Department of Biology, Queens College, and The Graduate School and University Center, CUNY, Flushing, NY 11367, USA
* Author for correspondence (e-mail: cathy_savage{at}qc.edu)
Accepted 18 July 2002
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SUMMARY |
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Key words: Body size, TGFß, BMP, Smad, Caenorhabditis elegans
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INTRODUCTION |
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Smads are separated into three categories, R-Smad, Co-Smad and anti-Smad.
R-Smad activity is regulated by the receptors by phosphorylation. Co-Smads are
not activated by phosphorylation, but cooperate with R-Smads to form a
functional complex. The anti-Smads negatively regulate the TGFß pathway,
and their expression is dependent upon TGFß signaling
(Massagué and Chen,
2000). The structure of a Smad comprises conserved MH1 and MH2
domains, separated by a variable linker region. The MH1 domain has the
DNA-binding region and nuclear localization sequence
(Shi et al., 1998
;
Xiao et al., 2000
). The MH2
domain contains an SSXS motif that is phosphorylated by type I receptor on the
last two serines (Souchelnytskyi et al.,
1997
). Once phosphorylated, the activated R-Smad forms a
heterodimer or heterotrimer with the Co-Smad
(Shi et al., 1997
;
Wu et al., 2001
;
Qin et al., 1999
;
Qin et al., 2001
).
In the nematode Caenorhabditis elegans, there are two
characterized TGFß-related pathways, the Dauer pathway and the Sma/Mab
(small/male tail abnormal) pathway
(Patterson and Padgett, 2000;
Savage-Dunn, 2001
). The Dauer
pathway controls entry into and exit from the dauer stage, an L3 larval stage
specialized for harsh environmental conditions. Entry into the dauer stage is
regulated by environmental cues, such as the availability of food, the
population density and temperature. The Dauer pathway is composed of the
ligand (daf-7), the type II receptor (daf-4), the type I
receptor (daf-1) and the Smads (daf-8, daf-14 and
daf-3). Loss of function of any factor except daf-3 results
in the dauer constitutive phenotype, in which worms enter dauer even under
favorable conditions (Estevez et al.,
1993
; Ren et al., 1998;
Gunther et al., 2000
;
Inoue and Thomas, 2000
).
However, the absence of daf-3 activity gives a dauer defective
phenotype, in which worms do not form dauers
(Patterson et al., 1997
).
The ligand for the Sma/Mab pathway, dbl-1, is related to
Drosophila dpp and vertebrate BMPs
(Suzuki et al., 1999;
Morita et al., 1999
). It
functions with the type II receptor daf-4 and the type I receptor
sma-6 (Estevez et al.,
1993
; Krishna et al.,
1999
). The Dauer and Sma/Mab pathways use a common type II
receptor, daf-4. In the Sma/Mab pathway, daf-4 and
sma-6 receptors activate the R-Smads, sma-2 and
sma-3. These are thought to form complexes with the Co-Smad,
sma-4, to propagate the signal into the nucleus
(Savage et al., 1996
;
Savage-Dunn et al., 2000
).
Loss-of-function mutations in any of the Sma/Mab pathway components result in
small body size. In addition, defects of the male tails are seen, including
sensory ray fusions and crumpled spicules. Both of the R-Smad proteins, SMA-2
and SMA-3, are crucial for pathway function, suggesting the formation of a
heteromeric complex containing two different R-Smad subunits
(Savage-Dunn et al.,
2000
).
The underlying cause of the small body size phenotype is still poorly
understood. In theory, the final size of an organ or organism can be
determined by the regulation of cell number, cell size, or both. Cell number
may be controlled by the regulation of cell division or of cell death. Cell
size is usually coordinated with the cell cycle, and often correlates with
ploidy (Galitski et al., 1999).
Mutants of the Sma/Mab pathway provide an opportunity to study the molecular
regulation of body size in a viable animal model. Previous reports
(Suzuki et al., 1999
;
Flemming et al., 2000
) and our
own unpublished results (R. T. and C. S.-D., unpublished) indicate that these
small mutants contain normal numbers of cells. Therefore, some or all cell
sizes must be reduced. We have addressed the cell and tissue specificity of
the Sma/Mab pathway regulation of body size in several ways. First, we
measured individual cell and organ sizes in small mutants to determine which
were reduced in size. Second, we characterized the expression and subcellular
localization of SMA-3. We have previously reported that sma-3 is
widely expressed, based on a transcriptional lacZ fusion
(Savage-Dunn et al., 2000
).
