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First published online 21 June 2006
doi: 10.1242/dev.02476
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1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY
14853, USA.
2 Department of Embryology, Carnegie Institution of Washington, 3520 San Martin
Drive, Baltimore, MD 21218, USA.
3 Departments of Pathology and Genetics, Stanford University School of Medicine,
300 Pasteur Drive-L235, Stanford, CA 94305, USA.
* Author for correspondence (e-mail: jl53{at}cornell.edu)
Accepted 31 May 2006
 |
SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mesoderm, Dorsoventral patterning, M lineage, TGFß, SMA-9, Schnurri, Sma/Mab, dbl-1, sma-2, sma-3, sma-4, sma-6, daf-4, C. elegans
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
The TGFß signaling pathway regulates many cellular and developmental
processes (Feng and Derynck,
2005; Massague et al.,
2000; Shi and Massague,
2003). The core components of the TGFß signaling pathway have
been well studied; these include type I and type II transmembrane receptors
and intracellular Smad proteins that transduce the TGFß signal from the
cell membrane to the nucleus (Feng and
Derynck, 2005; Shi and
Massague, 2003). Despite a general understanding of the underlying
signal transduction pathways, our understanding of the mechanisms involved in
regulating the specific signaling output in different cell types is far from
complete.
There are two canonical TGFß-related signaling pathways in C.
elegans: the Sma/Mab pathway that regulates body size and male tail
patterning; and the dauer pathway that controls formation of dauer larvae
(Patterson and Padgett, 2000;
Savage-Dunn, 2001). In
addition, a TGFß-related molecule, UNC-129, is required to guide pioneer
motor axons along the dorsoventral axis
(Colavita et al., 1998).
Studies in both Drosophila and vertebrates have shown that the
TGFß signaling pathway plays crucial roles in regulating dorsoventral
patterning (De Robertis and Kuroda,
2004). Despite the role of UNC-129 in guiding the dorsal migration
of motor axons, a role of the canonical TGFß signaling pathways in
dorsoventral patterning is unclear in C. elegans. DBL-1, the ligand
of the Sma/Mab pathway, has been proposed to specify dorsal cell fates in male
sensory ray patterning (Suzuki et al.,
1999). However, the fate transformations observed in
dbl-1 mutants (rays 5, 7 and 9 to rays 4, 6 and 8, respectively) can
equally be interpreted as a posterior to anterior transformation.
The C. elegans post-embryonic mesodermal lineage, the M lineage,
provides an excellent system with which to study dorsoventral patterning and
cell fate specification at single cell resolution. The M lineage arises from a
single precursor cell, the M mesoblast, which is born during embryogenesis
(Sulston and Horvitz, 1977).
During larval development, the M mesoblast first divides along the
dorsoventral axis to generate two daughter cells: M.d and M.v. These two cells
subsequently give rise to distinct dorsal and ventral cell types. The dorsal
cell, M.d, gives rise to six body wall muscles (BWMs) and two non-muscle
coelomocytes (CCs), whereas the ventral cell, M.v, gives rise to eight BWMs
and two sex myoblasts (SMs). The SMs subsequently migrate from the ventral
posterior to the presumptive vulval region where they each undergo three
rounds of cell division and differentiate into vulval and uterine muscles
(Fig. 1E).
|
). We have found that none of the TGFß pathway mutants
exhibit any M lineage defects; however, mutations in any of the core
components of the Sma/Mab TGFß signaling pathway suppressed the M lineage
defects of sma-9 mutants. These findings uncovered a role of the
Sma/Mab TGFß signaling pathway in patterning the M lineage along the
dorsoventral axis. Studies in Drosophila have shown that Shn is a
transcriptional repressor that forms a complex with the Smad proteins Mad and
Medea to repress transcription of genes such as brinker
(Marty et al., 2000;
Pyrowolakis et al., 2004). Our
data suggest that SMA-9 may act independently of complex formation with the
Smad proteins to antagonize their function, adding another potential mode of
action for the Shn family of proteins. |
MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Analyses were performed at 20°C, unless otherwise
noted. The following strains were used in this work.
LG I: rnt-1(e1241) (Link et
al., 1988), rnt-1(ok351)
(Lee et al., 2004).
LG II: sma-6(e1482) (Brenner,
1974), sma-6(wk7)
(Krishna et al., 1999).
LG III: lin-6(e1466) dpy-5(e61)/hT2[qIs48]
(Wang and Kimble, 2001),
cup-5(ar465) (Fares and
Greenwald, 2001), sma-2(e502) unc-32(e189)
(Manser and Wood, 1990),
sma-3(e491) unc-32(e189), sma-4(e729) and lon-1(e185)
(Brenner, 1974),
sma-3(wk28) (Savage-Dunn et al.,
2000), daf-4(e1364) unc-32(e189) and daf-4(m63)
(Riddle, 1977).
