Department of Biology, McGill University, 1205 Avenue Dr Penfield, Montréal, Québec H3A 1B1, Canada
* Author for correspondence (e-mail: siegfried.hekimi{at}mcgill.ca)
Accepted 30 August 2005
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SUMMARY |
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Key words: Defecation, Homeobox, Paired-like, Enteric muscles, dsc-1, clk-1
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Introduction |
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By relating behavioral outputs to patterns of gene expression and the
expression of unique differentiated cellular features, it is possible to study
the molecular, genetic and cellular bases of behavior in C. elegans.
For example, neural circuits regulating behaviors such as pharyngeal pumping,
egg-laying and locomotion have all been studied in this manner (reviewed by
Rankin, 2002;
Whittaker and Sternberg,
2004
). The neuromuscular system that regulates the Defecation
Motor Program (DMP) (Liu and Thomas,
1994
) is also beginning to be studied at this level. The DMP
consists of three distinct, stereotyped steps
(Thomas, 1990
): the posterior
body muscle contraction (pBoc), the anterior body muscle contraction (aBoc)
and the expulsion (Exp), which is the most complex step as it consists of the
coordinated contractions of two specialized enteric muscles, the intestinal
and anal depressor muscles, and probably the relaxation of a third enteric
muscle, the sphincter. The defecation cycle period, which is the interval
between two series of these three contractions, is very regular over time in
single animals, and from animal to animal, but is modulated by sensory inputs
such as touch (Liu and Thomas,
1994
), food (Liu and Thomas,
1994
) and temperature (Branicky
et al., 2001
).
Two GABAergic neurons, AVL and DVB, send processes to the enteric muscles
(the intestinal, sphincter and anal depressor muscles) and are required for
the proper execution of the DMP. Laser ablation studies have revealed that
these two neurons are partially redundant for enteric muscle contractions.
AVL, but not DVB, also appears to be required for the aBoc step
(McIntire et al., 1993b). The
pBoc step may be regulated by a non-neural pathway, as neuronal ablations have
failed to identify any neuron required for the pBoc (E. Jorgensen, personal
communication). Interestingly, the neurotransmitter GABA, normally an
inhibitory neurotransmitter, is an excitatory neurotransmitter for enteric
muscle contraction. Indeed, loss of the GABA biosynthetic enzyme glutamic acid
decarboxylase (GAD), encoded by the unc-25 gene, causes an expulsion
defective (or Exp) phenotype, which can be rescued by exogenous GABA
(Jin et al., 1999
;
McIntire et al., 1993a
). In
addition, mutations that disrupt GABA transport (unc-47, gat-1), and
presynaptic GABA release (unc-2) also produce an Exp phenotype
(Jiang et al., 2004
;
Mathews et al., 2003
;
McIntire et al., 1993a
;
McIntire et al., 1997
). The
excitatory effect of GABA on these muscles is mediated by exp-1, an
unusual cation-selective GABA receptor, which is expressed in the intestinal
and anal depressor muscles (Beg and
Jorgensen, 2003
). There is little known about the development and
specification of these muscles, except that they are non-striated muscles that
require Twist, encoded by the hlh-8 gene, for their formation. Loss
of hlh-8 also produces an Exp phenotype, because these muscles are
missing or are severely reduced in the mutant
(Corsi et al., 2000
).
In addition to mutants defective in the execution of the DMP steps, a
number of mutants that affect the periodicity of the defecation cycle have
been identified (e.g. Branicky et al.,
2001; Iwasaki et al.,
1995
). In general, these mutants are distinct from those affecting
the steps of the DMP, suggesting that the expression of the DMP is regulated
by mechanisms that are distinct from those required for maintaining the
periodicity of the DMP. The characterization of some of these genes has shown
that calcium-dependent signals in the intestine play an important role in
regulating the periodicity of the DMP. In particular, loss of itr-1
(dec-4/lef-1), which encodes the worm inositol triphosphate receptor
(IP3 receptor) and regulates calcium release from the endoplasmic
reticulum, slows down the defecation period
(Dal Santo et al., 1999
).
