle progression in C. elegans.Development 125,3585
-3597.
Hsu, D. R., Chuang, P. T. and Meyer, B. J.
(1995). DPY-30, a nuclear protein essential early in
embryogenesis for Caenorhabditis elegans dosage compensation.
Development 121,3323
-3334.
Inoue, T. and Thomas, J. (2000). Targets of TGF-ß signaling in Caenorhabditis elegans dauer formation. Dev. Biol. 217,192 -204.[CrossRef][Medline]
Jensen, R. (2001). Orthologs and paralogs - we need to get it right. Genome Biol. 2, interactions1002.1 - interactions1002.3.
Jia, K., Albert, P. and Riddle, D. (2002).
DAF-9, a cytochrome P450 regulating C. elegans Larval development and
adult longevity. Development
129,221
-231.
Krishna, S., Maduzia, L. and Padgett, R.
(1999). Specificity of TGFß signaling is conferred by
distinct type I receptors and their associated SMAD proteins in
Caenorhabditis elegans. Development
126,251
-260.
Luo, K., Stroschein, S. L., Wang, W., Chen, D., Martens, E.,
Zhou, S. and Zhou, Q. (1999). The Ski oncoprotein interacts
with the Smad proteins to repress TGFß signaling. Genes
Dev. 13,2196
-2206.
Maduro, M. and Pilgrim, D. (1995).
Identification and cloning of unc-119, a gene expressed in the
Caenorhabditis elegans nervous system.
Genetics 141,977
-988.
Marques, G., Bao, H., Haerry, T. E., Shimell, M. J., Duchek, P., Zhang, B. and O'Connor, M. B. (2002). The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function. Neuron 33,529 -543.[Medline]
Mello, C. C., Kramer, J., 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]
Miyazono, K., Suzuki, H. and Imamura, T. (2003). Regulation of TGF-ß signaling and its roles in progression of tumors. Cancer Sci. 94,230 -234.[Medline]
Morita, K., Chow, K. and Ueno, N. (1999).
Regulation of body length and male tail ray pattern formation of
Caenorhabditis elegans by a member of TGF-ß family.
Development 126,1337
-1347.
Moustakas, A. and Kardassis, D. (1998).
Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional
interactions between Sp1 and Smad family members. Proc. Natl. Acad.
Sci. USA 95,6733
-6738.
Nomura, T., Khan, M. M., Kaul, S. C., Dong, H. D., Wadhwa, R.,
Colmenares, C., Kohno, I. and Ishii, S. (1999). Ski is a
component of the histone deacetylase complex required for transcriptional
repression by Mad and thyroid hormone receptor. Genes
Dev. 13,412
-423.
Ogura, K., Shirakawa, M., Barnes, T. M., Hekimi, S. and Ohshima, Y. (1997). The UNC-14 protein required for axonal elongation and guidance in Caenorhabditis elegans interacts with the serine/threonine kinase UNC-51. Genes Dev. 11,1801 -1811.[Abstract]
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.
Okkema, P. G., Harrison, S. W., Plunger, V., Aryana, A. and
Fire, A. (1993). Sequence requirements for myosin gene
expression and regulation in Caenorhabditis elegans.Genetics 135,385
-404.
Patterson, G. I. and Padgett, R. W. (2000). TGFß-related pathways. Trends Genet. 16, 27-33.[CrossRef][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.
Ren, P., Lim, C.-S., Johnsen, R., Albert, P., Pilgrim, D. and
Riddle, D. (1996). Control of C. elegans larval
development by neuronal expression of a TGF-ß homolog.
Science 274,1389
-1391.
Riddle, D. and Albert, P. (1997). Genetics and environmental regulation of dauer larva development. In C. elegans II (ed. D. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess), pp. 739-768. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Schackwitz, W., Inoue, T. and Thomas, J. (1996). Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans. Neuron 17,719 -728.[Medline]
Shi, Y. and Massague, J. (2003). Mechanisms of TGF-ß signaling from cell membrane to the nucleus. Cell 113,685 -700.[Medline]
Shinagawa, T., Dong, H. D., Xu, M., Maekawa, T. and Ishii,
S. (2000). The sno gene, which encodes a component
of the histone deacetylase complex, acts as a tumor suppressor in mice.
EMBO J. 19,2280
-2291.
Shinagawa, T., Nomura, T., Colmenares, C., Ohira, M., Nakagawara, A. and Ishii, S. (2001). Increased susceptibility to tumorigenesis of ski-deficient heterozygous mice. Oncogene 20,8100 -8108.[CrossRef][Medline]
Stroschein, S. L., Wang, W., Zhou, S., Zhou, Q. and Luo, K.
(1999). Negative feedback regulation of TGF-ß signaling by
the SnoN oncoprotein. Science
286,771
-774.
Sun, Y., Liu, X., Eaton, E. N., Lane, W. S., Lodish, H. F. and Weinberg, R. A. (1999). Interaction of the Ski oncoprotein with Smad3 regulates TGF-ß signaling. Mol. Cell 4, 499-509.[Medline]
Thatcher, J., Haun, C. and Okkema, P. (1999).
The DAF-3 Smad binds DNA and represses gene expression in the
Caenorhabditis elegans pharynx. Development
126,97
-107.
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.
Trent, C., Tsung, N. and Horvitz, H. (1983).
Egg-laying defective mutants of the nematode C. elegans.Genetics 104,619
-647.
Ueki, N. and Hayman, M. J. (2003). Direct
interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for
Ski-mediated repression of TGF-ß signaling. J. Biol.
Chem. 278,32489
-32492.
Walhout, A. J. and Vidal, M. (2001). High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24,297 -306.[CrossRef][Medline]
Wang, W., Mariani, F. V., Harland, R. M. and Luo, K.
(2000). Ski represses bone morphogenic protein signaling in
Xenopus and mammalian cells. Proc. Natl. Acad. Sci.
USA 97,14394
-14399.
Wu, J. W., Krawitz, A. R., Chai, J., Li, W., Zhang, F., Luo, K. and Shi, Y. (2002). Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-ß signaling. Cell 111,357 -367.[Medline]
Xu, W., Angelis, K., Danielpour, D., Haddad, M. M., Bischof, O.,
Campisi, J., Stavnezer, E. and Medrano, E. E. (2000). Ski
acts as a co-repressor with Smad2 and Smad3 to regulate the response to type
ß transforming growth factor. Proc. Natl. Acad. Sci.
USA 97,5924
-5929.
Zheng, G., Teumer, J., Colmenares, C., Richmond, C. and Stavnezer, E. (1997). Identification of a core functional and structural domain of the v-Ski oncoprotein responsible for both transformation and myogenesis. Oncogene 15,459 -471.[CrossRef][Medline]