LG IV: daf-1(m213) and daf-1(m40)
(Riddle, 1977),
unc-129(ev554), unc-129(ev557) and unc-129(ev566)
(Colavita et al., 1998),
daf-14(m77) (Riddle et al.,
1981).
LG V: him-5(e1467) (Hodgkin et
al., 1979), dbl-1(wk70)
(Suzuki et al., 1999),
dbl-1(nk3) (Morita et al.,
1999).
LG X: dpy-6(e14) unc-9(e101), lon-2(e678)
(Brenner, 1974),
daf-3(e1376) and daf-3(mgDf90)
(Patterson et al., 1997),
sma-9 (wk55), sma-9(wk62), sma-9(wk71), sma-9(wk82), sma-9(qc1),
sma-9(qc3), sma-9(qc5), sma-9(qc6), sma-9(qc7), sma-9(qc8), sma-9(qc9),
sma-9(qc10) and sma-9(qc11)
(Liang et al., 2003),
sma-9(tm572) (gift from Shohei Mitani, Tokyo Women's Medical
University School of Medicine, Japan).
Integrated transgenic lines
LW0081: ccIs4438(intrinsic CC::gfp) III;
ayIs2(egl-15::gfp) IV; ayIs6(hlh-8p::gfp) X
(Jiang et al., 2005).
Intrinsic CC::gfp is a twist-derived coelomocyte marker
(Harfe et al., 1998b).
BW1935: unc-119(ed3) III; ctIs43[pDP#MM016B, pNY+nls, pMY-nls
(dbl-1::GFP)] him-5(e1490) V. (Suzuki
et al., 1999).
Additional cell-type-specific reporters for the M lineage were as described
in Kostas and Fire (Kostas and Fire,
2002).
Strains generated in this work
The lon-2(e678) sma-9(cc604) unc-9(e101) triple mutant was
generated from a lon-2(e678) egl-15(n484)/sma-9(cc604)
unc-9(e101) heterozygote by identifying Lon-non-Egl recombinants. Most of
the Lon-non-Egl recombinants segregated lon-2(e678) sma-9(cc604)
unc-9(e101) triple mutant animals. We then confirmed the presence of both
the lon-2(e678) mutation (deletion) and the sma-9(cc604)
mutation in the strain by PCR and sequencing. All other strains generated in
this work (see Table 4) were
generated through standard genetic crosses using appropriate balancers to
follow individual chromosomes.
|
). Two
recessive mutations, cc606 and cc604, lacked M-derived
coelomocytes. Both mapped to the X chromosome and failed to complement each
other. cc604 was further mapped between dpy-6 and
unc-9, and failed to complement two sma-9 alleles,
sma-9(wk55) and sma-9(wk62)
(Liang et al., 2003). The
molecular lesions of cc606 and cc604 were identified through
direct sequencing of PCR fragments encompassing the entire sma-9
genomic region. Detailed information on the primers used is available upon
request.
Isolation and genetic analysis of sma-9 suppressor mutations jj1 and jj3
arIs37 [secreted CC::gfp (gfp with a signal peptide driven by the myo-3
promoter taken up by the coelomocytes)] I; cup-5(ar465) III;
sma-9(cc604) X animals lacking M-derived coelomocytes were
mutagenized by EMS. Individual F1 animals and their clonal F2 progeny were
screened for the restoration of M lineage-derived coelomocytes by direct
visual examination using a fluorescence stereomicroscope. Approximately 2800
haploid genomes were examined. Seven recessive suppressor mutations
(jj1-jj7) were obtained. jj1 and jj3 were mapped
via snip-SNP mapping (Wicks et al.,
2001). The molecular lesions of jj1 and jj3 were
identified via direct sequencing of PCR fragments encompassing the entire
sma-6 and sma-3 genomic regions respectively. Detailed
information on the primers used is available upon request.
Plasmid constructs and transgenic lines
sma-9 reporter constructs
We generated three genomic-cDNA hybrid constructs, one for each C-terminal
isoform of sma-9 as described in Liang et al.
(Liang et al., 2003). These
constructs were referred to as C1 (Class I), C2 (Class II) and C3 (Class III).
C1 was generated by ligating the cDNA fragment from yk128a08 (Class
I) to a genomic fragment containing sequences from the predicted ATG to exon
20. C2 and C3 were generated by ligating a genomic fragment containing
sequences from the predicted ATG to exon 10 to cDNA fragments from
yk1103h10 (Class II) and yk328c9 (Class III), respectively.