Here, we provide a higher resolution expression pattern using SMA-3::GFP
translational fusions. We find that sma-3 is expressed in the
pharynx, intestine and hypodermis. Third, we used mosaic analysis and directed
expression of sma-3 to determine where it functions to regulate body
size. These experiments indicate that the hypodermis is the crucial tissue
involved in body size regulation.
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MATERIALS AND METHODS |
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Small mutants chosen were either known null mutants or the most severe
mutants available. sma-6(wk7) has an early stop codon in the
extracellular region that results in a null mutation
(Krishna et al., 1999). There
is an early termination in the DBL-1 bioactive domain in dbl-1(wk70)
mutants (Suzuki et al., 1999
).
An arginine in the beginning of the linker region in SMA-3 mutates into a stop
codon, suggesting sma-3(wk30) is a strong allele
(Savage-Dunn et al., 2000
). In
sma-2(e502), the mutation G372D disrupts a critical amino acid in the
sma-2 MH2 domain (Savage et al.,
1996
). The DNA sequence of the canonical sma-4(e729)
allele had not previously been determined. We therefore sequenced the
sma-4 gene from these mutants. Fragments of the sma-4 gene
were amplified by PCR and directly sequenced. sma-4(e729) contains a
single nucleotide substitution resulting in a Q246 (CAA) to stop (UAA)
mutation. Therefore, sma-4(e729) is an early termination
mutation.
Construction of sma-3 and GFP fusion genes
An 8 kb sma-3 genomic fragment was obtained from the cosmid R13F6
by digesting with PstI and subcloning into the vector pBLUESCRIPT SK+
(pCS29). This construct rescues sma-3(wk30) mutants. To create GFP
fusion constructs, MluI restriction sites were created in the
sma-3-coding region, after the start codon (pCS185) or before the
stop codon (pCS186) independently, by site-directed mutagenesis (MutaGene kit
from BioRad). A GFP MluI fragment from pPD118.90 (A. Fire) was
subcloned into the newly generated MluI sites, forming two
sma-3::gfp translational fusion constructs; N-terminal GFP (pCS170)
or C-terminal GFP (pCS171). We also created a sma-3 construct lacking
all coding sequences. An XhoI-MluI fragment from pCS185
containing sma-3 upstream sequences was ligated into
XhoI-MluI-digested pCS186 in which upstream and coding
regions had been removed leaving only downstream sequences. This
sma-3 construct (pCS210) therefore contains upstream and downstream
noncoding sequences but no coding region.
Fusion of tissue specific promoters and sma-3::gfp coding
region
The promoters used in tissue specific expression of sma-3::gfp
included elt-3, vha-7, dpy-7 (hypodermal); elt-2, vha-6
(intestine); myo-2 (pharynx); and the third isoform of tmy-1
(pharynx and intestine) (Gilleard et al.,
1999; Oka et al.,
2001
; Gilleard et al.,
1997
; Okkema et al.,
1993
; Fukushige et al.,
1998
; Anyanful et al.,
2001
). The elt-2 and elt-3 promoters were kindly
provided by R. W. Padgett's laboratory (HW373 and HW375). The others were
obtained by PCR using N2 genomic DNA as template from worm lysates. The PCR
primers used were:
After the PCR products were digested by the appropriate restriction enzymes, the promoters were cloned into pBLUESCRIPT SK+ vector. Next, a PstI fragment containing the sma-3::gfp(N)-coding region was excised from pCS170 (one PstI site derives from the GFP vector pPD118.90) and inserted into vectors containing the heterologous promoters. Thus, after translation, each of the protein products has GFP at the N terminus and SMA-3 at the C terminus.
Transformation and integration
Transformation of constructs into worms was carried out by microinjection
(Mello et al., 1991). Unless
otherwise stated, the injection solution contains 20 ng/µl of experimental
plasmid and 100 ng/µl pRF4 (rol-6 plasmid). The co-injection of
vha-6::sma-3 and myo-2::sma-3 includes 20 ng/µl of each.
The co-injection of C-terminal sma-3::gfp (pCS171) with pCS29, pCS170
or pCS210 contains 10 ng/µl of each experimental plasmid.