Furthermore, the same authors showed that calcium levels peak in the intestine
just before the first muscle contraction of the DMP, and that expression of
the itr-1 in the intestine is sufficient for normal rhythm
generation. Moreover, mutations in unc-43, the C. elegans
calcium-dependent calmodulin Kinase II (CaM Kinase II), which is widely
expressed in neurons, muscles and the intestine, result in multiple behavioral
defects, including defecation phenotypes
(Reiner et al., 1999
).
The characteristics of several other mutants also point to the intestine as
the main modulator of the DMP periodicity. For example, flr-1 and
flr-4 mutants, originally identified on the basis of their resistance
to fluoride (Katsura et al.,
1994), have very short defecation cycle lengths
(Iwasaki et al., 1995
).
Intestinal expression of both flr-1, which encodes an ion channel of
the degenerin/epithelial sodium channel superfamily, and flr-4, which
encodes a serine/threonine protein kinase, is sufficient to rescue their
respective defects (Take-Uchi et al.,
1998
; Take-Uchi et al.,
2005
). The elo-2 gene, which encodes a fatty acid
elongation enzyme that is expressed in the intestine, also appears to be
involved in regulating the defecation cycle length as RNAi-mediated
suppression of elo-2 results in a shortened defecation cycle length
(Kniazeva et al., 2003
).
Finally, loss of function of dsc-4, which encodes the worm microsomal
triglyceride transfer protein (MTP), and is probably required in the intestine
for lipid transport, also causes a shortened defecation cycle length
(Branicky et al., 2001
;
Shibata et al., 2003
).
We have studied the altered defecation cycle of the clk-1 mutants,
in which a number of behavioral, developmental and physiological features are
abnormal (Branicky et al.,
2001; Wong et al.,
1995
). clk-1 encodes a highly conserved protein that is
required for ubiquinone (UQ or CoQ) biosynthesis
(Ewbank et al., 1997
). We have
analyzed the defecation behavior of clk-1 mutants in some detail and
found that in the mutants the cycle length is both increased and insensitive
to changes in temperature. To investigate these defects further, we have
carried out a screen to identify suppressor mutations (which we call
dsc, for defecation suppressor of clk-1) and identified two
classes of suppressor mutants. The class I dsc mutants (which include
dsc-4) suppress both the lengthened defecation cycle and the
temperature insensitivity; the class II mutants (which include dsc-1)
suppress only the lengthened defecation cycle, but does not restore normal
reaction to changes in temperature
(Branicky et al., 2001
).
Here we report our characterization of dsc-1. We find that the dsc-1 mutants have a shortened defecation cycle length and are also expulsion defective. We cloned dsc-1 and found that it encodes a Paired-like homeobox transcription factor that is expressed in some sensory neurons and in the enteric muscles. dsc-1 is the first homeobox gene of its class that is found to be expressed in, and important for the differentiation of, non-neuronal cells in C. elegans. We show that expression of dsc-1 in the intestinal and anal depressor muscles alone is sufficient to rescue both the defecation cycle length and expulsion defects. However, a variety of experimental manipulations allow for the uncoupling of the effects of dsc-1 on these two phenotypes, suggesting a previously unanticipated role for the enteric muscles in regulating the defecation cycle length. We also show that dsc-1(+) activity is required for the expression of exp-1 in the intestinal and anal depressor muscles, and that the expulsion defect of the dsc-1 mutant is due to the loss of exp-1 expression.
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Materials and methods |
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The mutations used to refine the genetic position of dsc-1 include
lin-15(n765), flr-4(ut7) and unc-7(e5). dsc-1 was also
mapped relative to the mnDF20 deficiency using the SP278 strain (mnDp1 (X;V)/+
V; mnDf20 X). Other mutations and strains used in this study include
exp-1(sa6) I, EG1653 oxIs22 [Punc-49:UNC-49::GFP,
lin-15(+)] II (Bamber et al.,
1999), clk-1(qm30) III, unc-25(e156) III and
hlh-8(nr2061) X (Liu et al.,
1999
).
Defecation was scored as described in Branicky et al.
(Branicky et al., 2001). Each
animal was scored for five consecutive cycles at 20°C. To quantify the Exp
phenotype, animals were also scored for five consecutive defecation cycles,
which includes six consecutive posterior body contractions (pBocs), and the
number of pBocs followed by an expulsion was determined. Student's
t-test was used to determine statistical significance and was carried
out using Graphpad Prism 4.0 software.