A 3763 bp fragment immediately upstream of the predicted ATG was used as the
sma-9 promoter. To generate the GFP tagged constructs, GFP was added
to the C-terminal end of the sma-9-coding region. All genomic
fragments were generated through long-range PCR (Expand Long Template PCR
system, Roche) using cosmid T01A5 as template. The following constructs were
made.
pMX15: 3.7kb sma-9p::sma-9C1::unc-54 3'UTR
pMX16: 3.7kb sma-9p::sma-9C2::unc-54 3'UTR
pMX9: 3.7kb sma-9p::sma-9C1::gfp::unc-54 3'UTR
pMX10: 3.7kb sma-9p::sma-9C2::gfp::unc-54 3'UTR
pMX11: 3.7kb sma-9p::sma-9C3::gfp::unc-54 3'UTR
Forced expression constructs
pMLF28: hlh-8p::sma-9C1::unc-54 3'UTR
pMLF30: hlh-8p::sma-9C2::unc-54 3'UTR
pMLF27: hsp16p::sma-9C1::unc-54 3'UTR
pMLF29: hsp16p::sma-9C2::unc-54 3'UTR
pMLF31: hlh-8p::sma-3::unc-54 3'UTR
pMLF33: hlh-8p::sma9C1::gfp::unc-54 3'UTR
Detailed information on all these constructs is available upon request.
Transgenic lines were generated as described by Mello and Fire
(Mello and Fire, 1995).
Heat-shock experiments
Heat shock was applied to transgenic lines that carried the
hsp-16p::sma-9C1::unc-54 3'UTR construct (pMLF27) or
the hsp-16p::sma-9C2::unc-54 3'UTR construct (pMLF29)
in the sma-9(cc604) background. Stage-synchronized animals were
collected at each of the following stages based on hlh-8::gfp
expression: embryos, larvae at the 1-M, 2-M, 4-M, 6-M, 8-M, 10- to 16-M and
2-SM stages. L1 larvae usually stay at the 1-M stage for about 8 hours, with
subsequent cell divisions each taking about 2 hours leading to the 16-M stage
(Sulston and Horvitz, 1977).
We divided animals at the 1-M stage into different groups: 1-2 hours, 2-4
hours and 5-7 hours post-hatching. Animals of the same stage were transferred
to the same plate for heat-shock treatment (37°C for 1 hour). Animals were
examined post heat-shock to ensure that they were still at the indicated
stage. They were allowed to recover at 20°C. Their terminal M lineage
phenotypes were examined at the young adult stage. Non-heat-shocked and
non-transgenic but heat-shocked animals were used as controls.
RNAi
The following plasmids were used to generate dsRNAs used in the RNAi
experiments. All yk clones are gifts from Yuji Kohara.
sma-9: yk127d10 and pJKL619, which has the N terminus of sma-9 from yk1185a11 cloned into pBS II SK+.
sma-2: pJKL716, which contains exons 6-7 of sma-2 cloned into pBS II SK+.
sma-4: pMLF35, which contains exons 7-10 of sma-4 cloned into pBS II SK+.
dsRNA was injected into LW0081 or sma-9 mutant animals following
the protocol of Fire and colleagues (Fire
et al., 1998). Progeny of injected animals were scored for defects
in the M lineage.
Antibodies, immunostaining and microscopy
Animals were fixed following the protocol in Hurd and Kemphues
(Hurd and Kemphues, 2003).
Goat anti-GFP antibodies (Rockland Immunochemicals) and rat anti-MLS-2
antibodies (Jiang et al.,
2005) were both used in 1:500 dilution. Anti-SMA-9 antibodies
(CUMC-R2 TB2, recognizing the C2 isoform)
(Liang et al., 2003) were used
in 1:2000 dilution. All secondary antibodies were from Jackson Immunoresearch
Laboratories and used in 1:50 to 1:200 dilution. Differential interference
contrast and epifluorescence microscopy were performed using a Leica DMRA2
compound microscope. Images were captured by a Hamamatsu Orca-ER camera using
the Openlab software (version 4.0.1, Improvision). Subsequent image analysis
was performed using Adobe Photoshop 7.0.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Six lines of evidence suggest that these alleles are
loss or reduction of function mutations in the sma-9 locus. First,
all 13 previously described sma-9 alleles lacked M lineage-derived
CCs (see Table 1 for
wk55, data not shown for qc1, qc3, qc5, qc6, qc7, qc8, qc9, qc10,
qc11, wk62, wk71 and wk82). Two additional sma-9
alleles isolated from our ongoing screen for mutants affecting the M lineage,
jj25 and jj29, also lacked M lineage-derived CCs (data not
shown). Second, both cc606 and cc604 failed to complement
the M lineage and body size defects of two sma-9 alleles,
wk55 and wk62. Third, both cc606 and cc604
contained molecular lesions in the sma-9-coding region
(Fig. 2). cc606 and
cc604 each result in a change of a Gln codon to stop, at positions 78
and 1676, respectively [numbers based on the system of Liang et al.