We used -rays to integrate the sma-3::gfp(N) array into
sma-3(wk30) and the co-injected sma-3::gfp(C) with
sma-3 genomic and sma-3::gfp(C) with sma-3
non-coding arrays into N2. More than 100 L4 or young adult worms with each
extra-chromosomal array were picked. After exposure to a cesium source, the
worms were separated into 20 plates (five worms in each). After starvation,
the worms were chunked into new plates. We allowed the worms to recover for 2
days. Each worm carrying the array was picked into a separate plate. After one
generation, plates with 100% worms containing the reporter gene
(rol-6) were selected. The integrated arrays are
qcIs6[sma-3::gfp(N) + rol-6], qcIs12[sma-3::gfp(C) +
sma-3 + rol-6] and qcIs16[sma-3::gfp(C) +
sma-3no-code + rol-6].
Length measurements
To characterize the small phenotype, we measured seam cells, pharynx length
and body length in the same animals. The jam-1::gfp marker
(Mohler et al., 1998) was used
to visualize seam cells. This marker localizes to the adherens junctions
surrounding these lateral hypodermal cells. jam-1::gfp was also
introduced into sma-1, sma-3, sma-4 and dbl-1 mutants by
standard genetic crosses. L3 larvae were picked and the seam cells were
observed under fluorescence. The seam cell lineage is highly dynamic and
involves major changes in cell shape. We focused on worms containing
rectangularly shaped cells, after they completed their divisions. We looked
for worms with at least four consecutive cells ideal for measurement. Once
ideal worms were found, the seam cells were photographed using a 40x
objective. The pharynx and the entire worm were photographed using a 10x
objective. The measurements were performed using SigmaScan software.
To assess body length in transgenic animals, cultures were synchronized by bleaching gravid hermaphrodites in order to isolate eggs. These eggs were introduced onto new plates. The body length was measured after 96 hours, in the adult stage. Transgenic (rolling) worms were picked, mounted on slides and measured as described above.
Mosaic analysis
Mosaic analysis was performed with strains containing high copies of a
plasmid pTG96 (generously provided by Min Han). The plasmid contains a fusion
of the ubiquitously expressed, nuclear localized sur-5 with GFP
(Yochem et al., 1998). The
sur-5::gfp was co-injected with the sma-3 rescuing clone
pCS29 into sma-3(wk30) animals. Two lines were obtained, with strain
designations CS122 sma-3(wk30);qcEx26 and CS125
sma-3(wk30);qcEx27. The extragenic arrays have rescuing activity. The
strains were then observed under fluorescence for mosaicism at a magnification
of 400x. Both small and wild-type mosaic animals were analyzed.
Western blot
Worms were washed off non-starved plates and frozen at -80°C overnight.
The boiling buffer (8% SDS+20 mM DTT+100 mM pH 6.8 Tris+10 mM PMSF) was added
and the samples were boiled for 5 minutes. The protein concentration was
determined and equal amounts of total protein were loaded in each lane. After
running the SDS-PAGE gel, the proteins were transferred onto nitrocellulose
membrane. The membrane was blocked by 5% BSA in PBS and probed by rabbit
anti-GFP antibody (Clontech). The secondary antibody (anti-rabbit) and
detection solutions were from the ECL western blotting analysis system
(Amersham).
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RESULTS |
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We have measured two accessible tissues to determine the extent of cell
size reduction in small animals. The seam cells are lateral hypodermal blast
cells that donate daughter nuclei to the hypodermal syncytium after each cell
division, and eventually fuse in adults
(Sulston and Horvitz, 1977).
We took advantage of the jam-1::gfp marker
(Mohler et al., 1998
), which
localizes to the adherens junctions surrounding the seam cells. The seam cell
marker was crossed into sma mutant backgrounds and fluorescence was
observed in the L3 stage. By this stage, sma mutant worm length is
significantly different from wild type
(Savage-Dunn et al., 2000
); at
later stages the seam cells will fuse with each other and cannot be measured
individually. The seam cells in mutant animals are shorter in length than the
seam cells of wild-type animals (Table
1). In Sma/Mab mutants, the difference in seam cell length is
proportional to the difference in overall body length
(Fig. 1). It was possible that
this reduction in seam cell length was offset by an increase in width, so we
also measured seam cell area. Again, we found that seam cell area was reduced
in Sma/Mab mutants (Table 1).
