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First published online 13 June 2007
doi: 10.1242/dev.008268
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Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
* Author for correspondence (e-mail: rueyling.lin{at}utsouthwestern.edu)
Accepted 15 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: C. elegans, TCF/POP-1, ß-catenin/SYS-1, Cell fate specification
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Schnabel and Priess,
1997). After initial fate specification, each blastomere generates
specific terminally differentiated cell types via an invariant lineage.
Lineage analyses using genetic mutants suggest that the C. elegans
embryo invariant lineage is primarily composed of stepwise bifurcations of
blastomere specification at anterior-posterior (A-P) cell divisions
(Kaletta et al., 1997).
Mutations in the gene lit-1, which encodes a Nemo-like MAP kinase
(Meneghini et al., 1999;
Rocheleau et al., 1999),
suggest that the LIT-1 protein functions in at least six consecutive A-P
divisions to effect a binary switch whereby the posterior cell of two
equivalent cells assumes the posterior cell fate
(Kaletta et al., 1997). The
nuclear level of LIT-1 is higher in posterior cells where its activity is
required, compared with their anterior sisters
(Lo et al., 2004;
Takeshita and Sawa, 2005). The
A-P asymmetry of LIT-1 levels is reciprocal to that of the C. elegans
TCF protein, POP-1, which is detected at a higher level in the nuclei of
anterior cells of all A-P divisions examined
(Lin et al., 1998;
Lin et al., 1995). Both LIT-1
and POP-1 A-P asymmetries require the MAP kinase kinase kinase MOM-4, a C.
elegans ß-catenin WRM-1, and a lesser contribution from other
components of the Wnt pathway (Lin et al.,
1998; Lo et al.,
2004; Maduro et al.,
2002; Meneghini et al.,
1999; Park and Priess,
2003; Rocheleau et al.,
1999; Shin et al.,
1999; Thorpe et al.,
1997). WRM-1 has been shown to bind directly to LIT-1 and activate
its kinase activity in vitro (Rocheleau et
al., 1999). We have shown previously that specific phosphorylation
of POP-1 by LIT-1-WRM-1 promotes its interaction with a 14-3-3 protein,
leading to nuclear export (Lo et al.,
2004). Therefore, in posterior cells, as compared with their
anterior sisters, nuclear levels of LIT-1 are high and those of POP-1 are
low.
At the 4-cell stage, a Wnt/MAPK signal from P2 to EMS specifies the
posterior daughter of EMS, E, to become an endoderm precursor
(Goldstein, 1992). The
anterior daughter of EMS, MS, generates mesoderm. Most mutations in either the
Wnt or MAP kinase signaling pathways result in a non-fully penetrant
transformation of E to MS (the Mom phenotype)
(Kaletta et al., 1997;
Rocheleau et al., 1997;
Thorpe et al., 1997). Mutation
in pop-1 results in MS adopting the fate of E
(Lin et al., 1995). We and
others have shown that POP-1 both represses E fate in the MS blastomere
(Calvo et al., 2001;
Lin et al., 1995;
Rocheleau et al., 1997;
Thorpe et al., 1997) and
promotes endoderm formation from E (Maduro
et al., 2005b; Shetty et al.,
2005). We showed that Wnt/MAPK signaling converts POP-1 from a
repressor to an activator of target genes
(Shetty et al., 2005).
Activation of these target genes in E by POP-1 requires the N-terminal domain
of POP-1 and that the POP-1 nuclear level in E be lowered. The requirement for
the N-terminal domain of POP-1, similar in sequence to the N-terminal domains
of other TCF proteins involved in binding ß-catenin
(van de Wetering et al.,
1997), suggests a ß-catenin co-activator.
The C. elegans genome encodes four ß-catenin-related
proteins: HMP-2, BAR-1, WRM-1 and SYS-1. The hmp-2 mutant phenotype
suggests a function exclusively in cell adhesion
(Costa et al., 1998) and,
although BAR-1 can function as a POP-1 transcriptional coactivator in vitro, a
bar-1 likely null mutation has no observable embryonic defect
(Eisenmann et al., 1998).
WRM-1 is required for asymmetric cell fates in C. elegans
embryogenesis (Rocheleau et al.,
1997), but it has never been shown to physically interact with
POP-1, nor has it been shown to function as a TCF/POP-1 co-activator
(Kidd et al., 2005;
Korswagen et al., 2000;
Natarajan et al., 2001;
Rocheleau et al., 1999). SYS-1
is required for asymmetric divisions of the somatic gonad precursors
(Kidd et al., 2005;
Miskowski et al., 2001), and
can function as a POP-1 transcriptional coactivator in vitro, via interaction
with the N-terminal ß-catenin-binding domain of POP-1
(Kidd et al., 2005). Recently,
SYS-1 has been implicated in endoderm precursor specification
(Phillips et al., 2007).