(Liang et al., 2003)]. Fourth,
a sma-9-containing cosmid, T05A10, rescued the M lineage defects of
both cc606 and cc604 mutants
(Fig. 2B; data not shown).
Fifth, sma-9(RNAi) resulted in the same M lineage defects as those of
the sma-9 mutant alleles (Table
1). Sixth, affinity-purified anti-SMA-9 antibodies (CUMC-R2 TB2)
(Liang et al., 2003) failed to
detect any signal in sma-9(cc604) mutant animals (data not shown).
Together, these results provide strong evidence that cc606 and
cc604 are mutant alleles of sma-9 and that cc604
behaves as a strong reduction or loss-of-function allele. We therefore chose
cc604 for all of our analysis described below.
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), we
observed no defects in the division patterns up to the 16-M stage (data not
shown). At the 16-M stage, however, the two CC precursors on the dorsal side
of the animal (M.dlpa and M.drpa) behaved like their ventral counterparts
(M.vlpa and M.vrpa). Both cells (M.dlpa and M.drpa) underwent another round of
division to produce two body wall muscles (BWMs) and two sex myoblasts (SMs).
This resulted in the loss of two M-derived CCs and the production of two extra
SMs and two extra BWMs on the dorsal side of cc604 mutants
(Fig. 1B,F). The two SMs on the
dorsal side behaved as bona fide SMs. They migrated as normal SMs to the
future vulval region (albeit on the dorsal side most of the time), divided
three times to produce 16 sex muscle precursors, which then differentiated
into sixteen uterine and vulval muscles (um and vm)
(Fig. 1D,F; data not shown).
The differentiated cell types generated from the M lineage in cc604
mutants were confirmed using DIC optics in combination with cell type-specific
markers, including myo-3::gfp (labeling BWMs)
(Fire et al., 1998) (data not
shown), a twist-derived CC::gfp (labeling coelomocytes)
(Harfe et al., 1998b)
(Fig. 1A-D),
egl-15::gfp (labeling type I vulval muscles)
(Harfe et al., 1998a)
(Fig. 1C,D),
arg-1::gfp (labeling both type I and type II vulval muscles)
(Kostas and Fire, 2002) (data
not shown) and rgs-2::gfp (preferentially labeling uterine muscles)
(Dong et al., 2000) (data not
shown). Thus, sma-9 mutants exhibit a loss of dorsal, and a
duplication of ventral, M-derived cell fates. Outside of the M lineage, sma-9(cc604) mutants did not show defects in other mesodermally derived cells, including the number and pattern of embryonically derived BWMs, the enteric muscles and the head mesodermal cell, although a low penetrance (<10%) of distal tip cell migration defects (extra turns) was observed (data not shown).
A region containing a cluster of three zinc fingers in SMA-9 is critical for its function in regulating M lineage patterning and body size
sma-9 encodes a predicted large C2H2
zinc-finger protein with complex splicing isoforms that differ at both the N
and C termini (Liang et al.,
2003). As SMA-9 functions in the M lineage and in regulating body
size and male tail patterning, we asked whether some SMA-9 isoforms are
specifically required for proper M lineage development.
sma-9 is a complex locus that generates a variety of different
cDNA species, with alternative splicing contributing to potential
heterogeneity at both the N- and C-terminal regions of the protein
(Liang et al., 2003). No
full-length cDNA clones that encompass the entire predicted sma-9
locus have been isolated. Instead, there are cDNA clones that represent two
major N-terminal and three major C-terminal isoforms
(Liang et al., 2003). When we
generated artificial full-length cDNA constructs by piecing together different
combinations of the N- and C-terminal cDNA clones, none of these constructs
could rescue either the M lineage or body size defects of sma-9
mutants. A set of constructs was then made that would maintain what could be
an important physiological diversity at the N terminus, while constraining the
C-terminal structure (where the Zn fingers reside) to one of several isoforms.
These genomic-cDNA hybrid constructs contained genomic sequences from the N
terminus and cDNA sequences for each of the three different sma-9
C-terminal isoforms, which we termed C1, C2 and C3
(Fig. 2A, Materials and
methods). C1 is predicted to contain all seven zinc-finger (ZF1-7) domains. C2
contains a unique 70 amino acid domain instead of ZF6 and ZF7 at its
C-terminal end. C3 is the shortest isoform and lacks ZF3-7. For each of the
three constructs (C1, C2 and C3), we placed a GFP tag at the C terminus and
used the predicted sma-9 promoter to drive its expression
(Fig. 2A, Materials and
methods).
In the wild-type background, all three constructs showed similar GFP
expression pattern and subcellular localization: GFP was localized in nuclei
of a wide variety of cell types (Fig.
