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First published online 10 July 2006
doi: 10.1242/dev.02475
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1 Department of Biology, University of California, Riverside, Riverside, CA
92521, USA.
2 Graduate Program in Cell, Molecular and Developmental Biology, University of
California, Riverside, Riverside, CA 92521, USA.
Author for correspondence (e-mail:
mmaduro{at}citrus.ucr.edu)
Accepted 5 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mesoderm, C. elegans, tbx-35, Cell fate specification
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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600 million years ago
(Chen et al., 2004
). Mesoderm
in vertebrates includes such tissues as heart, blood, muscles and bone. In the
nematode C. elegans, the mesodermal cell MS is one of six founder
cells that generate the major tissues of the embryo
(Fig. 1). MS gives rise to many
mesodermal cell types, including cells of the posterior half of the pharynx,
one-third of the body muscle cells and four embryonically derived coelomocytes
(primitive blood-like cells) (Sulston et
al., 1983). MS also signals descendants of the AB lineage,
enabling them to make the remaining, largely anterior, half of the pharynx
(Priess et al., 1987). Early
events that specify MS are tied to the specification of its sister E through a
common gene regulatory network that intersects with Wnt/MAPK signaling
(Maduro and Rothman,
2002).
The bZIP/homeodomain protein SKN-1, at the top of this pathway, is crucial
to the specification of MS and E (Bowerman
et al., 1992). skn-1(-) embryos fail to specify MS all
the time, and E most of the time, and the mis-specified cells adopt the fate
of their lineal cousin C, which makes body muscle and hypodermal cells.
Ectopic accumulation of SKN-1 results in production of supernumerary MS and E
cell types at the expense of others
(Bowerman et al., 1993;
Bowerman et al., 1992;
Mello et al., 1992). At least
two activities restrict SKN-1 activity to EMS. In four-cell embryos, SKN-1
protein is detected at high levels in EMS and in its sister cell
P2, but at low levels in the two AB daughters ABa and ABp, owing to
activity of the CCCH zinc-finger protein MEX-1
(Bowerman et al., 1993). In
P2, PIE-1, also a CCCH zinc finger protein, blocks transcription
and, hence, activation of SKN-1 target genes
(Batchelder et al., 1999). As
shown in Fig. 1,
mex-1(-) embryos show a transformation of the AB4 cells
into MS-like precursors, while in pie-1(-) embryos, P2
adopts an EMS-like fate, resulting in a transformation of C to MS and
P3 to E (Mello et al.,
1992).
To promote specification of MS, SKN-1 activates transcription of the
divergent GATA factors med-1,2 in EMS
(Coroian et al., 2005;
Maduro et al., 2001). With
respect to MS fate, loss of med-1,2 leads to a similar phenotype as
loss of skn-1 (transformation of MS to C), but although
skn-1 mutant embryos lack pharynx entirely, med-1,2(-)
embryos still make AB-derived anterior pharynx
(Coroian et al., 2005;
Maduro et al., 2001). The
targets of MED-1,2 in MS that specify a mesoderm fate are not known, although
several candidate genes have been identified by bioinformatics and
transcriptome analysis (Broitman-Maduro et
al., 2005; Robertson et al.,
2004).
In the E cell, the MEDs contribute to the activation of the E-specifying
genes end-1,3, but are dispensable for E specification much of the
time (Broitman-Maduro et al.,
2005; Goszczynski and McGhee,
2005; Maduro et al.,
2001). We have previously reported that 50% of
med-1,2(RNAi) embryos lack endoderm
(Maduro et al., 2001).
However, a recent study has shown that zygotic loss of med-1 and
med-2 results in a weak or undetectable endoderm defect
(Goszczynski and McGhee,
2005). We have since found that a significant maternal
contribution of the MED genes exists, explaining this discrepancy (M.F.M.,
G.B.-M., I. Mengarelli and J. Rothman, unpublished). In addition to MED-1,2,
we and others have further shown that Caudal/PAL-1 and the Wnt effector
TCF/POP-1 also contribute to E specification
(Maduro et al., 2005b;
Shetty et al., 2005).