Positional cloning of dsc-1
dsc-1 had previously been mapped to the right arm of LG X, between
unc-3 and lin-15
(Branicky et al., 2001). Using
2-point, 3-point and deficiency mapping strategies, we further refined the
genetic position of dsc-1 and determined that it is between the two
cloned genes pag-3 and unc-7 (see
www.wormbase.org).
The six cosmids that correspond to this region were injected into the
dsc-1 mutants as a single pool, in pairs, and singly, and it was
determined that the dsc-1 mutants could be rescued by all injection
mixes containing the C18B12 cosmid. The three predicted genes on the C18B12
cosmid were amplified from the cosmid by PCR and were tested for rescuing
activity. A PCR product corresponding to the predicted gene C18B12.3 (from
30798 to 24611 on C18B12) was able to rescue the dsc-1 mutants. All
cosmids and PCR products were injected at a concentration of 50 ng/µl with
the co-injection marker ttx-3::gfp
(Hobert et al., 1999
) at a
concentration of 150 ng/µl.
To determine the nature of the qm133 mutation, genomic DNA was extracted from dsc-1(qm133) mutants and the predicted coding region was sequenced from both strands. We identified a G-to-A transition at position 695 of the coding region relative to the ATG (position 26250 of the C18B12 cosmid) resulting in an R-to-H substitution at residue 232 of the protein.
RT-PCR
First-strand cDNA libraries were generated by the reverse transcription of
total RNA extracted from adult worms, using a polydT primer and reverse
transcriptase. The dsc-1 transcript was amplified from the library
with primers corresponding to the predicted transcript and primers
complementary to the polyA tail. The dsc-1 transcript contains five
exons and is 933 bp long. The 3' UTR is 284 bp long, and probably uses
one of two possible AAUAAA polyadenylation signals, located 14 or 18 bases
upstream of the cleavage site. The dsc-1 transcript does not seem to
be trans-spliced, as the transcript could not be amplified using primers
corresponding to either the SL1 or the SL2 splice-leader sequences, although
there is a consensus trans-splice site located 14 bases upstream of the start
codon.
Construction of plasmids and transgenic strains
For transcriptional and translational dsc-1::gfp reporter fusions,
portions of the dsc-1 promoter and genomic region were amplified from
the C18B12 cosmid with primers containing synthetic PstI and
NheI sites, and were cloned into the PstI and XbaI
sites of the pPD95.77 vector, kindly provided by A. Fire. Unless otherwise
indicated, constructs were injected into N2 and dsc-1 at a
concentration of 50 ng/µl along with the transformation marker
pRF4[rol-6(su1006)] or ttx-3::gfp at a concentration of 150
ng/µl.
Pdsc-1::gfp
The insert contains bases 30284-28330 of C18B12, which includes 2 kb
upstream of the initiating ATG of dsc-1 and the first 18 bp.
Pdsc-1::dsc-1::gfp
The insert contains bases 30284-25275 of C18B12, which includes 2 kb
upstream of the initiating ATG of dsc-1 and the entire genomic
sequence, excluding the stop codon.
For this construct, gfp expression could be observed in 4/4 lines,
all of which were rescued. However, rescued lines also produced 30%
non-rescued transgenic worms.
Nde-box::dsc-1::gfp
The Nde box is a concatamerized element of the ceh-24 promoter
that was previously found (Harfe and Fire,
1998) and has been used to drive expression specifically in the
enteric muscles (Bulow et al.,
2002
). It was amplified from the pBH10.21 clone and was cloned
into the PstI and XbaI sites of the pPD95.77 vector. (The
PstI site was introduced in the primer and the XbaI site was
internal to the PCR product.) The dsc-1 cDNA (19 to +930) was
amplified from a cDNA library with primers containing synthetic KpnI
sites, and was cloned into the KpnI site of pPD95.77. This construct
was injected into dsc-1 at concentrations of 25 and 100 ng/µl
along with the transformation marker ttx-3::gfp (at concentrations of
175 and 100 ng/µl). For this construct, gfp expression could only
be observed in 3/5 transgenic lines; all three were rescued. However, rescued
lines also produced
30% non-rescued transgenic worms.
Pexp-1::gfp (pAB04)
This was kindly provided by E. Jorgensen
(Beg and Jorgensen, 2003).