3A), similar to what we have previously reported using C2
isoform-specific anti-SMA-9 antibodies
(Liang et al., 2003). Both the
Cl and C2 constructs, but not the C3 construct, rescued the M lineage and body
size defects of sma-9(cc604) mutants
(Fig. 2B). The rescue
efficiency for both C1 and C2 was even better than that for the
sma-9-containing cosmid T01A5
(Fig. 2B). These results
suggest that the structures unique to the C1 and C2 isoforms, including ZF6-7
(for C1) and the 70 amino acid domain (for C2), are not crucial for SMA-9
function (Fig. 2B). Instead,
the structures missing in the C3 isoform but present in both C1 and C2,
including the ZF3-5 zinc-finger cluster, are apparently required for proper
SMA-9 function in regulating both body size and M lineage patterning.
|
SMA-9 functions in the M lineage for its proper patterning
We used the rescuing constructs described above to examine functional
requirements for SMA-9 in the M lineage. To first obtain a detailed expression
pattern, we double stained transgenic animals carrying the C1::GFP construct
with antibodies against GFP and antibodies against an M-lineage specific
marker MLS-2 (Jiang et al.,
2005). We detected GFP signals in the M lineage at both the 1-M
and 2-M stages (Fig. 3B-D) and
up to the 8-M stage (data not shown). Notably, both within and outside of the
M lineage, the dorsal and ventral cells showed similar distribution and
intensity of the SMA-9::GFP signals.
To test if SMA-9 functions within the M lineage to regulate its proper patterning, we replaced the sma-9 promoter with the M lineage-specific hlh-8 promoter in the C1 and C2 rescuing constructs. We confirmed that these constructs are expressed only in the M lineage by examining the expression pattern of GFP in transgenic animals carrying the hlh-8p::C1::GFP construct (data not shown). As shown in Fig. 2B, both constructs specifically rescued the M lineage defects without rescuing the body size defects of sma-9(cc604) mutants. Although these M lineage-specific rescue results cannot exclude the possibility that SMA-9 activity outside of the M lineage could contribute to patterning of the M lineage, they certainly illustrate the ability of SMA-9 produced within the M lineage to establish proper patterning.
SMA-9 is sufficient to act after the first cell division of the M lineage for proper cell-fate specification
To determine the crucial time period when SMA-9 function is required for
the proper development of the M lineage, we generated transgenic lines of
sma-9(cc604) animals carrying either C1 or C2 SMA-9 constructs driven
by a heat-shock inducible promoter. These animals were heat-shocked at
distinct stages during larval development (1-M, 2-M etc.
Fig. 1E) and assayed for rescue
of the M lineage defects of cc604 mutants (see Materials and methods
for heat-shock conditions).
As shown in Table 2, heat-shocking between the 1-M and 6-M stage resulted in rescue of the M lineage defects of cc604 mutants. The most efficient rescue was observed when animals were heat-shocked between the 2-M (C1: 93%, n=27; C2: 80%, n=5) and 4-M (C1: 100%, n=6; C2: 88%, n=8) stages. No rescue was observed when animals were heat-shocked at or after the 8-M stage (Table 2). Both the C1 and C2 SMA-9 isoforms behaved similarly, although the rescuing efficiency for C1 appeared higher than that of C2 (Table 2). As rescue was observed even when SMA-9 expression was induced after the 4-M stage (at the 6-M stage, C1: 43%, n=7; C2: 17%, n=6), we concluded that SMA-9 is sufficient to act after the first cell division of the M lineage for its proper cell-fate specification. The lack of rescue in animals heat-shocked at or after the 8-M stage suggests that SMA-9 function is most probably required prior to the 8-M stage for proper M lineage development.
|
). To test whether SMA-9 functions in a similar manner in the
M lineage, we examined the M lineage phenotypes in mutants of known components
of the TGFß signaling pathway in C. elegans. As shown in
Table 3, none of the mutants
examined showed any M lineage defects, suggesting that SMA-9 might function
through different mechanisms in the M lineage than its function in regulating
body size and male tail patterning.
|
). jj1 maps to chromosome II between -6.25 mu and
+1.85 mu; jj3 maps to chromosome III between -1.45 mu and +5.38 mu.
Subsequent out-crossing of jj1 and jj3 from the
sma-9(cc604) mutation revealed that jj1 and jj3
homozygous animals had a wild-type M lineage but a small body size.