The MS and E cells are made different from each other by a molecular
switching system that functions in the MS/E decision as well as many other
asymmetric cell divisions in C. elegans development
(Kaletta et al., 1997;
Lin et al., 1998). The
posterior daughter of EMS is specified to become the endodermal precursor E as
a result of a cell-cell interaction between EMS and its sister P2
(Goldstein, 1992). An `MS'
fate is the default state for an EMS daughter cell, as an isolated EMS cell
will divide symmetrically to produce two MS-like cells. Depletion of the
components of this signal, an overlapping Wnt/MAPK/Src pathway, results in a
similar phenotype in which E adopts an MS-like fate
(Bei et al., 2002;
Rocheleau et al., 1997;
Thorpe et al., 1997). In the
MS cell, POP-1 is essential to repress end-1,3, as evidenced by the
MS to E transformation displayed by pop-1(-) embryos
(Lin et al., 1995). In the E
cell, modification of POP-1 by the WRM-1/LIT-1 kinase blocks the repressive
activity of POP-1, allowing POP-1 to become an activator of endoderm
(Lo et al., 2004;
Rocheleau et al., 1999;
Shetty et al., 2005).
We report here that the MED-1,2 target gene tbx-35, which encodes a member of the T-box class of transcriptional regulators, specifies the MS fate. Restriction of MED-dependent activation of tbx-35 to MS results from Wnt-dependent POP-1-independent repression in E, implicating an unknown mechanism that blocks MS fate in the E cell. Our results identify an important regulator in the suite of blastomere-identity genes in the C. elegans embryo, and link the mesoderm-specifying activity of med-1,2 with MS-specific activation of tbx-35.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The balanced tbx-35(tm1789) strain MS516 was made by injecting
heterozygotes with a mixture of cosmid ZK177, an unc-119::CFP
reporter (pMM809) and the rol-6D plasmid pRF4
(Mello et al., 1991). The
phenotype of tbx-35(tm1789) embryos lacking the array persisted after
three backcrosses of MS516 to N2. Rescue with a fragment containing only
tbx-35 was obtained by injecting MS516 with a 2.4 kb PCR product
containing tbx-35(+) and the unc-119::YFP::lacZ reporter
pMM531, and obtaining lines that had lost the unc-119::CFP/Rol array
in favor of the unc-119::YFP array. PCR was used to validate the
genotypes of tm1789-derived strains and to confirm the absence of a
wild-type copy of tbx-35 in the tm618 and tm1789
strains.
Microscopy and imaging
All micrographs were obtained on an Olympus BX-71 microscope equipped with
a Canon Digital Rebel 350D and LMscope adapter (Micro Tech Lab, Austria). For
fluorescence micrographs, images acquired from different focal planes were
stacked with Adobe Photoshop 7.
Plasmids and cloning
A tbx-35::GFP reporter was constructed by cloning a 650 bp PCR
product, containing 605 bp upstream of the tbx-35 start codon, into
plasmid pPD95.67. A hs-tbx-35 construct (pGB223) was built by cloning
a PCR-amplified genomic fragment containing the coding region and introns into
plasmid pPD49.83. A cDNA-derived heat shock construct (pWH95) contained a
transversion resulting in an E42V substitution. Expression from this construct
was confirmed by in situ hybridization. Oligonucleotide sequences and cloning
details are available on request.
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) or by growth on E. coli HT115 bacteria
engineered to accumulate dsRNA (Timmons
and Fire, 1998). For RNAi by injection, embryos were collected
starting at 24 hours after parental injection. For RNAi by ingestion,
synchronized L2 animals were fed transgenic HT115 for a minimum of 2 days. In
our hands, these treatments produce >99% phenocopy embryos, consistent with
published phenotypes. RNAi targeted to multiple genes was performed by
injection, controlling for depletion of each targeted gene by parallel single
RNAi experiments (Maduro et al.,
2001). Control GFP dsRNA injections produced 4% arrested embryos
(n=591) that did not resemble tbx-35(tm1789). The additional
early-arrest embryos seen with tbx-35(RNAi) appear to be due to an
off-target effect, rather than a maternal contribution: a smaller
tbx-35 dsRNA produced no lethality, no germline transcripts were
observed by in situ hybridization and two germline mosaic MS516 mothers
produced 100% arrested embryos with the same tbx-35(-) phenotypes
described (data not shown).
In situ hybridization
Detection of endogenous mRNAs was performed as described
(Coroian et al., 2005).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wilson and Conlon,
2002). TBX-35 shares 25-28% identity with other metazoan Tbx
proteins within the DNA-binding domain
(Fig. 2B). The Tbx domain of
its closest C. elegans homolog, TBX-37, shows more relatedness with
other Tbx proteins (32-37% identity), while the more canonical C.
elegans Tbx protein MAB-9 shares 52% identity across the same region
(Woollard and Hodgkin, 2000).
