Division of Basic Sciences, Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
Accepted 24 April 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: C. elegans, Frizzled, polarity, POP-1, Wnt
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
POP-1 is related to TCF/Pangolin, a transcriptional effector of the
canonical Wnt signaling pathway, and POP-1 has been shown to function in
canonical Wnt signaling during larval development
(Lin et al., 1995;
Herman, 2001
). However, POP-1
asymmetry in the early embryo is regulated by a non-canonical Wnt pathway,
with parallel input from a mitogen-activated protein kinase (MAPK) pathway
(for a review, see Korswagen,
2002
). Components of these pathways include MOM-2/Wnt,
MOM-5/Frizzled, WRM-1/beta-catenin, MOM-4/MAPKKK/TAK1 and LIT-1/Nemo. Sister
cells show POP-1 asymmetry because they differ in their nucleo/cytoplasmic
distributions of POP-1 (Maduro et al.,
2002
). Studies with cultured vertebrate cells suggest that
WRM-1/beta-catenin can activate LIT-1/Nemo, resulting in phosphorylated POP-1
that accumulates in the cytoplasm
(Rocheleau et al., 1999
).
How is the a/p polarity system established? The most detailed experimental
studies to date have focused on the development of the EMS cell
(Goldstein, 1992;
Goldstein, 1993
). EMS divides
into an anterior, mesodermal precursor and a posterior, endodermal precursor.
This a/p polarity is induced during the 4-cell stage of embryogenesis by a
neighboring cell called P2. For example, removing P2
causes both EMS daughters to have anterior fates (high POP-1) and
repositioning P2 on the opposite surface of EMS reverses the
polarity of the division.
Relatively little is known about the cellular events that establish a/p
polarity in other embryonic cells. At the 2-cell stage, the posterior cell is
called P1 (the parent of P2 and EMS) and the anterior
cell is called AB (see Fig. 1).
Within the AB lineage, POP-1 asymmetry is first detectable after the third
division of AB, when there are four a/p sister pairs of AB descendants
(Lin et al., 1998); for
convenience we refer to this stage as the AB8 stage. POP-1 function
is essential for a/p differences in cell fate within each of the four sister
pairs of AB8 cells (Lin et al.,
1998
). Previous studies on how a/p differences are generated in
the AB lineage have reached contradictory conclusions. In one set of
experiments, AB was separated from P1 and allowed to develop to the
AB16 stage (Wittmann et al.,
1997
). As many as eight of the AB16 cells expressed a
transgenic marker that normally is expressed in the eight posterior
AB16 cells, suggesting that AB has an inherent a/p polarity. In a
different study, videomicroscopy was used to follow AB development after
killing P1 or P1 descendants
(Hutter and Schnabel, 1995
).
Several AB8 cells showed posterior to anterior transformations in
fate after killing P1, but not P1 descendants,
suggesting that P1 induces an a/p polarity in AB that is maintained
in a latent form until the AB8 stage.
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell isolations
Individual embryonic cells were isolated from devitellinized embryos by
gently drawing embryos in and out of a drawn-out capillary needle as described
previously (Edgar, 1995). Cells
were cultured in medium consisting of 5% L-15 (Gibco), 10% fetal calf serum
(Gibco), and 4.7% sucrose, adjusted to 320-330 mOsm. To devitellinize embryos,
eggs were placed in hypochlorite solution [6% NaOCl, 2.5 N KOH] on an inverted
microscope slide for 3.5 minutes, then rinsed three times with 0.25 M HEPES,
pH 7.0 before transfer to culture medium.
Immunostaining and analysis of embryos and cultured blastomeres
Isolated cells were micropipetted directly into a drop of fixative [2%
paraformaldehyde, 60 mM PIPES, 25 mM HEPES (pH 6.8), 10 mM EGTA, 2 mM
MgCl2] on a poly-L-lysine (Sigma)-coated glass slide. After five
minutes, excess fixative was removed and slides were placed in -20°C
acetone for 5 minutes. Cells were rinsed twice in Tris-Tween [100 mM Tris-HCl
(pH 7.5), 200 mM NaCl, 0.1% Tween], then incubated with 10% normal goat serum
(Gibco) in Tris-Tween at room temperature for 30 minutes. Cells were then
incubated overnight at room temperature with primary antisera. Fixation and
immunostaining of intact embryos was as described previously
(Lin et al., 1998). The
following dilutions of antibodies/antisera were used: anti POP-1 [1:2500 mouse
mABRL2 (P4G4) (Lin et al.,
1998
)]; midbody staining [1:50 rabbit anti-PKL-2 (kindly provided
by M. Land)]; P-granules [1:17000 rabbit anti-PGL-1
(Kawasaki et al., 1998
)].