Nde-box::exp-1
This fusion was created using a PCR approach essentially as described
previously (Hobert, 2002); the
construct is not a GFP fusion, so as not to interfere with the function of
EXP-1. The Nde-box portion was amplified from the pBH10.21 clone and the
exp-1 gene (from 60 to +4128, which includes the UTRs) from
genomic DNA. The fusion PCR product was injected in exp-1 and
dsc-1 mutants at a concentration of
1 ng/µl together with the
transformation marker ttx-3::gfp at a concentration of 100 ng/µl.
For both backgrounds 4/4 lines were rescued. Rescued lines produced
10%
non-rescued transgenic worms.
RNAi
A portion of the dsc-1 cDNA (+33 to + 933) was amplified from a
first-strand cDNA library with primers containing synthetic NheI and
PstI restriction sites and was cloned into the corresponding sites in
the pPD129.36 vector, kindly provided by A. Fire. The cDNA flanked by the T7
promoter sites was then amplified from the clone and was used as the template
for in vitro transcription (Promega Ribomax). Double-stranded RNA was injected
into wild-type animals at a concentration of 1 µg/µl as described
(Fire et al., 1998
). The F1
progeny were scored for their defecation cycle length and number of pBocs
associated with expulsions.
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Results |
---|
dsc-1 encodes a Q50 Paired-like homeodomain protein
dsc-1 was previously mapped to LGX between unc-3 and
lin-15 (Branicky et al.,
2001). Using 2- and 3-point mapping strategies we determined that
dsc-1 is tightly linked to, and to the left of both unc-7
and flr-4. Using the chromosomal deficiency mnDf20, which
deletes pag-3 but not dsc-1, we determined that
dsc-1 is right of pag-3. The six cosmids that cover the
region between pag-3 and flr-4 were injected into
dsc-1 mutants as a single pool, then in pairs and then singly. All
injection mixes containing the C18B12 cosmid were capable of rescuing all
phenotypes of the dsc-1 mutants. Of the three predicted genes on the
C18B12 cosmid, a PCR product corresponding to only one, C18B12.3, was capable
of rescuing dsc-1 mutants (Fig.
1).
We sequenced the coding region of C18B12.3 and found a G-to-A point mutation that results in an R-to-H substitution at residue 232 of the protein (Fig. 3B). In addition, a strain harboring a deletion in the C18B12.3 gene, (C18B12.3(tm241)), phenocopied and failed to complement the dsc-1(qm133) mutant (Fig. 1 and data not shown). Finally, injection of double-stranded RNA corresponding to the C18B12.3 coding region into wild-type animals also phenocopied the dsc-1(qm133) mutation (Fig. 1). Together these observations indicate that dsc-1 corresponds to the predicted gene C18B12.3 and suggest that the dsc-1(qm133) mutation may be a genetic null.
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dsc-1 is expressed in enteric muscles |
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We found that both the transcriptional and translational fusions are expressed in a subset of bilateral sensory neurons. Based on the positions of the cell bodies and processes we believe we have identified these neurons as AWA, AWB, AWC, ASE, FLP and PVD (Fig. 4A,B,D,E). GFP expression could also occasionally be observed in a sixth pair of unidentified neurons, located to the anterior of AWC (not shown). As expected, we observed expression of the transcriptional fusion in both the cell bodies and axons, and in the cytoplasm as well as in the nucleus, whereas expression of the translational fusion was mostly confined to the nucleus.
In addition to expression in neurons, we also observed expression in the enteric muscles. Expression of the transcriptional fusion could be observed only in the sphincter muscle (Fig. 4C), whereas expression of the translational fusion was observed in the nuclei of the two intestinal muscles (IM) and the anal depressor muscle (AD), but not in the sphincter. This suggests that sequences upstream and internal to the dsc-1 gene might participate in a combinatorial code of expression that identifies as a group all the muscles that are involved in defecation, and that there are sequences in the coding region that prevent expression of dsc-1 in the sphincter and promote expression of dsc-1 in the IM and AD (see Discussion).