Whereas animals homozygous for a reduction-of-function mutation are sterile,
sys-1(RNAi) resulted in a very low penetrance gutless phenotype.
We show here that SYS-1 is a limiting coactivator for POP-1 in the activation of Wnt/MAPK-responsive genes in the E blastomere. SYS-1 exhibits a reiterated asymmetry that is reciprocal to the reiterated asymmetry of nuclear POP-1 through all A-P divisions examined. We show that the SYS-1-to-POP-1 ratio appears critical for both anterior and posterior cell fates at multiple divisions: a high ratio drives the posterior cell fate, whereas a low ratio drives the anterior cell fate. SYS-1 and POP-1 levels are regulated in opposite directions by two pathways known to regulate endoderm specification: SYS-1 levels are increased primarily by the MOM-2/MOM-5/APR-1 pathway, whereas nuclear POP-1 levels are decreased primarily by the MOM-4/LIT-1/WRM-1 pathway. Together, these two pathways efficiently increase the SYS-1-to-POP-1 ratio in the posterior cell, promoting asymmetric cell fates.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). TX932
[sys-1(q544)/fog-3(q470)(I); teIs3(Pmed-1gfp::pop-1)(V)]
(Miskowski et al., 2001).
TX964 [teIs98(Ppie-1gfp::sys-1)]. JM139
[Ppho-1 NLS::gfp::H2B] (a gift from J. McGhee and J. Yan,
University of Calgary, Alberta, Canada). teIs98 was generated by
microparticle bombardment (Praitis et al.,
2001). All other strains were obtained from the
Caenorhabditis Genetics Center (CGC).
RNA interference (RNAi)
sys-1 RNAi was performed by feeding L3 larvae with RNAi bacteria
followed by injection. We obtained 3-4% gutless embryos upon
sys-1(RNAi) into JK2761 [sys-1(q544)/unc-29(e1072)
fog-3(q470)(I)] but not wild-type N2. RNAi experiments were performed by
injection for imb-4, skn-1, med-1/med-2, apr-1, lit-1 and
mom-5 (Rogers et al.,
2002) and by feeding for wrm-1, pop-1, skn-1, rpn-8, pas-4,
pbs-2, ran-3 and ran-4
(Timmons and Fire, 1998). RNAi
by feeding for each of the genes listed here resulted in nearly 100% dead
embryos. For pop-1 mild depletion, pop-1-feeding RNAi
bacteria were diluted with HT115 bacteria. Embryos laid 16-30 hours after
injection or 48-60 hours after feeding were collected and either allowed to
differentiate for 12 more hours, a period long enough for wild-type control
embryos to hatch, or processed for imaging.
Analysis of embryos and imaging
Expression of Wnt target genes upon sys-1 depletion was analyzed
as described previously (Shetty et al.,
2005). Embryos were collected from mom mutant or RNAi
hermaphrodites, and either assayed for GFP and scored later for gut formation,
or fixed for immunofluorescence. teEx321 was crossed into JM139
hermaphrodites. Embryos were collected and assayed first for GFP::SYS-1 in
early embryos, then PHO-1 reporter GFP in the newly hatched larvae.
GFP::SYS-1 was analyzed by imaging of live embryos or by immunofluorescence
using anti-GFP (Invitrogen) and anti-POP-1 antibodies
(Lin et al., 1998). Images
were collected as stacks along the z-axis with 3 microns between
adjacent images (
16 slices per specimen). 16-bit images were collected
with raw pixel values within the linear range of the CCD camera and processed
and quantified using ImageJ (Lo et al.,
2004; Rogers et al.,
2002). Owing to variation in expression levels from embryo to
embryo, for embryos with abolished GFP::SYS-1 asymmetry we could not determine
whether the level of GFP::SYS-1 was elevated in anterior cells, decreased in
posterior cells, or both.
Intestinal cells were identified by their birefringent gut-specific
granules under polarized optics and by staining with the monoclonal antibody
ICB4 (Kemphues et al., 1988).
Pharyngeal tissues were identified based on morphology using DIC and
immunofluorescence using the monoclonal antibody 3NB12
(Priess and Thomson, 1987).
Pharyngeal staining was imaged as a stack along the z-axis to
facilitate the counting of pm7 cells.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Shetty et al.,
2005; Zhu et al.,
1997). Depletion of sys-1 by RNAi resulted in a reduction
in the expression levels of both sdz-23 and end-1 reporter
genes in all embryos examined (n>200), similar to that observed
when pop-1 is removed (Fig.