For a negative control, we used sma-1 mutants. sma-1 mutants
are also small, but they follow a different growth pattern than the TGFß
Sma/Mab mutants (Savage-Dunn et al.,
2000
). At the L3 stage, sma-1 body length is less than
the Sma/Mab pathway mutants, but the mean length and area of the seam cells is
larger (Table 1).
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We also measured the length of the pharynx in the same worms. Interestingly, in the TGFß mutants the pharynx is smaller than in N2, but only slightly so, with the ratios varying between 0.93 and 0.96 of wild type (Table 1, Fig. 1). In sma-1, the pharynx is 33% smaller than wild-type. Thus, in the small mutants examined, different tissues are reduced in size by different proportions. In the TGFß Sma/Mab mutants, but not in sma-1 animals, the seam cell size is proportional to the body size.
Levels of functional activity of sma-3::gfp translational
fusions
To determine the expression pattern and subcellular localization of SMA-3
Smad protein, we made two kinds of sma-3::gfp translational fusion
gene constructs. In pCS170, GFP is inserted at the N terminus, and in pCS171,
GFP is inserted at the C terminus. After transformation into
sma-3(wk30), the rescuing ability was assessed. Rescue of body size
was assessed by measuring worm length 96 hours after embryo collection. Rescue
of male tail patterning was assessed by determining the frequency of sensory
ray fusions. In the wild-type C. elegans male tail are nine bilateral
pairs of sensory organs, the sensory rays
(Sulston et al., 1980). Each
ray is characterized by its unique position, morphology and neurotransmitter
usage. The Sma/Mab pathway plays a role in the specification of rays 5, 7 and
9. In mutants, these rays often display characteristics of rays 4, 6 and 8,
respectively, resulting in readily observable fusions between rays 4-5, 6-7
and 8-9 (Savage et al., 1996
;
Suzuki et al., 1999
;
Morita et al., 1999
;
Krishna et al., 1999
).
The N-terminal sma-3::gfp construct is functional, restoring most
of the body length (Table 2)
and rescuing the male tail sensory ray pattern
(Table 3). However, the
C-terminal construct has very little rescuing activity in body length
(Table 2) and it only partially
rescues male tail ray fusions (Table
3). When the extra-chromosomal array with the C-terminal construct
is crossed into a wild-type background, it even shows a slight inhibition in
body length, but does not affect male tail development (Tables
2 and
3), suggesting that it can
interfere with wild-type SMA-3 function. The R-Smad C-terminal SSXS motif is
the site of phosphorylation and may participate in intermolecular interactions
(Wu et al., 2001;
Qin et al., 2001
). The
C-terminal insertion of GFP may disrupt some of these interactions.
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sma-3::gfp fusion gene expression pattern and protein
localization
Both the N-terminal functional and the C-terminal nonfunctional
sma-3::gfp constructs show the same pattern of expression, but the
level of fluorescence of the C-terminal construct is much higher. In
Fig. 2, we show the expression
of the sma-3::gfp C-terminal construct. Expression begins late in
embryogenesis, and continues through larval stages into adulthood. In larvae,
expression is strong in the hypodermis, pharynx and intestine. sma-3
expression in the hypodermis is seen throughout the large hypodermal syncytium
hyp7, but not in the lateral hypodermal blast cells (the seam cells). Nuclear
accumulation in all of these tissues is strong. This nuclear localization does
not depend on the activity of sma-6, however (see
Fig. 6E,F).
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Expression of the N-terminal construct is similar, although much weaker,
even after integration (Fig.
3A). Again, the nuclear fluorescence is prominent in the pharynx,
intestine and hypodermis. We asked whether the nuclear accumulation depends on
the activity of other components in the pathway. When the integrated
N-terminal construct array (qcIs6) was crossed into
sma-4(e729) (Fig. 3C)
or sma-2(e502) (Fig.
3E) mutant backgrounds, the nuclear localization did not change
significantly. This result is consist with previous reports that R-Smad
nuclear translocation does not require complex formation with a co-Smad
partner (Liu et al., 1997).
When the array is crossed into sma-6(wk7) mutants, the protein became
evenly distributed between the cytoplasm and the nucleus in many but not all
animals (Fig. 3G). Thus, the
nuclear accumulation of SMA-3::GFP is enhanced by but not dependent on
activation by the type I receptor. Determining whether this extensive nuclear
localization is characteristic of the endogenous SMA-3 protein must await the
development of SMA-3 antibodies.