Secondary antibodies were conjugated to either Cy-3 (Jackson ImmunoResearch
Laboratories) or FITC (Tago). Cells were stained with DAPI
(4,6-diamidino-2-phenylindole) at 60 ng/ml for five minutes. POP-1 was scored
only when the PKL-2 staining pattern provided an unambiguous indication of
sister pairs, thus some results are scored by the number of sister pairs
rather than by the number of experiments.
dsRNA-mediated interference (RNAi)
Standard techniques were used to synthesize double-stranded RNA (dsRNA)
from T7 promoter-tagged, PCR-amplified genomic DNA for mom-2, mom-5,
goa-1, and gpa-16. PCR primers were chosen to span exons and
generate fragments between 0.5 and 2 kb in size. L4 or young adult
hermaphrodites were soaked overnight with dsRNA
(Tabara et al., 1998).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Why does P2 signaling fail to alter POP-1 levels in the ABp
daughters? Because P2 has been shown to interact with ABp, but not
EMS, through a separate, Notch-related signaling pathway, we wondered whether
this pathway precluded POP-1 asymmetry. However, embryos that were depleted of
the receptor GLP-1/Notch by glp-1(RNAi) did not show POP-1 asymmetry
at the ABp division, similar to wild-type embryos (0/5). A second possibility
is that the transverse divisions of the AB2 cells, perpendicular to
P2, prevent POP-1 asymmetry. A previous study noted that EMS may
fail to respond to induction if it divides perpendicular to, rather than in
line with, P2, although this analysis is complicated by the fact
that signaling from P2 normally orients the EMS division
(Goldstein, 1995). To examine
whether division orientation influenced POP-1 asymmetry, we used RNAi to
inhibit the functions of the G alpha proteins encoded by goa-1 and
gpa-16 (see Materials and Methods); such embryos appear to have
random spindle orientations (Gotta and
Ahringer, 2001
; Zwaal et al.,
1996
). We observed that a/p, but not transverse, divisions of EMS
resulted in POP-1 asymmetry (6/6 and 0/5, respectively;
Fig. 4A). Similarly, a/p, but
not transverse, divisions of the AB2 cells resulted in POP-1
asymmetry (4/4 and 0/4, respectively; Fig.
4A). When the functions of goa-1 and gpa-16 were
inhibited in mom-2(or42) mutant embryos, POP-1 asymmetry was not
detectable after either an a/p, or transverse, division of the AB2
daughters (0/12 and 0/6, respectively). As a second method for altering the
AB2 division axis, a laser microbeam was used to fuse the
AB2 daughters together immediately after their birth. As a fused
AB2 cell enters mitosis, it often develops two largely separate
spindles that orient a/p. After the tetrapolar division, the posterior
daughters usually showed lower levels of nuclear POP-1 than the anterior
daughters (16/18 sister pairs; Fig.
4C).
|
P1 descendants induce high/low POP-1 polarity
The AB2 polarization assay described above (long red arrow in
Fig. 1) was used to examine the
signaling properties of several cells in the early embryo. We found that graft
AB2, AB4, AB8, P1 and EMS cells
could not induce POP-1 asymmetry in this assay
(Table 1). In contrast, the
P1 descendants E, C and P3 appeared equivalent to
P2 in their ability to induce POP-1 asymmetry with high/low
polarity (Table 1). The
P1 descendant MS usually induced POP-1 asymmetry, however the
asymmetry was markedly less than that observed with the other P1
descendants (data not shown).
|
|
Videomicroscopy was used to examine the cell contacts and spindle orientations of the four AB4 cells in live embryos (n=12); these cells are named ABal, ABar, ABpl and ABpr. ABal contacts only MS, and its spindle normally orients toward MS. ABpr and ABpl contact MS, E and C simultaneously, however their spindles orient toward E and often move slightly toward E before cell division (Fig. 5A,C). Finally, ABar contacts C and MS, and its spindle is oriented approximately between C and MS. The observation that three of the AB4 cells contact multiple signaling cells simultaneously suggests that the AB4 cells distinguish, or integrate, the various signals. We used devitellinized 8-cell embryos to test whether an AB4 cell could distinguish between signals from MS and E. In devitellinized embryos, the spindles of the AB4 cells usually orient toward MS (see Fig. 1), and the AB4 daughters that show POP-1 asymmetry invariably have high/low POP-1 polarity with respect to the position of MS. We therefore grafted an E cell onto one of the AB4 cells of a host embryo at a site either opposite, or orthogonal, to the site of contact with MS (yellow arrow in Fig. 1). In each of six experiments, the spindle of the AB4 cell aligned toward the graft E, rather than the host MS, and divided with high/low POP-1 polarity relative to the graft E (Fig. 5I,J). We conclude that signaling from E predominates over signaling from MS. Similarly, we infer that signaling from C may predominate over MS signaling in normal development because POP-1 polarity in ABar is high/low relative to C, rather than MS.