1A,C,E,F; data not shown). However, sys-1-depletion did
not result in derepression of either E-specific reporter in the MS lineage
(Fig. 1A-D), or in defects in
GFP::POP-1 asymmetry (data not shown). These results suggest that
sys-1 functions in activation of Wnt target gene expression in E,
downstream or independent of the regulation of nuclear POP-1 levels.
sys-1(RNAi) resulted in nearly 100% dead embryos arrested without
proper morphogenesis, but nonetheless with differentiated tissue types, such
as epidermal, pharyngeal and muscle cells
(Fig. 1H). Despite pleiotropic
terminal phenotypes, 3-4% of sys-1(RNAi) embryos were consistently
observed to lack intestine (n=497,
Fig. 1I), a percentage of
gutless similar to that observed in pop-1(RNAi) embryos
(Maduro et al., 2005b). The
maternal transcription factor SKN-1 functions in parallel to the POP-1 pathway
to specify endoderm (Maduro et al.,
2005b). Depletion of pop-1 in the skn-1(zu67)
mutant background enhanced the skn-1(zu67) gutless phenotype from 64%
to almost 100%. sys-1(RNAi) also enhanced the skn-1(zu67)
gutless phenotype to 100% (n=68), consistent with SYS-1 functioning
in the same pathway as POP-1 in endoderm specification.
Elevated POP-1 or reduced SYS-1 levels enhance the Mom phenotype, whereas elevated SYS-1 levels suppress it
sys-1(RNAi) produced a strong enhancement of the gutless phenotype
in all mom mutants examined (Table
1). A striking example is the enhancement of lit-1(t1534)
from 0% (n=116) to 100% (n=126) gutless. Conversely, we
observed a strong suppression of the gutless phenotype in most mom
mutants or RNAi embryos carrying a transgene expressing GFP::SYS-1 in either
all [teIs98(Ppie-1gfp::sys-1)] or the EMS
[teEx321(Pmed-1gfp::sys-1)] lineages. For example, the
penetrance of the Mom phenotype in apr-1(RNAi) and
mom-4(ne19) embryos dropped from 29% (n=70) and 40%
(n=65) to 0% (n=20) and 1% (n=67), respectively, in
the teEx321 background. Both these SYS-1 reduction-of-function and
overexpression results suggest that the level of SYS-1 is important for
endoderm specification.
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). We observed a strong enhancement of the Mom
phenotype with teIs3 (Table
1), which increased the percentage of gutless embryos from 29%
(n=70) and 43% (n=65) to 71% (n=62) and 75%
(n=56), respectively, for apr- 1(RNAi) and
mom-4(ne19). This effect is similar to that observed for depletion of
sys-1 and opposite to that observed following an increase in SYS-1
levels. These results demonstrate that both POP-1 and SYS-1 levels are
important for endoderm specification. A high level of SYS-1 promotes, whereas
a low level of SYS-1 or a high level of POP-1 antagonizes, the endoderm
fate.
teIs98(Ppie-1gfp::sys-1) suppressed the gutless
phenotype in skn-1(RNAi) embryos
(Table 1), suggesting that the
effect upon the gutless phenotype reflects an altered POP-1 activity and not
an altered SKN-1-dependent pathway. The penetrance of the gutless phenotype
dropped from 76% (n=163) in skn-1(RNAi) embryos to 16%
(n=215) following skn-1(RNAi) in teIs98. This
SKN-1-independent suppression was also observed in mom-2(or42) and
mom-5(or57) mutant backgrounds
(Table 1). Because expression
of GFP::SYS-1 in teEx321(Pmed-1gfp::sys-1) depends on
SKN-1 activity, we inactivated the SKN-1 pathway by RNAi of med-1 and
med-2, genes encoding transcription factors downstream of
skn-1, as it has been shown that depletion of both med-1 and
med-2 by RNAi results in gutless embryos
(Maduro et al., 2001). We
performed med-1/2(RNAi) in mom-2(or42), mom-4(ne19), mom-2(or42);
teEx321 and mom-4(ne19); teEx321 embryos. In every case we
examined, teEx321 suppressed the gutless phenotype when
med-1 and med-2 were depleted by RNAi
(Table 1).
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Nuclear SYS-1 levels are higher in E than MS, reciprocal to nuclear POP-1
Localization of GFP::SYS-1 in the MS and E blastomeres and their
descendants in teEx321(Pmed-1gfp::sys-1) embryos was
determined by immunofluorescence using an anti-GFP antibody. We observed a
dynamic subcellular localization of GFP::SYS-1 throughout the cell cycle (see
Fig. S1 in the supplementary material), including centrosomal accumulation at
metaphase. During interphase, we observed a higher overall level of nuclear
and cytoplasmic GFP in E versus MS. It has also recently been reported that
VENUS-tagged full-length SYS-1 associates with centrosomes and is at a higher
level in E than in MS (Phillips et al.,
2007). The GFP::SYS-1 asymmetry is reciprocal to POP-1 nuclear
asymmetry (Lin et al., 1995)
(Fig. 2A-D).