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Mosaic analysis of sma-3
As SMA-3 expresses in the pharynx, intestine and hypodermis, we wished to
determine the tissue in which its activity is crucial for regulation of body
size. We first addressed this by mosaic analysis. The sma-3 rescuing
genomic fragment was injected with a ubiquitously expressed nuclear localized
SUR-5::GFP marker (Yochem et al.,
1998) into sma-3(wk30) mutants. Animals inheriting the
array are wild-type in length, while those without the array are small.
Animals were screened for rare loss of the extrachromosomal array in somatic
tissues (Table 4). Each tissue
was scored as positive if any cells of the tissue expressed the construct.
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We find that animals with loss of the array in the pharynx or the intestine have wild-type body size (Table 4). Rare small worms have also been isolated with expression in the intestine and/or the pharynx. Therefore, expression of SMA-3 in the intestine and the pharynx is neither necessary nor sufficient for regulation of body size. However, expression of sma-3 in the hypodermis appears to be vital for restoration of wild-type body size. We have not observed a mosaic worm of wild-type length without sma-3 expression in the hypodermis. Conversely, we have not seen a small worm with hypodermal expression.
Effects of tissue-specific expression of sma-3 on body
length
To confirm the conclusions of the mosaic analysis and apply a quantitative
measure of body length, we created transgenic animals with sma-3
expression driven by heterologous promoters. Constructs were made with
tissue-specific promoters and the sma-3::gfp N-terminal fusion. Four
types of expression patterns were used. For hypodermal expression, we used
elt-3::sma-3 (Gilleard et al.,
1999), vha-7::sma-3
(Oka et al., 2001
) and
dpy-7::sma-3 (Gilleard et al.,
1997
). These hypodermal promoters function in hyp7 but not in the
seam cells. For pharyngeal expression, we chose myo-2::sma-3
(Okkema et al., 1993
). For
intestinal expression, we used elt-2::sma-3
(Fukushige et al., 1998
) and
vha-6::sma-3 (Oka et al.,
2001
). Finally, for simultaneous expression in the pharynx and
intestine, we used tmy-1 isoform III
(Anyanful et al., 2001
) as well
as a combined injection of myo-2::sma-3 and vha-6::sma-3. We
confirmed the expression of SMA-3::GFP in the specified tissues by direct
fluorescence (Fig. 4).
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The hypodermal expression of sma-3 from any of the three hypodermal promoters restores body length to the same extent as the sma-3 native promoter (Table 2). When sma-3 is only expressed in intestine, there is no effect on the body length. The pharyngeal expression or the co-expression in pharynx and intestine do not increase the body length significantly. Therefore, sma-3 expression in the hypodermis is sufficient for the regulation of body length.
SMA-3 protein accumulation
We have found that the nonfunctional sma-3::gfp C-terminal
construct gives a stronger fluorescent signal than the functional N-terminal
construct (Fig. 5A-D). This
difference in intensity was consistent in at least three independent lines for
each construct (data not shown). We also tried lower (10 µg/ml) or higher
(40 µg/ml) concentrations of the construct in injection mixtures, but the
intensity of fluorescence was not different from that with 20 µg/ml (data
not shown). The reduced fluorescence of the sma-3::gfp(N) construct
could be due to changes in protein folding that affect GFP fluorescence or to
reduced protein accumulation.
|
We hypothesized that the difference in fluorescence is due to differing levels of accumulation of the sma-3::gfp(N) and sma-3::gfp(C) fusion proteins. There are two possible causes of such differences in accumulation. First, the C-terminal construct could be inherently more stable. Second, SMA-3 degradation could be induced by sma-3 activity in a negative-feedback loop. To solve this puzzle, we simply mixed the two sma-3::gfp constructs together (10 µg/ml each) and microinjected into sma-3(wk30) mutants. This mixture can rescue the sma-3 mutant, in both body length and male tail patterning (Tables 2 and 3). In other words, the mixture provides sma-3 activity. However, the level of fluorescence is low, although the C-terminal construct is present (Fig. 5E,F). This implies that after adding the functional sma-3 construct, the C-terminal fusion protein could be degraded by an unknown factor. This result also contradicts the model that the difference in fluorescence is due to differences in protein folding that affect GFP fluorescence.