Hence, TBX-35 is a fairly divergent Tbx factor.
tbx-35 is a direct target of MED-1
Our biochemical analysis of the MED-1 binding sites in end-1 and
end-3 identified the consensus site RAGTATAC, four of which occur in
the tbx-35 promoter (Fig.
2A) (Broitman-Maduro et al.,
2005). Three additional closely-related RGGTATAC sites, which can
bind MED-1 at a lower affinity (our unpublished observations) are also found
in close proximity. We and others have previously reported the early MS
lineage expression of a tbx-35::GFP reporter, as shown in
Fig. 3A,B
(Broitman-Maduro et al., 2005;
Robertson et al., 2004). Using
whole-mount in situ hybridization, we confirmed activation of endogenous
tbx-35 in the MS cell (Fig.
3G). Consistent with positive regulation of tbx-35,
overexpression of MED-1 is sufficient to promote widespread activation of a
tbx-35 reporter (Broitman-Maduro
et al., 2005). SKN-1 protein is also present in MS, but only a
single site matching the SKN-1 RTCAT consensus is present in the
tbx-35 promoter, 31 bp upstream of the start codon
(Blackwell et al., 1994;
Bowerman et al., 1993). As bona
fide SKN-1 targets contain clusters of several overlapping sites in the
5' flanking region (An and Blackwell,
2003; Coroian et al.,
2005; Maduro et al.,
2001), it is unlikely that SKN-1 directly regulates
tbx-35.
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). We confirmed a direct interaction of
tbx-35 with MED-1 in vitro by gel shift assay using the MED-1
DNA-binding domain (DBD) and a 190 bp fragment of the tbx-35 promoter
(Fig. 2C). Competition with an
oligonucleotide segment of the end-1 promoter containing a single MED
site abolished this shift, whereas a competitor with a mutated MED site
competed only weakly. As MED-1 and MED-2 are 98% identical and functionally
redundant, as evidenced by the ability of either med-1 or
med-2 to rescue the lethality of med-1,2(-) embryos
(Coroian et al., 2005), we
conclude that both MED-1 and MED-2 activate tbx-35.
tbx-35 is expressed in cells that have MS identity
As embryonic activation of tbx-35 is restricted to MS, we assessed
association of tbx-35 with `MS fate' by evaluating its expression in
mutant backgrounds that generate ectopic MS-like cells. A tbx-35::GFP
reporter is activated ectopically in the AB lineage in mex-1(RNAi)
embryos and in the C lineage in pie-1(RNAi) embryos
(Fig. 3C,D), consistent with
the position of ectopic MS-like cells made in these mutants
(Mello et al., 1992). We
confirmed by in situ hybridization that tbx-35 mRNAs are found in the
AB lineage in mex-1(RNAi) embryos
(Fig. 3H). Depletion of the
ß-catenin WRM-1 results in the ectopic activation of tbx-35::GFP
in the early E lineage (Fig.
3E), as anticipated by the E to MS transformation in such animals
(Rocheleau et al., 1997).
Finally, tbx-35 is activated in both E and MS in embryos mutant for
lit-1 (Fig. 3I), which
encodes a Nemo-like kinase required to transduce the Wnt/MAPK signal that
distinguishes E from MS (Rocheleau et al.,
1999). We conclude that tbx-35 expression is associated
with cells that adopt the MS fate.
Wnt/MAPK-dependent restriction of tbx-35 to MS does not require POP-1
As the Wnt/MAPK effector TCF/POP-1 is an activator in E, and a repressor in
MS, it is not known how Wnt/MAPK signaling might direct an inverse pattern for
tbx-35 regulation, namely repression in E and activation in MS.