Similar to POP-1 asymmetry, the reciprocal GFP::SYS-1 asymmetry was also found to be reiterated in subsequent A-P divisions in the EMS lineage (Fig. 2E-L). MS and its descendents divide along the A-P axis five times during embryogenesis. By following the first three rounds of A-P divisions in the MS lineage in fixed embryos, we observed a higher level of cytoplasmic and nuclear GFP in all posterior cells compared with their anterior sisters (Fig. 2E-H; data not shown). E divides A-P then left-right, after which the four E descendents undergo several more rounds of A-P divisions to generate the entire intestine. We observed asymmetric GFP::SYS-1 in all A-P divisions of the E lineage (Fig. 2E-H,K-L). GFP::SYS-1 levels were observed to be equal only in the two pairs of sisters derived from the left-right divisions of Ea and Ep (Fig. 2I,J).
AB, the anterior blastomere of a 2-cell embryo, first undergoes a dorsal-ventral then a left-right division, before dividing anterior-posteriorly five times. In early teIs98(Ppie-1gfp::sys-1) embryos, in which GFP::SYS-1 is expressed in all lineages, we observed GFP::SYS-1 asymmetry in all AB descendants that derived from A-P divisions that we could identify, including those generating 8, 16 and 32 AB cells (referred to as AB8, AB16 and AB32 cells). This reiterated reciprocal asymmetry of nuclear POP-1 and GFP::SYS-1 levels suggests that SYS-1 functions as a coactivator for POP-1 in the posterior cell following multiple A-P divisions in multiple lineages.
Differential regulation of SYS-1 and POP-1 A-P asymmetry by the Wnt and MAPK pathways
We depleted a component in the Wnt or MAPK pathways via genetic mutation or
RNAi and assayed the effect in the EMS lineage. We scored the MSa/MSp and
Ea/Ep pairs of sisters because stronger GFP::SYS-1 staining permitted easier
scoring (Fig. 2E,F;
Fig. 3Ba-c). GFP::SYS-1
asymmetry between MSa/MSp and Ea/Ep pairs of sisters was abolished or
defective in most mom-2(or42), mom-5(RNAi), mom-5(or57) or
apr-1(RNAi) embryos (Fig.
3Aa-d;
3Bd-f;
Table 2). Double staining with
anti-POP-1 antibody also showed subtle defects in nuclear POP-1 asymmetry,
although the POP-1 asymmetry defect tended to be less penetrant, with some
asymmetry often still observed between sister blastomeres
(Table 2). Both GFP::SYS-1 and
nuclear POP-1 asymmetries were restored in these mutants following the 2E/4MS
stage. Conversely, we observed no or very little defect in GFP::SYS-1
asymmetry in mom-4(ne19), lit-1(RNAi), lit-1(t1512) or
wrm-1(RNAi) embryos, despite a complete abolishment of POP-1
asymmetry (Fig. 3Ae-h;
3Bg-i;
Table 2).
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SYS-1 asymmetry is dependent on the proteasome but not nuclear export
Is GFP::SYS-1 asymmetry regulated, as it is for POP-1, via nuclear export?
Three genes, imb-4, ran-3 and ran-4, which have been shown
to function in nuclear export, were individually depleted by RNAi in
teEx321(Pmed-1gfp::sys-1). All (n=18)
imb-4(RNAi) and 50% of ran-3(RNAi) and
ran-4(RNAi) embryos (n=15 and 11, respectively) exhibited
exclusively nuclear GFP::SYS-1, consistent with these three genes functioning
in GFP::SYS-1 nuclear export. However, GFP::SYS-1 levels were still observed
to be different between A-P sisters in all embryos depleted of any of these
three genes (Fig. 3Ai,j; data
not shown). This result, and the cytoplasmic GFP::SYS-1 observed in
pop-1 mutant embryos, suggest that neither nuclear export nor
differential subcellular distribution accounts for the GFP::SYS-1 asymmetry
between A-P sisters.
GFP::SYS-1 asymmetry is lost, however, by depletion of components of the
proteasome. When three components of the proteasome, rpn-8, pas-4,
pbs-2 were individually depleted, we observed in each case a similar
level of GFP::SYS-1 in almost all GFP-expressing blastomeres
(Fig. 3Cd; data not shown).