MOM-2/Wnt and POP-1 asymmetry at the AB8 stage
Consistent with previous results by others, we found that mom-2
mutants at the AB8 stage had a partial loss of POP-1 asymmetry
between sister pairs of AB8 cells
(Table 2) (see also
Lin et al., 1998;
Meneghini et al., 1999
). To
examine the role of MOM-2/Wnt in signaling, we isolated individual cells from
mom-2(or42) mutant embryos to use as graft cells in the
AB2 polarization assay with a wild-type host embryo. Graft
P2 or C cells from a mom-2(or42) mutant were unable to
induce POP-1 asymmetry in this assay, whereas the MS cell sometimes induced
weak POP-1 asymmetry (Table 1;
the E cell adopts an MS-like fate in mom-2 mutants, and was not
tested separately). We next grafted either a C or MS cell from a
mom-2(or42) mutant onto an isolated, wild-type AB4 cell.
Similar to the above results, C did not align the AB4 spindle (0/6)
and did not induce POP-1 asymmetry between the AB4 daughters
(Table 1). In contrast, MS
aligned the AB4 spindle (15/15) and induced high/low POP-1 polarity
with either a wild-type or reduced level of POP-1 asymmetry
(Fig. 5G and
Table 1). We conclude that
MOM-2/Wnt is essential for signaling from C, but not MS. As described above,
in wild-type development one AB4 cell (ABar) contacts MS and C
simultaneously, and the ABar daughters show high/low POP-1 polarity with
respect to C. In mom-2(or42) mutant embryos, POP-1 asymmetry often
was not detectable between the daughters of ABar (7/13 embryos). However, when
present, the POP-1 polarity was reversed and oriented high/low with respect to
MS (6/6). Thus, MS, rather than C, may orient the polarity of the ABar
division in mom-2 mutant embryos.
|
POP-1 asymmetry beyond the AB8 stage
The above results provide evidence that P1 descendants play an
important role in generating POP-1 asymmetry between the AB8 sister
pairs, a stage when POP-1 plays a critical role in specifying a/p differences
in cell fate. Surprisingly, we found that if an isolated AB cell was allowed
to develop to the AB16 or AB32 stages, most of the
resulting sister pairs showed POP-1 asymmetry (80%, 45 sister pairs and 97%,
107 sister pairs, respectively; Fig.
3C,D). Although POP-1 asymmetry was never observed between the
daughters of an isolated EMS cell (0%, 8 sister pairs), we often observed
POP-1 asymmetry between the granddaughters and great-granddaughters (56%, 18
sister pairs and 95%, 40 sister pairs, respectively). Therefore AB and EMS
descendants after the AB8 stage have an ability to generate POP-1
asymmetry that appears to be independent of prior exposure to the signaling
cells P2, MS, E, P3 or C.
We wanted to determine whether POP-1 asymmetry in sister pairs of AB16 cells required signaling between AB descendants. For these experiments we separated AB from P1, then separated each of the successive descendants of AB immediately after each cell division. The sequentially isolated cell was allowed to divide one additional time before staining the resulting sister pair for POP-1. As expected, POP-1 asymmetry was not observed between sequentially isolated sister pairs of AB4 or AB8 cells (0/11 and 0/10, respectively). In contrast, POP-1 asymmetry was usually present between sequentially isolated sister pairs of AB16 and AB32 cells (18/35 and 29/29, respectively; Fig. 3E,F).
MOM-5/Frizzled, but not MOM-2/Wnt, is essential for isolated AB
descendants to develop POP-1 asymmetry
Intact mom-2(or42) mutant embryos show a variable reduction or
loss of POP-1 asymmetry between the EMS daughters and between sister pairs of
AB8 cells (Table 2).
However, mom-2(or42) mutants and mom-2(RNAi) embryos
analyzed after the AB8 stage showed strong POP-1 asymmetry between
most sister pairs of AB or EMS descendants
(Fig. 2C,D;
Table 2 and data not shown).