Indeed, in pop-1(RNAi) and pop-1(zu189) mutants,
tbx-35::GFP persisted in the MS lineage alone
(Fig. 3F; data not shown). By
in situ hybridization, we observed the same MS-specific activation of
endogenous tbx-35 in pop-1(RNAi) embryos
(Fig. 3J). We conclude that
nuclear differences between MS and E exist in the absence of pop-1,
showing that POP-1 is not required for at least one property of the MS cell
(activation of tbx-35). We further conclude that an uncharacterized
Wnt/MAPK-dependent mechanism must exist to repress tbx-35 in E (see
Discussion).
tbx-35 is an essential gene
The foregoing studies establish that tbx-35 is activated at the
correct time and place to specify MS downstream of MED-1,2. To assess the
developmental requirement for tbx-35, we attempted to obtain
tbx-35(RNAi) embryos by gonadal injection of tbx-35 dsRNA
(Fire et al., 1998). Although
78% (107/138) of tbx-35(RNAi) embryos were viable, 22% (31/138)
underwent developmental arrest. The majority had fewer than 100 cells,
apparently caused by a nonspecific effect (see Materials and methods), but
three embryos completed morphogenesis and lacked the MS-derived region of the
pharynx (not shown). As this proportion of mutants is impracticably small (2%
of total), we were fortunate to obtain two tbx-35 chromosomal
mutations (tm618 and tm1789) from the laboratory of Shohei
Mitani (Tokyo, Japan). The tm618 allele is a 1276 bp deletion/13 bp
insertion that removes 59 C-terminal codons and the putative 3'UTR
(Fig. 2A). tm1789 is a
922 bp deletion that removes much of the 5' flanking region, including
five out of seven MED-1 binding sites, and 91 N-terminal codons that include
one-third of the conserved T-box domain. Hence, tm1789 may be a null
mutant. Indeed, although tm618 homozygotes are viable and demonstrate
no obvious phenotype, tm1789 embryos either fail to hatch or hatch as
inviable larvae.
To test whether the lethality of tm1789 results from loss of
tbx-35 function, we constructed a homozygous tm1789 strain
balanced with an extrachromosomal array consisting of cosmid ZK177 and an
unc-119::CFP reporter (Maduro and
Pilgrim, 1995). We obtained a line, MS516, in which all viable
animals exhibit unc-119::CFP expression, and which segregates
40% inviable animals that lack reporter expression, confirming rescue by
ZK177. We were further able to rescue with a 2.4 kbp PCR product containing
tbx-35 with 734 bp of 5' flanking sequence and 374 bp
downstream of the predicted polyA site, allowing us to conclude that the
lethality of tm1789 results from loss of tbx-35 function
(see Materials and methods).
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25% of
tbx-35(-) embryos elongate to only 1.5-fold
(Fig. 4D), 60% elongate to
twofold and 15% elongate to threefold. The least-elongated embryos are similar
to med-1,2(-) embryos in three respects: med-1,2(-) embryos
arrest at between onefold and twofold; approximately one-third of
med-1,2(-) and tbx-35(-) embryos contain internal cavities,
similar to the hypodermis-lined cavities seen in skn-1(-) embryos;
and the pharynx is abnormally small and lacks a grinder, a marker of
MS-derived pharynx (Bowerman et al.,
1992; Maduro et al.,
2001). However, although some med-1,2(-) embryos lack
endoderm (Coroian et al.,
2005; Goszczynski and McGhee,
2005; Maduro et al.,
2001), all tbx-35(-) homozygotes make endoderm (not
shown).
We examined pharynx production in tbx-35(-) embryos using a
ceh-22 reporter to mark pharynx muscle
(Table 1)
(Okkema and Fire, 1994).
Although wild-type embryos display a normal pharynx with ABa-derived and
MS-derived regions, tbx-35(-) embryos appear to lack the posterior,
MS-derived region (Fig. 4B,E).
Indeed, in situ hybridization of the pharynx-specifying gene pha-4
and the pharyngeal myosin gene myo-2 revealed smaller,
anterior-specific domains of expression in tbx-35(-) embryos when
compared with wild type (Fig.
5A,B,E,F) (Gaudet and Mango,
2002; Okkema et al.,
1993). Production of ABa-derived pharynx requires a
Notch/GLP-1-mediated cell-cell interaction between MS and the AB lineage
(Priess et al., 1987). To
determine if the remaining pharynx in tbx-35(-) embryos is ABa
derived, we combined the glp-1(or178) mutation with
tbx-35(tm1789). Although tbx-35(+); glp-1(-) embryos
contained an average of 5.7±0.2 (n=74) pharynx muscle cells
(Fig. 4C), tbx-35(-);
glp-1(-) embryos had a mean of 0.3±0.1 (n=74)
(Fig. 4F). Therefore, loss of
tbx-35 greatly reduces, but does not completely eliminate, MS-derived
pharynx. The slightly leaky nature of these tbx-35(-) defects
contrasts with med-1,2(-) embryos, which never elongate past twofold
and contain no MS-derived pharynx (Maduro
et al., 2001).
|
). At
1.5-fold elongation, MS-derived muscles are found at the anterior end of
the embryo (arrowheads in Fig.