This loss of GFP::SYS-1 asymmetry is not due to a loss of embryonic polarity
in RNAi embryos per se. PIE-1 in wild-type embryos is localized to germ cell
precursors (Fig. 3Ca-c)
(Mello et al., 1996), which
requires proper establishment of embryonic polarity and proteasome-mediated
degradation of PIE-1 segregated to somatic cells
(DeRenzo et al., 2003). Under
our RNAi conditions, we detected in all cases (n=6) GFP::SYS-1
equally distributed between A-P sister blastomeres, and PIE-1 primarily in the
germline precursor (Fig.
3Cd-f). We also detected a small amount of PIE-1 in somatic cells,
consistent with a defect in proteasome-mediated degradation in these RNAi
embryos. These results argue that GFP::SYS-1 asymmetry is regulated via
proteasomal degradation.
Elevated SYS-1 expression results in an MS-to-E fate transformation
Approximately 50% (n=71) of GFP-positive
teEx321(Pmed-1gfp::sys-1) embryos either failed to hatch
or died as L1 larvae. Immunofluorescence analyses detected no embryos lacking
gut (n>1000). Instead, we observed a small number of embryos (3%,
n>500) with approximately twice the number of intestinal cells and
missing the posterior pharynx (Fig.
4). This phenocopies pop-1(zu189) mutant embryos
(Lin et al., 1995), consistent
with an MS-to-E fate transformation.
This pop-1 phenocopy was greatly enhanced following mild reduction
of pop-1 by RNAi. Under pop-1 RNAi conditions in which two
control strains, N2 and teIs18(Psdz-23gfp::H2B)
(Shetty et al., 2005),
produced only 13% (n=82) and 29% (n=104) pop-1-like
embryos, respectively, teEx321(Pmed-1gfp::sys-1)
GFP-positive embryos produced 75% (n=49) pop-1-like embryos.
As elevated SYS-1 levels result in an MS-to-E fate transformation, this result
strongly suggests that the SYS-1 level is limiting with respect to POP-1 in
MS, precluding MS from making endoderm.
The SYS-1-to-POP-1 ratio is also critical for other A-P divisions
The SYS-1-to-POP-1 ratio regulates reiterating, asymmetric cell fate
changes at other A-P divisions within the MS and E lineages.
The MS lineage
The C. elegans pharynx consists of eight muscle types (pm1 through
pm8) arranged as eight consecutive rings with three cells in each ring
(Fig. 5A)
(Albertson and Thomson, 1976).
An antibody, 3NB12, recognizing pm3, pm4, pm5 and pm7 cells, generates a
characteristic staining gap in wild-type pharynx between pm5 and pm7, owing to
the inability of this antibody to stain pm6 cells
(Fig. 5B)
(Priess and Thomson,
1987).
The three pm6 and three pm7 cells are all derived from the MS lineage. One
pm6 and two pm7 cells are derived from MSa, whereas two pm6 and a single pm7
cell are derived from MSp (Sulston et al.,
1983). Based upon the known lineage, an MSa-to-MSp fate change
would be predicted to result in the MSa-derived pm6 cell (MSaapappa)
and the MSa-derived pm7 cells (MSaaaappp and MSaapaapp) adopting
the fate of MSpapappa, MSpaaappp and MSpapaapp,
respectively (see Table S1 in the supplementary material). The net result
would be the absence of one 3NB12-positive pm7 cell and the presence of one
additional 3NB12-negative pm6 cell at the terminal bulb. The same lineage
calculations for an MSp-to-MSa fate change predict one additional
3NB12-positive pm7 muscle cell and the absence of one 3NB12-negative pm6
muscle cell (Fig. 5B and see
Table S1 in the supplementary material).
Approximately 10% (n=1000) of the
teEx321(Pmed-1gfp::sys-1) embryos exhibited a decrease in
the number of pm7 muscle cells upon 3NB12 staining
(Fig. 5B). Instead of the three
pm7 cells always observed in wild-type embryos, we often observed only two pm7
cells in teEx321 embryos. On the contrary, 10% of embryos and
larvae derived from sys-1(q544/+); teIs3(Pmed-1gfp::pop-1)
had four 3NB12-positive cells and a smaller area than in the wild type
unstained by 3NB12 in the terminal bulb
(Fig. 5B). We did not observe
any teEx321 embryos with extra pm7 muscle cells or progeny of
sys-1(q544/+); teIs3 with missing pm7 muscle cells
(n>1000). Although we cannot be certain that an extra pm7 cell is
generated at the expense of a pm6 cell, or that a missing pm7 cell has become
a pm6 cell, the phenotype observed for teEx321 is consistent with the
lineage prediction for an MSa-to-MSp fate change. Likewise, the
sys-1(q544/+); teIs3 phenotype is consistent with the predicted
MSp-to-MSa fate change.