Complementation assays between jj1 and a sma mutation on
chromosome II [sma-6(e1482)], and between jj3 and three
sma mutations on chromosome III [sma-2(e502), sma-3(e491)
and sma-4(e729)] showed that jj1 failed to complement
sma-6(e1482) and jj3 failed to complement
sma-3(e491). We further identified the molecular lesions in
jj1 and jj3. jj1 contains a G to A change in sma-6,
resulting in a Trp (TGG) to stop (TAG) mutation at amino acid 328. This
mutation is predicted to result in a truncated SMA-6 protein lacking the
intracellular kinase domain and is probably a null allele of sma-6.
jj3 contains a T to A substitution at the beginning of intron 9 in
sma-3, which is likely to cause aberrant splicing, and results in a
truncation of the highly conserved MH2 domain. Thus, jj3 represents a
likely null allele of sma-3.
|
;
Krishna et al., 1999;
Savage-Dunn et al., 2000). We
also generated double mutants between sma-3(jj3) and two other
alleles of sma-9: sma-9(wk55) and sma-9(cc606). As shown in
Table 4, all these double
mutants exhibited a wild-type M lineage while retaining a small body size,
suggesting that the genetic interactions between sma-3 and
sma-9 and between sma-6 and sma-9 are not allele
specific.
SMA-9 specifically antagonizes the Sma/Mab TGFß signaling pathway in regulating dorsal M-derived cell fates
sma-3 and sma-6 both encode components of the Sma/Mab
TGFß signaling pathway. SMA-3 is one of the regulatory Smad proteins and
SMA-6 is the type I receptor (Savage-Dunn,
2001). The genetic interaction between sma-9 and these
two genes suggested that SMA-9 may participate in regulating the output of the
Sma/Mab TGFß signaling pathway in the M lineage. To test this hypothesis,
we generated double mutants between sma-9(cc604) and available
mutations in other core members of the Sma/Mab TGFß signaling pathway.
These include the Sma/Mab ligand DBL-1, the type II receptor DAF-4, the other
regulatory Smad (SMA-2) and the co-Smad, SMA-4
(Estevez et al., 1993;
Morita et al., 1999;
Savage et al., 1996;
Savage-Dunn, 2001;
Suzuki et al., 1999). None of
the genes encoding these components when mutated display any M lineage defects
on their own (Table 3).
However, mutations in any of them completely or nearly completely suppress the
M lineage defect of sma-9(cc604) mutants. All double mutants with
sma-9(cc604) were small, indicating that the small body size defect
of sma-9(cc604) mutants was not suppressed
(Table 4). In the case of
dbl-1, we observed a low level of suppression (14%) of
sma-9(cc604) M lineage defects in the dbl-1(wk70)/+
background (Table 4). This
suggests that simply lowering the level of DBL-1 is sufficient to rescue the M
lineage phenotypes of sma-9 mutants. Because mutations in the core
components of the Sma/Mab TGFß signaling pathway all suppressed the M
lineage defects of sma-9 mutants, we conclude that SMA-9 functions to
antagonize the core Sma/Mab TGFß signaling pathway for the correct
patterning of the M lineage.
The inability of the TGFß mutants to suppress the body size defects of
sma-9 mutants suggests that the mechanism of SMA-9 function in the M
lineage might be different from its function in regulating body size. We
further examined this by generating double mutants between
sma-9(cc604) and mutations in additional genes that appear to
function in regulating body size, including lon-1(e185), two alleles
of rnt-1,e1241 and ok351, and lon-2(e678)
(Ji et al., 2004;
Maduzia et al., 2002;
Morita et al., 2002;
Brenner, 1974). lon-1
and rnt-1 function downstream (Ji
et al., 2004; Maduzia et al.,
2002), while lon-2 is proposed to function upstream
(Brenner, 1974), of the core
Sma/Mab TGFß pathway. We found that neither of the two rnt-1
alleles suppresses the M lineage defects of sma-9, and that
lon-1(e185) suppresses the M lineage defects of sma-9 at a
very low penetrance (7%) (Table
4). However, lon-2(e678), a predicted null allele of
lon-2 (R. Padgett, personal communication), suppresses the M lineage
defects of sma-9 at a relatively high penetrance (88%)
(Table 4). We also noticed that
the lon-2(e678) sma-9(cc604) double mutant animals appeared wild type
with respect to body size (Table
4). Although we do not understand why the lon-2 mutation
would suppress the M lineage defects of sma-9 mutants, the M lineage
and the body size phenotypes of lon-2(e678) sma-9(cc604) mutants that
we observed suggest that SMA-9 and LON-2 are likely to function in parallel to
each other. Collectively, our data also indicate that not all genes involved
in body size regulation are involved in M lineage patterning.
To further determine if the interaction between sma-9 and the
Sma/Mab TGFß pathway is specific, we generated double mutants between
sma-9(cc604) and daf-1(m40). daf-1 encodes the type I
receptor unique to the TGFß pathway in controlling dauer development
(Georgi et al., 1990). We
found that daf-1(m40) does not suppress the M lineage defects of
sma-9(cc604) (Table
4). Similarly, egl-20(n585) and lin-17(n671),
mutations in the Wnt signaling pathway
(Maloof et al., 1999;
Sawa et al., 1996), do not
suppress the M lineage defects of sma-9(cc604) (data not shown).