4H) (Sulston et al.,
1983). Similarly staged tbx-35(-) embryos lack these
muscles, and instead show an accumulation of extra muscle cells in the middle
of the embryo (Fig. 4K). The
same pattern is observed with expression of the body muscle myosin gene
myo-3 by in situ hybridization
(Fig. 5C,G). The occurrence of
an apparent ectopic region of muscle cells suggests that MS still makes muscle
in tbx-35(-) mutants. To test whether these are `MS type' or `C type'
muscle cells, we depleted Caudal/PAL-1 by RNAi to specifically block formation
of C- and D-type muscle (Hunter and
Kenyon, 1996). Although pal-1(RNAi) blocks formation of
C- and D-derived muscle, showing a lack of hlh-1 expression where
these muscles should be (arrows in Fig.
4I), nearly all muscle is absent in tbx-35(-);
pal-1(RNAi) embryos (Fig.
4L). As MS-derived pharynx and body muscle are greatly reduced in
tbx-35(-) embryos, we conclude that tbx-35 is required to
make the majority of the descendants of MS.
The production of ectopic C-type muscle in tbx-35(-) mutants is
anticipated from the observation that both skn-1(-) and
med-1,2(-) embryos display a transformation of MS into a C-like cell
(Bowerman et al., 1992;
Hunter and Kenyon, 1996;
Maduro et al., 2001). We
examined early tbx-35(-) embryos for evidence of ectopic activation
of zygotic pal-1, a marker for the early C lineage
(Baugh et al., 2005). By in
situ hybridization, we observed zygotic pal-1 transcripts in only the
early C lineage in wild-type (Fig.
5D) and in both the C and MS lineages in 30% of
tbx-35(-) embryos (Fig.
5H). We observed similar ectopic expression in tbx-35(-);
pal-1::YFP embryos (data not shown). With both approaches, ectopic
pal-1 signal in the MS lineage occurred slightly later than that of
the C lineage, and with variable intensity. We conclude that the MS blastomere
in tbx-35(-) embryos exhibits a transformation of MS to C of varying
expressivity.
tbx-35 is required for ectopic MS-like fates
The results thus far suggest tbx-35 plays an important role in MS
specification. We next evaluated the requirement for tbx-35 in
specification of abnormal MS-like cells that arise in specific mutant
backgrounds. In pie-1(-) embryos, C adopts an MS-like fate, resulting
in embryos in which nearly all muscle cells are derived from MS
(Mello et al., 1992). As loss
of tbx-35 leads to apparent production of C-type muscle from MS, we
simultaneously depleted pie-1 and pal-1 by RNAi to ensure
the generation of only MS-type muscle in these embryos. We found that although
tbx-35(+); pie-1(RNAi); pal-1(RNAi) embryos generated an average of
29.4±0.9 (n=25) muscle cells, tbx-35(-); pie-1(RNAi);
pal-1(RNAi) embryos made only 4.4±0.6 (n=25) muscle cells
(Fig. 6A,D), consistent with a
requirement for tbx-35 function in the mis-specification of the C
cell as `MS' in pie-1 mutants.
|
). Large
numbers of pharynx cells (30.0±1.8, n=26) were seen in
tbx-35(+); mex-1(RNAi); pie-1(RNAi) embryos
(Fig. 6B), which were nearly
eliminated (1.0±0.2 cells, n=38) when tbx-35 was
mutant (Fig. 6E). Production of
MS-type muscle in a mex-1(-); pie-1(-) background was also found to
be strongly dependent on tbx-35
(Table 1). We further found
that the number of cells expressing the hypodermal marker lin-26
(Labouesse et al., 1996) was
increased in a tbx-35(-); mex-1(-); pie-1(-) background over
tbx-35(+); mex-1(-); pie-1(-)
(Fig. 6C,F). As C makes
hypodermal cells, this result is consistent the transformation of at least
some MS-like cells to C-like cells in the absence of tbx-35.
|
). To
block formation of AB-derived pharynx, we used a glp-1(or178)
mutation. We found that although tbx-35(+); glp-1(or178); wrm-1(RNAi)
embryos make 22.6±0.7 pharynx cells (n=42), tbx-35(-);
glp-1(or178); wrm-1(RNAi) embryos make only 5.6±0.7 cells
(n=49). We conclude that tbx-35 is crucial for specification
of MS fate in both its normal context and in genetic backgrounds that result
in production of ectopic MS-like cells.