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). The four cells that comprise ring 1 are two pairs of
sisters (Ealaaa and Ealaap, Earaaa and Earaap), which derive from the most
anterior lineage of Eal and Ear, respectively
(Fig. 5C). These cells would be
the most sensitive to any anterior-to-posterior fate transformation within the
E lineage. All fate transformations within the E lineage should still give
rise to intestinal cells. Fortunately, intestinal cells that derive from
different branches of the E lineage exhibit different properties
(Fukushige et al., 2005;
Schroeder and McGhee, 1998).
For example, the gut phosphoesterase PHO-1, which is expressed strongly in all
other gut cells, is expressed weakly in the six cells that make up the first
two intestinal rings (Fig. 5D)
(Fukushige et al., 2005). We
observed that 100% (n=22) of
teEx321(Pmed-1gfp::sys-1) GFP-positive progeny exhibited
bright PHO-1 reporter GFP fluorescence in at least some of the four
anterior-most intestinal cells, compared with 0% (n=10) of wild-type
embryos (Fig. 5D). The first
gut ring is also responsible for attaching intestine anteriorly to the
pharyngeal/intestinal valve cells. We observed 13% (n=71) of
teEx321 GFP-positive progeny with intestine unattached to the pharynx
(not shown). These results are consistent with lineage defects among the cells
comprising ring 1. In addition, MSa also gives rise to the pm8 muscle cell,
which attaches the pharynx to the pharyngeal/intestinal valve cells
(Sulston et al., 1983).
Therefore, pm8 lineage defects could also contribute to the unattached
anterior intestine phenotype observed in teEx321 progeny.
|
), would
be the most sensitive to any posterior-to-anterior fate transformation within
the E lineage. In 23% (n=105) of larvae derived from
sys-1(q544/+); teIs3(Pmed-1gfp::pop-1), the posterior of
the intestine was not attached to the rectum (not shown). No defect in
intestinal attachment to pharynx was observed (n>1000). Similarly,
no defect in intestinal attachment to rectum was observed in
teEx321(Pmed-1gfp::sys-1) embryos (n>1000).
Staining with antibody ICB4 (Kemphues et
al., 1988) showed that, although the majority of embryos derived
from teEx321 and sys-1(q544/+); teIs3 had a disorganized
intestine, they had twenty, or close to twenty, intestinal cells
(n=50). Together, our results show that a high SYS-1-to-POP-1 ratio inhibits the anterior cell fate, whereas a low ratio inhibits the posterior fate in multiple A-P divisions during C. elegans embryogenesis.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SYS-1 functions as a limiting coactivator for POP-1 in the E blastomere
Nuclear POP-1 asymmetry has been observed in several asymmetric A-P sisters
whose differential cell fates require MAPK and/or Wnt signaling
(Herman, 2001;
Lin et al., 1998;
Lin et al., 1995;
Siegfried et al., 2004).
Signaling and POP-1 activation are required for the posterior cell fate and in
all cases these cells have a lower level of nuclear POP-1
(Herman, 2001;
Maduro et al., 2005b;
Shetty et al., 2005;
Siegfried and Kimble, 2002).
The requirement for lowered levels of nuclear POP-1 in cells in which POP-1
functions as an activator suggested a model invoking a co-activator for POP-1,
the amount of which is limiting with respect to POP-1
(Herman, 2001;
Kidd et al., 2005;
Shetty et al., 2005;
Siegfried et al., 2004;
Phillips et al., 2007). In
cells with high nuclear POP-1 levels, only a small portion of POP-1 would be
bound to the limiting co-activator, with the majority free to bind to
co-repressor(s) resulting in transcriptional repression of target genes. In
addition, POP-1 could be qualitatively different between asymmetric sisters,
also promoting preferential interaction of POP-1 with co-activator in
Wnt/MAPK-responsive cells.
We show here that the ß-catenin SYS-1, like the TCF protein POP-1, is important for endoderm fate. Our data argue that the SYS-1-to-POP-1 ratio, more so than the absolute SYS-1 and POP-1 levels, is critical in E blastomere fate specification. A high SYS-1-to-POP-1 ratio promotes, whereas a low SYS-1-to-POP-1 ratio antagonizes, E fate. In the most convincing demonstration, simply increasing SYS-1 levels by introducing extra copies of gfp::sys-1 was sufficient to transform MS blastomere fate to endoderm, or E fate. These results strongly support the model that SYS-1 levels are limiting with respect to POP-1 levels in the specification of endoderm fate.
Activation of Wnt target genes by coordinate regulation of ß-catenin/SYS-1 and TCF/POP-1
A high SYS-1-to-POP-1 ratio can be achieved by decreasing the level of
POP-1, increasing the level of SYS-1, or both. Our results demonstrate that
GFP::SYS-1 and POP-1 levels are simultaneously regulated in opposite
directions. Whereas the MOM-4/LIT-1/WRM-1 pathway plays the major role in
lowering nuclear POP-1 levels, GFP::SYS-1 asymmetry is regulated primarily by
the MOM-2/MOM-5/APR-1 pathway, with very little or no input from wrm-1,
lit-1 or mom-4 (Fig.