We further tested this model by co-injecting the nonfunctional sma-3::gfp(C) (10 µg/ml) construct either with the functional sma-3 genomic fragment (pCS29) (10 µg/ml) or with a sma-3 construct in which the coding region had been deleted (pCS210) (10 µg/ml) that would not have SMA-3 activity. In the co-injection with sma-3 genomic sequences, only a trace amount of GFP fluorescence can be seen (Fig. 6A,B). However, in the co-injection with pCS210, the level of fluorescence remains high (Fig. 6G,H). Thus, under a variety of conditions, sma-3::gfp fluorescence levels negatively correlate with SMA-3 activity levels. Finally, we addressed the question of whether a negative-feedback loop requiring other components of the pathway regulates SMA-3 protein accumulation. In fact, an extrachromosomal array carrying sma-3::gfp(C) and the sma-3 genomic fragment shows increased levels of fluorescence in a sma-4 or sma-6 mutant background, suggesting that the feedback is dependent on an intact signaling pathway (Fig. 6C-F).
We verified these results in a western blot using anti-GFP antibody. The extrachromosomal arrays were integrated: qcIs12 contains sma-3::gfp(C) and sma-3 genomic, qcIs16 contains sma-3::gfp(C) and sma-3 non-coding genomic. qcIs12 was crossed into the sma-4(e729) and sma-6(wk7) mutant backgrounds to determine whether loss of SMA-3::GFP(C) protein accumulation requires active Co-Smad and type I receptor, respectively. Consistent with the results from fluorescence in whole worms, SMA-3::GFP from qcIs12 in the N2 background is almost undetectable (lane 1). By contrast, a strong band is detectable in the qcIs16 strain (lane 4). In the sma-4 (lane 2) and sma-6 (lane 3) backgrounds, SMA-3::GFP levels from qcIs12 increase relative to the levels in a wild-type background. These results are consistent with a negative-feedback loop regulating SMA-3 protein accumulation.
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DISCUSSION |
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We have previously shown that the Sma/Mab pathway functions
postembryonically (Savage-Dunn et al.,
2000), in contrast to the embryonic requirement for sma-1
(McKeown et al., 1998
). In
this study, we have addressed the tissue specificity of the Sma/Mab pathway in
body size regulation and find that the hypodermis is the crucial
TGFß-responsive tissue involved in body size regulation. The hypodermis
of C. elegans forms the outer layer of cells surrounding the animal,
and it secretes the cuticle (Johnstone,
2000
). The largest region of the hypodermis is made up of a single
multinucleate syncytium, hyp7 (Sulston and
Horvitz, 1977
). Two lateral rows of hypodermal blast cells, the
seam cells, divide during each larval stage to form one daughter cell that
fuses with hyp7 and one that remains in the seam. After fusion with hyp7,
these nuclei undergo endoreduplication
(Hedgecock and White, 1985
;
Flemming et al., 2000
).
Additional smaller hypodermal cells are present in the head (hyp1-hyp6) and
the tail (hyp8-hyp12).
We conclude that Sma/Mab signal transduction functions in the hypodermis to
regulate body size based on (1) the SMA-3 expression pattern; (2) cell and
organ size measurements; (3) sma-3 mosaic analysis; and (4) directed
expression of sma-3. We have examined SMA-3 Smad expression and
subcellular localization using SMA-3::GFP fusion constructs. SMA-3 is
expressed in the pharynx, intestine and hypodermis. This expression pattern
coincides with the expression pattern of the type I receptor SMA-6
(Krishna et al., 1999;
Yoshida et al., 2001
). In cell
size measurements, we find that the seam cells in Sma/Mab mutants, but not the
pharynx, show a reduction in size similar to the reduction in the body length.
The relative maintenance of pharynx length in Sma/Mab mutants suggests that
the expression of Sma/Mab signaling components in the pharynx may serve an
unidentified role. We used mosaic analysis and tissue-specific expression of
sma-3 to determine in which tissues it is required for body size. Our
results indicate that SMA-3 function in the hypodermis is necessary and
sufficient for body size regulation. The hypodermal requirement for SMA-3 is
consistent with the results from similar experiments on the receptors
sma-6 (Yoshida et al.,
2001
) and daf-4 (Inoue et al., 2000).