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), we tested whether TBX-35 can reprogram non-MS cells
to become mesoderm precursors, similar to the ability of MED-1 to promote the
generation of ectopic pharynx and endoderm
(Maduro et al., 2001). A heat
shock (hs) tbx-35 transgene was constructed and introduced into the
ceh-22 and hlh-1 reporter strains. We used
skn-1(RNAi) and skn-1(RNAi); pal-1(RNAi) backgrounds to
block specification of pharynx and muscle precursors, respectively
(Bowerman et al., 1992;
Hunter and Kenyon, 1996). As
shown in Fig. 7, expression of
hs-tbx-35 can promote development of pharynx and muscle in a
significant proportion of embryos (30% in the case of pharynx). Heat shock of
skn-1(RNAi) embryos carrying a hs-tbx-35[E42V] transgene,
containing a missense change in a conserved amino acid
(Fig. 2B), resulted in
embryonic arrest without production of pharynx (n>50). We conclude
that high levels of TBX-35 are sufficient to specify MS fates. As this
activity requires a conserved amino acid in its predicted DNA-binding domain,
TBX-35 appears to function as a classical transcriptional activator.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gaudet and Mango,
2002).
TBX-35-independent MS fates
Our results suggest that skn-1(-) and med-1,2(-) embryos
fail to make MS-derived cell types because they do not activate
tbx-35. Although loss of med-1,2 leads to an almost complete
loss of MS-derived tissues and embryonic lethality, even in embryos that make
a gut (Bowerman et al., 1992;
Maduro et al., 2001), some
tbx-35(tm1789) mutants elongate to threefold and even hatch before
arresting. As no tbx-35(-) embryos escape lethality, there is
probably no redundant activity that can completely compensate for the absence
of TBX-35 function.
Do additional regulators specify at least some MS-like fates? Our
observation that MS lineage activation of zygotic pal-1 in
tbx-35(-) embryos is of variable intensity is consistent with the
existence of other MS fate-promoting, C-repressing regulators. At least two
more genes, hlh-25 and hlh-27, encoding homologs of the bHLH
family of transcription factors, are activated in MS at the same time as
tbx-35 (Broitman-Maduro et al.,
2005). Overexpression of hlh-25 can specify some early
cells as muscle progenitors, suggesting that HLH-25/27 may participate
directly in muscle specification, or indirectly in other aspects of muscle
inductions known to involve MS
(Broitman-Maduro et al., 2005;
Schnabel, 1995).
Alternatively, targets of TBX-35 may be able to respond directly to residual
MED-1,2, or other activators, present in the early MS lineage.
Mesoderm versus endoderm: a simpler network for a more complex lineage?
The embryonic MS and E lineages are remarkably different. The E lineage
generates 20 cells of a single type (gut), while MS generates 80 cells of
various types, including pharynx, muscle, coelomocytes and cells of the
somatic gonad (Sulston et al.,
1983). E specification requires an extrinsic induction, transduced
through an overlapping Wnt/MAPK/Src pathway, while activity of TCF/POP-1,
Caudal/PAL-1, SKN-1 and MED-1,2 all contribute to E specification
(Bei et al., 2002;
Goldstein, 1992;
Maduro et al., 2005b;
Rocheleau et al., 1997;
Shetty et al., 2005;
Thorpe et al., 1997). By
contrast, MS identity is an intrinsic property of an EMS daughter
(Goldstein, 1992), and our
data show that MS may be specified by the relatively simple linear pathway,
SKN-1
MED-1,2TBX-35.
Although specification of the MS cell appears to be comparatively simple,
the development of its descendants requires the deployment of multiple gene
batteries. For one, a complex gene network is deployed by the regulator
FoxA/PHA-4 to specify the developing pharynx, an organ that itself is composed
of different cell types such as neurons and muscle
(Gaudet and Mango, 2002;
Sulston et al., 1983). As
MS-derived pharynx cells arise from only two of the four granddaughters of MS
(MSaa and MSpa), additional factors must restrict pharynx fate to these
sublineages (Sulston et al.,
1983). As TCF/POP-1 displays nuclear anteroposterior differences
in these cells (Lin et al.,
1998; Maduro et al.,
2002), the Wnt/MAPK pathway is likely to be involved in
differential activation of pharynx potential within the MS lineages. The most
obvious conclusion from the differences between MS and E, then, is that the
complexity of the lineage elaborated by an early embryonic cell is not
necessarily correlated with the nature of the gene networks that specify the
progenitor itself.