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Expression of dsc-1(+) in two enteric muscles is sufficient to rescue the defecation defects of dsc-1 mutants |
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dsc-1(+) is required for the expression of exp-1 |
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We found that expression of an exp-1::gfp transcriptional reporter could not be detected in the IM and AD in either dsc-1(qm133) or dsc-1(tm241) backgrounds, while gfp expression could be observed when the same transgene was transferred into a dsc-1(+) background (Fig. 6A,B and data not shown). exp-1::gfp expression could also be restored when a dsc-1-rescuing construct was introduced into the dsc-1 mutant lines (data not shown). In addition, we observed that dsc-1(+) was not required for the expression of exp-1 in the PDA neuron, a neuron that is known to express exp-1 but is not known to have a role in defecation. Taken together, these results indicate that dsc-1(+) is required for the expression of exp-1 in an enteric-muscle-specific manner.
As a control, we also examined the expression of unc-49 in
dsc-1 mutants (Fig.
6C,D). unc-49 encodes a typical anion-gated GABA channel
(Bamber et al., 1999), which is
expressed in the sphincter, and should promote relaxation of this muscle in
response to GABA. However, the exact role of the sphincter in hermaphrodite
defecation is less clear than for the other two muscles. Given that
unc-49 mutants are not expulsion defective
(McIntire et al., 1993a
), the
sphincter probably plays only a minor role in the expulsion step in
hermaphrodites. However, in males there is a remodeling of the system in
adulthood such that the expulsion step is achieved by relaxation of the
sphincter, rather than by contraction of the IM and AD
(Reiner and Thomas, 1995
). The
expression of unc-49 does not appear to be in any way altered in
dsc-1 mutants, which is consistent with the expression pattern of
dsc-1. Moreover, we found that dsc-1 mutant males do not
have the Con phenotype that is associated with the Exp phenotype
(Fig. 2F). This further
supports a requirement for dsc-1 only in the IM and AD muscles, and
only for the expression of genes such as exp-1, that act in the IM
and AD. As has previously been reported
(Reiner and Thomas, 1995
), we
found that exp-1 males are also not Con, whereas unc-25
males are severely Con. This is consistent with a requirement for GABA for the
expulsion in males, not by acting on EXP-1 to promote contraction of the IM
and AD, but rather by acting on UNC-49 to promote relaxation of the
sphincter.
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The dsc-1 expulsion defect is due to the absence of EXP-1 |
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|
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dsc-1 regulates the defecation cycle length |
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|
Secondly, we examined the defecation cycle lengths of other Exp mutants in
both clk-1(+) and clk-1(qm30) backgrounds
(Fig. 7). Our survey included
unc-25, a mutant of GAD (Jin et
al., 1999), hlh-8, a mutant that affects the development
of the enteric muscles (Corsi et al.,
2000
), and exp-1, which, as described above, affects the
enteric muscle contraction in response to GABA
(Beg and Jorgensen, 2003
). All
mutants showed the oscillation in cycle length characteristic of Exp mutants
(see legend of Fig. 7). We
found that only exp-1, like dsc-1, significantly shortened
the average cycle length in a wild-type background. The others
(unc-25 and hlh-8) significantly increased the average cycle
length (Fig. 7A), which
suggests that an expulsion defect per se does not necessarily lead to an
average shortening of the defecation cycle length. Moreover, we found that, by
contrast to what is observed with dsc-1 mutants, none of the Exp
mutants, including exp-1, could suppress the lengthened defecation
cycle of clk-1 mutants (Fig.
7B).
Thirdly, we observed that overexpression of dsc-1 can
significantly increase the defecation cycle length. The transgenic lines that
express dsc-1, either under the control of the dsc-1
promoter or the Nde-box, which presumably leads to overexpression, have
significantly longer defecation cycle lengths than the wild type
(Fig. 5C;
Fig. 1B). As a loss of
dsc-1 activity results in a shortened defecation cycle length, this
suggests that the level of dsc-1(+) expression is somehow involved in
setting the defecation rate. A similar observation has been made with the
itr-1 gene, except that the effect is reversed (loss-of-function
mutations slow down, whereas overexpression speeds up, the cycle)
(Dal Santo et al., 1999).
Fourthly, we found that dsc-1 can also significantly shorten the lengthened defecation cycle of the itr-1(sa73) mutant (data not shown). This again supports a role for dsc-1 in cycle length regulation, and suggests that dsc-1 affects the cycle length by a mechanism that is independent of the regulation that depends on calcium signaling in the gut. However, given that itr-1(sa73) is not a null allele, potential interactions between dsc-1 and itr-1 need to be further investigated.