6). These observations offer an explanation for the synergy
observed by us and others between mutations in these two groups of genes:
whereas mom-2, mom-5 and mom-4 mutations alone have an
incompletely penetrant gutless phenotype, mom-2; mom-4 and mom-5;
mom-4 embryos have a 100% gutless phenotype
(Rocheleau et al., 1997;
Rocheleau et al., 1999;
Thorpe et al., 1997). Defects
in altering the level of either SYS-1 or POP-1 alone would not result in as
dramatic a change of the SYS-1-to-POP-1 ratio as defects in altering both.
Consistent with our results, Phillips et al. have recently shown that SYS-1
asymmetry in somatic gonad precursors is MOM-5-dependent, but LIT-1- and
WRM-1-independent (Phillips et al.,
2007). lit-1 or wrm-1 mutants, unlike
mom-4 mutants, produce 100% gutless embryos. It is possible that the
LIT-1/WRM-1 kinase regulates POP-1 transcriptional activity, in addition to
its role in regulating POP-1 nuclear levels. Supporting this possibility,
phosphorylation of TCF proteins by nemo-like kinase (NLK) has been reported to
inhibit the DNA-binding activity of TCFs
(Ishitani et al., 2003) and to
enhance transcription of Wnt targets
(Thorpe and Moon, 2004). We
propose that these two pathways specify endoderm fate by collaborating to very
efficiently elevate the SYS-1-to-POP-1 ratio in the E blastomere. Furthermore,
we propose that this redundancy helps to insulate a genetic system used in
multiple A-P divisions during C. elegans embryogenesis from
deleterious mutations, as well as ensuring that cell fate decisions are robust
in a rapidly dividing embryo that nevertheless maintains an invariant
lineage.
Studies in Drosophila also suggest that the ß-catenin/TCF
ratio can play an important role in Wnt signaling. wingless and
armadillo (arm) mutant phenotypes can be partially
suppressed by a reduction in Drosophila TCF (dTCF; also known as PAN
- Flybase) activity, whereas the phenotype of a weak wingless allele
is enhanced by overexpression of wild-type dTCF
(Cavallo et al., 1998). It
appears, therefore, that lower levels of dTCF, or higher levels of ARM,
analogous to what we show here, contribute to Wnt signal strength. However,
whereas regulation of ß-catenin levels by Wnt is well documented in
vertebrates and Drosophila, possible regulation of TCF levels by the
MAPK pathway is less well documented. A recent report that NLK promotes TCF
degradation by facilitating its interaction with an E3 ligase
(Yamada et al., 2006),
suggests that simultaneous regulation of ß-catenin and TCF levels in
opposite directions might not be unique to the C. elegans embryo.
The two ß-catenins, WRM-1 and SYS-1
It has been suggested that the cellular functions carried out by a single
ß-catenin in vertebrates and flies are carried out by multiple
ß-catenins in worms (Korswagen et
al., 2000). ß-catenin has been shown to undergo
importin-independent nuclear import, perhaps by directly binding to the
nuclear pore complex through its armadillo repeats, which resemble the heat
repeats of importin (Fagotto et al.,
1998). ß-catenin nuclear localization has been shown to then
facilitate nuclear import of LEF1 and TCF4 by direct binding
(Asally and Yoneda, 2005;
Hsu et al., 2006). It is
possible that WRM-1 is a specialized ß-catenin that functions as a
nuclear import receptor for LIT-1, or additional factors, in response to
Wnt/MAPK signaling during C. elegans embryogenesis.
It is interesting that two ß-catenins in C. elegans, SYS-1
and WRM-1, both function in endoderm precursor specification, exhibit an
elevated level in the posterior cell of all A-P divisions, function in
elevating the SYS-1-to-POP-1 ratio, have seemingly very different biological
activities and are regulated through different mechanisms. Our current data
are consistent with SYS-1 A-P asymmetry being regulated by differential
degradation between sister cells. This resembles the downregulation of
vertebrate ß-catenins in the absence of Wnt signaling
(Krieghoff et al., 2006), but
is different from the situation for WRM-1, the asymmetric levels of which are
dependent on IMB-4-mediated nuclear export
(Nakamura et al., 2005).
Recent work has shown that cortically localized WRM-1 represses its nuclear
accumulation (Mizumoto and Sawa,
2007). It is interesting to note that GFP::SYS-1 also localized to
centrosomes and differentially localized to cortex. However, the functional
significance, if any, of centrosomal and cortical SYS-1 localization requires
further investigation.