These results indicate a specific role for SMA-9 in modulating the core
Sma/Mab TGFß pathway in M lineage patterning.
Antagonism between SMA-9 and the Sma/Mab TGFß signaling pathway occurs within the M lineage
To test whether the antagonism between SMA-9 and the TGFß pathway is
occurring within M lineage cells, we used the M lineage-specific
hlh-8 promoter to express sma-3 exclusively in the M lineage
of sma-3(jj3); sma-9(cc604) double mutants. If SMA-3 functions cell
autonomously in the M lineage, we would expect that introducing sma-3
specifically in the M lineage would eliminate the suppression phenotype and
restore the sma-9 mutant phenotype. We found that in four independent
lines, greater than 92% (n=279) of transgenic animals were missing
both M-derived CCs, indicating that expressing sma-3 exclusively in
the M lineage is sufficient to eliminate suppression
(Fig. 4C,
Table 4). These data suggest
that the interaction between the Sma/Mab TGFß pathway and SMA-9 occurs
cell-autonomously within the M lineage.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
SMA-9 has previously been shown to function in regulating body size and
male tail patterning (Liang et al.,
2003). Our finding adds another example to the multiple functions
of SMA-9 in C. elegans. SMA-9 is a homolog of the Drosophila
protein Schnurri (Shn) (Liang et al.,
2003). Like SMA-9, Shn has multiple functions during
Drosophila development, such as dorsoventral patterning of the early
embryo, proliferation and differentiation in the wing, and maintenance of
germline stem cells (Arora et al.,
1995; Grieder et al.,
1995; Shivdasani and Ingham,
2003; Staehling-Hampton et
al., 1995; Torres-Vazquez et
al., 2000; Xie and Spradling,
1998). Our data provides evidence for a conserved function of
SMA-9 and Shn in dorsoventral patterning. In both animals, SMA-9 and Shn are
required to specify dorsal cell fates. Vertebrates have multiple Shn/SMA-9
homologs, including Shn1, Shn2 and Shn3
(Fan and Maniatis, 1990;
Gascoigne, 2001;
Jin et al., 2006;
Lallemand et al., 2002;
Nakamura et al., 1990;
Oukka et al., 2002;
Seeler et al., 1994;
Takagi et al., 2001;
van 't Veer et al., 1992);
however, no reports have shown their functions in dorsoventral patterning. Our
results raise the possibility that one or more members of this protein family
also function in dorsoventral patterning in vertebrates.
sma-9 has multiple splicing isoforms
(Liang et al., 2003). We have
found that SMA-9 with either of two different C-terminal ends (C1 or C2) can
rescue the M lineage and body size defects of sma-9 mutants
(Fig. 2). These results suggest
that the structures unique to either C1 or C2 are not crucial, although each
alone could be sufficient, for SMA-9 function in regulating the M lineage and
body size. The lack of rescue by the C3 isoform suggests that the common
motifs shared by C1 and C2, including the three zinc fingers (ZF3-5), are
crucial for SMA-9 function. Understanding how these zinc fingers contribute to
SMA-9 function will help us better understand how SMA-9 regulates M lineage
patterning.
SMA-9 unexpectedly antagonizes the Sma/Mab TGFß signaling pathway in patterning and fate specification in the M lineage
Previous studies have shown that sma-9 mutants exhibit a small
body size and defects in male tail patterning and that these defects are
similar, but not identical, to those exhibited by mutations in the core
Sma/Mab TGFß signaling pathway (Liang
et al., 2003). Furthermore, sma-9 acts downstream of the
DBL-1 ligand in regulating both body size and male tail patterning
(Liang et al., 2003). These
studies clearly showed a contributory role for SMA-9 in the TGFß response
pathway. In this work, we uncovered a novel and unexpected role for the
Sma/Mab TGFß signaling pathway that appears to be antagonized by SMA-9.
We have shown that none of the core Sma/Mab TGFß signaling pathway
members have any M-lineage defects when mutated
(Table 3). However, mutations
in any of them suppress the M lineage defects of sma-9 without
suppressing its body size defect (Table
4). Furthermore, this suppression is specific to the Sma/Mab
TGFß signaling pathway and it is occurring specifically in the M lineage
(Table 4).
There are two possible scenarios for how SMA-9 might antagonize the TGFß signaling pathway in M lineage development. One possibility is that SMA-9 functions upstream of the ligand DBL-1 to reduce its expression or activity. However, we have shown that both SMA-9 and SMA-3 function within the M lineage for its correct patterning (Figs 3, 4). Thus, it is unlikely that SMA-9 functions upstream of DBL-1 in regulating M lineage patterning, unless DBL-1 functions in an autocrine manner. Supporting this notion, we have found that DBL-1 expression is not altered in sma-9(cc604) mutants (data not shown).