Although the seam cells are reduced in size in Sma/Mab mutants, the effect
on the seam cells may not be cell autonomous, as neither sma-3 (this
study) nor sma-6 (Yoshida et al.,
2001) expression is detected in these cells. Furthermore, we have
shown that sma-3 expression from hyp7-specific promoters, which also
do not express in the seam cells, is sufficient to rescue the body size in a
sma-3 mutant (Table
2). Because the seam cells and hyp7 are joined by gap junctions
(D. Hall, personal communication) and adherens junctions
(Mohler et al., 1998
), there
could be communication between these tissues without postulating additional
extracellular signals. We speculate that in addition to the seam cells, the
large hypodermal syncytium hyp7 may be reduced in volume in the small mutants.
This hypothesis could be tested by electron microscopy. As dbl-1 is
expressed primarily in the nervous system
(Suzuki et al., 1999
), these
results suggest a model in which postembryonic growth of hypodermal cells is
regulated by TGFß-related signaling from the nervous system to the
hypodermis.
One important question that remains to be addressed is why these cells are
smaller. Several possibilities may be considered. One is that reduced DNA
content leads to smaller size. The nuclei of the C. elegans intestine
and hypodermis normally undergo endoreduplication during larval growth
(Hedgecock and White, 1985).
In late adulthood the hypodermal nuclei in wild type have an average ploidy of
10.7C, and some nuclei have gone through two rounds of endoreduplication
(Flemming et al., 2000
).
Furthermore, Flemming et al., found reduced ploidy in hypodermal cells of
daf-4 and sma-2 mutants, 5.8 and 7.0 respectively. They
proposed that this may be the reason for the smaller body size of Sma/Mab
mutants, as ploidy can control cell size
(Galitski et al., 1999
). It is
not yet clear, however, whether changes in ploidy are sufficient to explain
the changes in body size.
Another mechanism that could contribute to smaller cell size is reduced
protein synthesis. To test this hypothesis, we tried growing worms on the
protein synthesis inhibitor cycloheximide to see if we could phenocopy the
small defect. At concentrations in which the worms could grow, however, they
were normal in size. A third possibility is a change in the cell cycle. By
speeding up the cell cycle, small cells could be generated such as is seen in
yeast wee mutants (Futcher,
1996). Because the small mutants do not develop more quickly,
however, any cell cycle defect could only be in some stage(s) of the cell
cycle. Finally, metabolic changes could decrease cell size. In
Drosophila (but not in C. elegans) mutations in insulin
signaling result in small cells and small animals
(Oldham et al., 2000
).
Similarly, some change in sugar or fat metabolism could underlie the small
phenotype.
SMA-3 protein accumulation may be regulated by a feedback loop
Using different SMA-3::GFP fusion constructs, we have obtained evidence
that SMA-3 protein accumulation is negatively regulated by the level of SMA-3
and Sma/Mab pathway activity. Furthermore, a negative-feedback loop is
consistent with the lack of effect of overexpressing sma-3. The
overexpression of dbl-1 ligand induces a long phenotype and male tail
sensory ray defects (Suzuki et al.,
1999), while the overexpression of sma-3 does not (data
not shown). In other systems, it has been shown that Smads are degraded by the
activity of Smurf E3 ubiquitin ligases
(Zhu et al., 1999
;
Zhang et al., 2001
;
Podos et al., 2001
). In human
or Xenopus, Smurf-1 and Smurf-2 induce R-Smad degradation by the
ubiquitin pathway (Zhu et al.,
1999
; Zhang et al.,
2001
). Through interaction with the anti-Smad Smad7, Smurf-1 can
also induce the degradation of type I receptor
(Ebisawa et al., 2001
). Smurf-2
enhances the degradation of type I receptor
(Kavsak et al., 2000
) or SnoN
oncogene (Bonni et al., 2001
).
The target is selected by the Smad with which it interacts. A Smurf gene,
Dsmurf (lack FlyBase) is found in
Drosophila, where it negatively regulates dpp signaling in
embryonic dorsoventral patterning (Podos
et al., 2001
). In C. elegans, we find several open
reading frames with homology to human or Xenopus Smurf genes,
allowing the possibility that one or more of these genes functions in the
Sma/Mab pathway. It will be interesting to determine whether a Smurf gene
participates in a negative feedback loop.
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ACKNOWLEDGMENTS |
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