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Discussion |
---|
Caenorhabditis elegans Prd-like genes have mostly been studied for
their roles in the nervous system and, with one exception, have all been shown
to have roles in specifying neuron sub-type identity: that is, particular
aspects of neuronal function such as choice of neurotransmitter synthesis,
connectivity or sensory abilities. Briefly, unc-30, the worm Rx
family ortholog, specifies the identities of 19 GABAergic neurons
(Eastman et al., 1999;
Jin et al., 1999
). The worm
Otx family orthologs ttx-1, ceh-36 and ceh-37 specify the
identities of the ASH, the AWB and AWC, and the ASE sensory neurons,
respectively (Lanjuin et al.,
2003
; Satterlee et al.,
2001
). ceh-10 specifies the identity of the AIY
interneuron (Altun-Gultekin et al.,
2001
; Svendsen and McGhee,
1995
). unc-4 specifies the identities of the VA
motoneurons (Miller et al.,
1992
; White et al.,
1992
). ceh-17, a worm Prx family member, also functions
in a subset of neurons, but appears to be required for axonal outgrowth rather
than subtype identity (Pujol et al.,
2000
).
Remarkably, the function of some of these genes is precisely conserved in
vertebrates. For example, vertebrate orthologs of unc-30, ceh-36 and
ceh-37, and ceh-17 (Pitx2, Otx1 and Phox2b, respectively)
are all expressed in cell types similar to those in which they are expressed
in the worm, and can complement the mutant worm genes
(Lanjuin et al., 2003;
Pujol et al., 2000
;
Westmoreland et al., 2001
).
However, this does not mean that they are not involved in other processes as
well. For example, Pitx2 is also involved in the development of the pituitary
gland, craniofacial region, eyes, heart, abdominal viscera and limbs
(Gage et al., 1999
;
Kitamura et al., 1999
;
Lin et al., 1999
;
Liu et al., 2001
).
dsc-1 encodes a Q50 Prd-like protein, which does not appear to
have any clear ortholog in any species but C. briggsae. It is
expressed in a few sensory neurons (Fig.
4), as well as in the non-striated enteric muscles. We have
determined that dsc-1 is required in three of these muscles for the
expression of exp-1, a GABA-gated cation channel. Our analysis
suggests that, as in other systems, Prd-like genes in C. elegans may
play key developmental roles in non-neuronal tissues. We speculate that other
Prd-like genes in C. elegans might also be involved in specifying
neurotransmitter sensitivity. Indeed, unc-42, another Prd-like gene
in C. elegans that is without a clear vertebrate ortholog, appears to
be required for regulating glutamate receptor expression in the nervous system
(Baran et al., 1999).
Enteric muscle development and function in C. elegans
Muscles in worms are of two broad types. The main class of muscles is the
striated muscles, the so-called `body-wall muscles', which attach to the outer
body wall and contract longitudinally for locomotion. The second class is the
non-striated muscles, which are a diverse group of single-sarcomere muscles.
These muscles include the pharyngeal muscles, which are used for feeding, the
uterine and vulval muscles, which are used for egg-laying, the male-tail
associated muscles, which are used for mating, and the enteric muscles we have
discussed here (the AD, IM and sphincter), which are required for the
expulsion part of the defecation behavior
(www.wormatlas.org).
Only very few genes required for the development of the enteric muscles
have been identified so far. The most extensively characterized is
hlh-8, the C. elegans homolog of Twist, a basic
helix-loop-helix (bHLH) transcription factor that has an evolutionarily
conserved role in mesodermal patterning
(Corsi et al., 2000;
Harfe et al., 1998
).
hlh-8 mutants have defects in the patterning of the post-embryonic
mesodermal lineages, and in the development of the IM and AD muscles, which
develop embryonically. One downstream target of hlh-8 in the AD
muscle is the NK-2 class homeobox gene ceh-24, whose expression is
dependant on hlh-8(+) activity. However, ceh-24 mutants do
not appear to have expulsion defects
(Harfe and Fire, 1998
),
suggesting that the activity of ceh-24 is not necessary for the main
aspects of muscle function. hlh-8 is not required for the development
of the sphincter muscle (Corsi et al.,
2000
). In fact, although a number of genes have been reported to
be expressed in the sphincter (WormBase Release WS147,
www.wormbase.org),
none has been shown to be required for its development.