A second possibility is that SMA-9 acts to repress TGFß target gene
expression. Previous studies in Drosophila have shown that Shn
mediates BMP signaling by forming a repressor complex with the Smad proteins
Mad and Medea to repress gene expression
(Marty et al., 2000;
Muller et al., 2003;
Pyrowolakis et al., 2004).
Recently, it has been shown that mouse Shn-2 can bind to Smad1/4 to activate
PPAR 
2 gene expression during adipocyte
differentiation (Jin et al.,
2006). We have found that the suppression of the sma-9 M
lineage phenotype by sma-3 is not allele specific
(Table 4). Additionally,
mutations in sma-2 and sma-4 as well as RNAi knockdown of
these two genes all suppressed the M lineage defects of sma-9 mutants
(Table 4). Although these
observations cannot completely rule out the possibility that SMA-9 functions
by forming a complex with SMA-2, SMA-3 or SMA-4 in the M lineage, we favor the
model that SMA-9 functions in parallel to the TGFß pathway and directly
binds to the regulatory regions of TGFß targets in the M lineage to block
the actions of the Smad proteins. Consistent with this, studies by Wang and
colleagues (Wang et al., 2005)
failed to detect a direct interaction between SMA-9 and SMA-2, SMA-3 or SMA-4
using the yeast two-hybrid system.
The antagonism between SMA-9 and the core Sma/Mab TGFß signaling
pathway may not be restricted to the M lineage. Liang and colleagues
(Liang et al., 2003) have
previously reported that sma-9; Sma/Mab pathway double mutants
exhibit phenotypes similar to those of Sma/Mab pathway single mutant in terms
of male ray patterning (Liang et al.,
2003). In particular, the moderate reduction in the frequency of
serotonergic fate expression in R9B and weak ectopic expression of this fate
in R5B and R7B phenotypes of sma-9 mutants were completely suppressed
by Sma/Mab pathway mutants. This was interpreted to suggest that SMA-9 acts
downstream of the Sma/Mab pathway (Liang
et al., 2003). In light of our current findings, these results may
actually suggest that SMA-9 antagonizes the function of the Sma/Mab pathway in
male ray patterning. Further molecular analysis of SMA-9 and TGFß pathway
targets will shed light on how SMA-9 antagonizes the function of the TGFß
signaling pathway in various cell types.
One surprising finding from our studies is that the lon-2 mutation
also suppresses the M lineage defects of sma-9 mutants
(Table 4). LON-2 has been
previously proposed to function upstream, and possibly as a negative
regulator, of the Smad proteins in regulating body size as sma-2;
lon-2 double mutant animals are small
(Brenner, 1974). We have found
that a null allele of lon-2, e678 (R. Padgett, personal
communication), partially suppresses the M lineage defects of
sma-9(cc604) mutants (Table
4). Furthermore, lon-2(e678) sma-9(cc604) double mutant
animals appear to have a wild-type body size
(Table 4). These observations
indicate that SMA-9 and LON-2 could function in parallel pathways and that
SMA-9 functions differently from the Smad proteins in regulating body size and
M lineage patterning.
TGFß signaling and early M lineage patterning and fate specification
SMs and CCs are descendants of two M daughter cells, M.v and M.d, after the
first cell division in the M lineage (Fig.
1). Because forced expression of sma-9 between the 2-M
and 6-M stages using a heat-shock inducible promoter can rescue the M lineage
defects of sma-9 mutants (Table
2), we believe that dorsoventral asymmetry of the M lineage is not
established during the first division of the M mesoblast. Instead, M.v and M.d
are probably born with the same potential to generate either the ventral SM
fate or the dorsal CC fate. The different developmental fate of these two
cells is probably due to influences of the different environments that these
two cells reside in. We believe that the decision of cells becoming either the
CC or SM precursors is most likely made by the 8-M stage because forced
expression of SMA-9 after the 8-M stage fails to rescue the M lineage defects
of sma-9 mutants (Table
2). As SMA-9 is not asymmetrically expressed in the M lineage, we
favor the model that the Sma/Mab TGFß signaling pathway acts to promote
SM cell fate dorsally in the M lineage, and that this action is antagonized by
SMA-9, resulting in CCs being formed. We are currently investigating when and
where the Sma/Mab TGFß signaling pathway is activated with respect to the
M lineage.
SMA-9 and the Sma/Mab TGFß signaling pathway alone cannot be
exclusively responsible for specifying all the fates of the M lineage,
especially in the ventral M lineage. Previous studies (e.g.
Greenwald et al., 1983) have
shown that LIN-12/Notch is required for the proper formation of the ventral CC
fates. It will be interesting to determine how SMA-9, the Sma/Mab TGFß
signaling pathway and the LIN-12/Notch signaling pathway are integrated to
properly specify the correct dorsal versus ventral cell fates in the M
lineage.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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