Pathways regulating endoderm
Analyses of the expression of the endoderm-specifying genes, end-1
and end-3, demonstrated that both the SKN-1/MED-1 and the POP-1 (and
now SYS-1) pathways contribute to the high level expression of these endoderm
genes (Maduro et al., 2005b;
Shetty et al., 2005). We
propose that transcriptional activation of end-1 and end-3
by either the SKN-1/MED-1 or POP-1/SYS-1 pathway alone is sufficient to
specify endoderm. In MS, where the SYS-1-to-POP-1 ratio is low, POP-1, in
conjunction with co-repressor(s), overrides transcriptional activation by
SKN-1/MED-1, resulting in no end-1 or end-3 expression and a
lack of endoderm fate. In E, a high SYS-1-to-POP-1 ratio allows POP-1 to
function as an activator, further elevating end-1 and end-3
transcription. Embryos with reduced expression of end-1 and
end-3 as a result of skn-1 depletion would be expected to be
sensitive to alterations in the SYS-1-to-POP-1 ratio, consistent with the
observed synergy between skn-1 and mom-2, mom-4 or
mom-5 mutations (Thorpe et al.,
1997), and the suppression of skn-1(zu67) by
teIs98(Ppie-1gfp::sys-1) (this study).
SYS-1 and POP-1 function in MS
POP-1 is also required for MS fate specification. However, the role of
POP-1 in MS specification is not simply repression of endoderm fate. In
embryos in which the endoderm-specifying genes end-1 and
end-3, as well as pop-1, are deleted, the MS blastomere
generates neither pharynx nor intestine
(Maduro et al., 2005a;
Zhu et al., 1997). This
indicates that POP-1 plays an important and direct role in specifying MS fate,
independent of its function in endoderm gene repression. We reported
previously that in a large percentage of embryos with artificially elevated
nuclear POP-1 levels in E, E also generates neither pharynx nor intestine
(Lo et al., 2004). Therefore,
a high level of nuclear POP-1 in E, although sufficient to repress endoderm,
is not always sufficient to promote mesoderm. A low level (but not complete
absence) of SYS-1 and a high level of nuclear POP-1 would appear to be
important for the activation of genes specifying the MS blastomere. Other
factors in addition to SYS-1 might also act with POP-1 to specify MS fate. A
candidate for such a factor is the T-box protein, TBX-35, which has recently
been shown to regulate MS-derived cell fates
(Broitman-Maduro et al., 2006).
Future studies will elaborate the roles of POP-1, SYS-1, TBX-35 and other
factors in MS fate specification.
SYS-1 and POP-1 function in reiterated A-P cell fate decisions
We show that in multiple A-P divisions, a low SYS-1-to-POP-1 ratio promotes
the anterior cell fate, whereas a high SYS-1-to-POP-1 ratio promotes the
posterior fate. This suggests that the SYS-1-to-POP-1 ratio might have a broad
role in controlling stepwise A-P binary decisions throughout C.
elegans embryogenesis. However, we did not observe the collapse of the
affected lineage to either the anterior-most or posterior-most cell fate by
overexpressing or removing SYS-1 or POP-1. We anticipated that all transgenic
strains isolated expressing altered levels of SYS-1 and/or POP-1 were likely
to have a non-fully penetrant defect in all divisions in which these proteins
might function. Full collapse of affected lineages would be expected to be
100% lethal and therefore not able to be isolated as a strain. In addition,
for transgenes utilizing the med-1 promoter to drive expression in
the E and MS lineages, transcription only occurs early in the lineage
(Maduro et al., 2001;
Robertson et al., 2004).
Therefore, there is expected to be a progressive decrease in GFP::SYS-1 levels
and a less severely altered SYS-1-to-POP-1 ratio as the embryo develops. It is
also possible that the SYS-1-to-POP-1 ratio might regulate only a subset of
cell divisions, despite exhibiting asymmetric levels in all A-P divisions.
Although asymmetric SYS-1 and nuclear POP-1 levels in AB8 cells are
dependent on Wnt/MOM-2, some of these AB cells do not contact P2 or its
descendants, which are the only cells that have been shown to be Wnt signaling
cells at this stage. It has recently been proposed that Wnt signaling from P2
and its descendants can influence the polarity of blastomeres that they do not
directly contact, because Wnt signaling can be passed on from cell to cell
(Bischoff and Schnabel, 2006).
However, the question remains: what initiates and regulates the asymmetry for
both POP-1 and SYS-1 after the AB8 stage, as both occur in mom-2
mutant embryos. Future studies involving blastomere isolation and
reassociation will be required to address this question.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/14/2685/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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