Here we have shown that dsc-1 is required for an aspect of terminal differentiation of the AD and IM muscles; that is, the specification of their sensitivity to GABA. Like hlh-8, dsc-1 does not appear to have a role in the sphincter, although it does contain some elements in its promoter that can drive its expression there. Indeed, the transcriptional dsc-1::gfp reporter is expressed exclusively in the sphincter (Fig. 4C); conversely, the translational reporter is expressed only in the AD and the IM (Fig. 4F). This suggests that there are elements in the dsc-1 promoter that direct expression to the sphincter, while there are elements in the intronic and/or exonic regions that prevent expression in the sphincter but instead promote expression in the AD and IM. We speculate that dsc-1 participates in a combinatorial code of expression that identifies all muscles that are involved in defecation as a group, but also ensures that exp-1 is only expressed in the muscles that need to contract during defecation.
The roles of dsc-1 in defecation
Two types of defecation mutants have been identified in C.
elegans: those that affect the aBoc and/or Exp step, and those that
affect periodicity. In general, these two classes of mutants are distinct,
although a few mutants fall into both classes (e.g. the flr mutants
and the unc-43(gf) mutant)
(Reiner et al., 1999;
Take-Uchi et al., 1998
;
Take-Uchi et al., 2005
).
Mutants that affect both the aBoc and Exp steps (defining the aex
genes) have general synaptic transmission defects
(Doi and Iwasaki, 2002
;
Iwasaki et al., 1997
).
Similarly, there is a class of dominant Exp mutants, also egg-laying
defective, that affect another general property, which is muscle excitability
(Reiner et al., 1995
). Most of
the recessive Exp mutants reported are defective in either the function of the
GABAergic DVB neuron (Hobert et al.,
1999
), the synthesis or transport of GABA
(McIntire et al., 1993a
;
McIntire et al., 1993b
), or
the response of the enteric muscles to GABA
(Bamber et al., 1999
;
Beg and Jorgensen, 2003
).
Consistently, we find that dsc-1 is required for an aspect of GABA
neurotransmission; that is, the expression of the appropriate GABA receptor in
the IM and AD.
As described in the Introduction, a number of genes affecting the
periodicity of the defecation cycle have been identified: in particular,
itr-1 (dec-4/lef-1), elo-2, dsc-4, flr-1 and
flr-4. Although these genes appear to affect diverse processes, all
affect defecation by acting in the gut, and presumably by altering some of its
properties (Dal Santo et al.,
1999; Kniazeva et al.,
2003
; Shibata et al.,
2003
; Take-Uchi et al.,
1998
; Take-Uchi et al.,
2005
). By contrast, we have found that dsc-1 modulates
the defecation cycle length by acting in the enteric muscles. As the gut
appears to control the rhythm of muscle contraction in this system, we
speculate that dsc-1 is required for a property of these muscles that
allows them to act in a feedback mechanism from the muscles to the gut. One
type of feedback mechanism that is operating in this system works through gut
distention. Indeed, a lack of enteric muscle contractions leads to a
progressive shortening of the defecation cycle until the contents of the gut
are forcefully expulsed by pressure alone, and is then followed by a few
longer cycles. We speculate that dsc-1 is necessary for a feedback
from the muscles that may involve a humoral signal that acts to coordinate the
calcium signal generated in the gut with the timed contractions of the various
muscles types. In this context, it is interesting to note that the
flr-1 and flr-4 mutants, which have a shortened defecation
cycle length, are also Exp. Furthermore, expression of both genes in the
intestine alone is sufficient to rescue both the cycle length and the Exp
defects. Possibly these genes could somehow be involved in transducing the
putative humoral signal from the muscle to the gut.
Interestingly, we have found that dsc-1 suppresses the lengthened defecation cycle of clk-1(qm30) mutants. Although exp-1 is a target of dsc-1 in the IM and AD muscle for the expulsion step of the defecation behavior, dsc-1 probably acts through different targets for cycle length regulation. Indeed mutation of exp-1 cannot suppress the lengthened defecation cycle of clk-1. The identification of the downstream effectors of dsc-1 are likely to give new insights into the slow defecation phenotype of clk-1 mutants and, more generally, into mechanisms that regulate the defecation cycle length in C. elegans.
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ACKNOWLEDGMENTS |
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