Wnt-dependent repression of mesoderm
We have found that tbx-35 is repressed in E in a
Wnt/MAPK-dependent manner, as depletion of ß-catenin/WRM-1 or Nemo/LIT-1
results in ectopic activation of tbx-35 in E. In the absence of
pop-1, activation of tbx-35 persists in the MS cell, even
though the endoderm genes end-1 and end-3 become activated
in both MS and E (Maduro et al.,
2005a). The persistence of tbx-35 expression in MS in
pop-1(-) embryos, and the E to C transformation in
end-1,3(-) embryos, suggest that repression of tbx-35 in E
is not achieved by END-1,3. As MS (like E) adopts an endoderm fate in
pop-1(-) embryos, END-1,3 outcompete TBX-35 when both are present
together in MS. Hence, depletion of a Wnt-dependent repressor of
tbx-35 might not demonstrate loss of endoderm. In Xenopus,
HMG proteins of the Sox family can directly interact with ß-catenin,
blocking activation of Wnt target genes by depleting this necessary TCF
co-activator (Zorn et al.,
1999), although in the case of the MS-E decision, such a mechanism
would probably block endoderm specification itself. The C. elegans
genome encodes at least three Sox-like genes (indexed in WormBase, release
WS156), one of which (sox-1) we have shown to be expressed in the
early MS and E lineages (Broitman-Maduro
et al., 2005); however, the role of any of these in MS or E
specification is not yet known, and as C. elegans continually
surprises the development field with unexpected Wnt/MAPK features
(Kidd, 3rd et al., 2005), we
can make no reliable predictions. The identification of such an effector would
provide an additional mechanism by which Wnt/MAPK-dependent nuclear
differences could be established in C. elegans development
(Kaletta et al., 1997;
Rocheleau et al., 1999).
T-box genes in mesoderm development
The C. elegans pharynx appears to be an organ whose development
requires the activity of multiple Tbx factors in different lineages. Although
tbx-35 specifies MS, the redundant genes tbx-37 and
tbx-38 are required for the GLP-1-mediated induction that specifies
the ABa-derived precursors of the largely anterior half of the pharynx
(Good et al., 2004). More
recently, yet another Tbx gene, tbx-2, was found to be required for
development of ABa-derived pharynx muscles
(Chowdhuri et al., 2006).
Tbx regulators are essential for heart development in vertebrates; in
humans, mutations in Tbx genes lead to congenital cardiac defects
(Plageman and Yutzey, 2005).
Recently, Tbx genes have been found to be important for development of heart
in Drosophila, in which the Tbx genes Dorsocross, midline
and H15 function in the specification and formation of cardiac cells
(Miskolczi-McCallum et al.,
2005; Reim and Frasch,
2005). Although C. elegans lacks a circulatory system,
its pharynx has some similarities to a heart, as it is a pumping organ
containing contractile muscle that is distinct from body muscle
(Avery and Shtonda, 2003;
Okkema et al., 1993). Indeed,
pharynx developmental defects in C. elegans ceh-22 mutants can be
rescued by expression of its vertebrate homolog, the heart specification gene
Nkx2.5 (Haun et al.,
1998). As MS descendants produce most of the posterior pharynx
(and MS induces the specification of the remainder from the AB lineage), is
the specification of MS by a Tbx factor truly an example of homology?
Primitive bilaterians are thought to have evolved regions of contractile
mesoderm that functioned as a primordial heart
(Bishopric, 2005).
Specification of cells within such a tissue territory may have been controlled
directly by Tbx regulators. Extant organisms such as C. elegans, in
which cell fates are specified early, might possess Tbx gene cascades into
which other layers of regulation have been intercalated
(Erwin and Davidson, 2002).
Hence, the use of tbx-35 to specify MS may reflect its derivation
from a more direct role for Tbx proteins in direct activation of genes in
differentiated mesoderm.
|
ACKNOWLEDGMENTS |
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Footnotes |
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