POP-1 polarity was abnormal in these mutants, for example, many transverse
sister pairs showed POP-1 asymmetry, and some a/p sister pairs had low/high
polarity. We sequentially isolated AB8 cells from
mom-2(or42) embryos and from mom-1(or10); mom-2(RNAi) double
mutant embryos, then allowed these cells to divide once or twice in culture
before immunostaining for POP-1. The mom-1 gene encodes the only
C. elegans protein related to Prc, and should thus be required for
secretion of MOM-2 and other Wnt family members
(Kadowaki et al., 1996;
Rocheleau et al., 1997
). We
found that the daughters and granddaughters of the sequentially isolated
AB8 cells usually showed robust POP-1 asymmetry
(Table 3). Thus, MOM-2/Wnt is
not essential for POP-1 asymmetry after the AB8 stage, but has a
role in POP-1 polarity.
|
Interactions resulting in low/high POP-1 polarity
A wild-type, sequentially isolated AB16 cell can divide into a
pair of sister cells with POP-1 asymmetry. However, when two adjacent
AB16 cells were allowed to divide, their spindles almost invariably
aligned to generate a line of two sister pairs with low/high-high/low POP-1
polarity (Fig. 3G). This was
the case irrespective of whether two sequentially isolated AB16
cells were combined and allowed to divide once (19/21 experiments), or a
sequentially isolated AB8 cell was allowed to divide twice (30/32).
Identical results were obtained for the sequentially isolated daughters of the
EMS cell (5/5). These results suggest that each parental cell aligned the
spindle of the other, with a reciprocal induction of low/high POP-1 polarity.
Thus, these cells induce low/high POP-1 in the distal/proximal daughters of
the responding cell, in contrast to the high/low POP-1 induced by MOM-2/Wnt
signaling.
We observed two additional examples in which low/high POP-1 polarity appeared to be induced in the distal/proximal daughters of a responding cell. First, experiments combining an AB4 cell with an MS, E or C cell, or combining an AB16 cell with a C cell, resulted in a high/low-high/low pattern of POP-1 polarity (Fig. 5G,H). This pattern is consistent with the hypothesis that the AB4 cell induced low/high POP-1 polarity in the MS daughters, whereas MS simultaneously induced high/low POP-1 polarity in the AB4 daughters. The second example involved POP-1 polarity in C and P3, the daughters of the P2 cell (see legend to Fig. 6 for details). In wild-type or mom-2(or42) mutant embryos, C is born proximal to ABp and has high POP-1, whereas P3 is distal and has low POP-1 (Fig. 6A, and see Fig. 1). In a devitellinized embryo, in which P2 does not contact ABp, we found that POP-1 polarity was reversed in the P2 daughters (Fig. 6C). We observed the same polarity reversal when isolated EMS and P2 cells were combined and allowed to divide (Fig. 6E). However, the normal pattern of POP-1 polarity was restored after ABp and ABa cells were grafted onto the P2 cell of a devitellinized wild-type host embryo (position of blue arrow in Fig. 1). This was the case irrespective of whether the graft ABp and ABa cells originated from wild-type embryos (10/10), or from mom-2(or42) mutants (5/5; Fig. 6G). Thus, AB2 cells can induce low/high POP-1 polarity in the P2 daughters.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At the next round of cell division, each AB4 cell divides into
daughters with POP-1 asymmetry, as do the subsequent AB8 and
AB16 cells. Previous studies reached contradictory conclusions
about whether asymmetry in the AB lineage was determined intrinsically or
through cell interactions (see Introduction). Our present study provides a
resolution of this paradox. We observed POP-1 asymmetry in sister pairs of
AB16 cells, but not AB8 cells, which were derived from
an isolated AB. These results are consistent with those from a previous study
that killing P1 caused posterior to anterior fate transformations
at the AB8 stage; POP-1 remains high in all of the AB8
cells after P1 is removed, so all cells should adopt anterior fates
(Hutter and Schnabel, 1995).
Our results also support the study of Wittmann et al., which suggested AB
descendants had an intrinsic asymmetry independent of P1; their
study used a transgenic marker that is expressed at the AB16 stage
(Wittmann et al., 1997
). Thus,
cell interactions are essential for POP-1 asymmetry at, but not after, the
AB8 stage. Reported examples of AB8 cells that correctly
adopted posterior fates after killing or removing P1 may represent
cells in transition between the two modes of generating POP-1 asymmetry
(Gendreau et al., 1994
;
Hutter and Schnabel,
1995
).
POP-1 asymmetry at the AB8 stage
Although we propose that cell interactions determine POP-1 asymmetry in the
early AB lineage, our results argue against a previous model that the primary
interaction is between AB and P1 (latent polarity model), rather
than between AB descendants and P1 descendants
(Hutter and Schnabel, 1995).
We have shown that exposing AB to P1 is not sufficient to generate
POP-1 asymmetry at the AB8 stage, and that P1 does not
provide signaling activity in our assays. In the previous study, laser
irradiation of the P1 descendants did not prevent a/p polarity at
the AB8 stage, suggesting that the P1 descendants were
not essential for polarization (Hutter and
Schnabel, 1995
). However, laser-irradiation does not effectively
prevent P2 from signaling EMS, nor does it prevent P2
from signaling ABp through a separate, Notch-related signaling pathway
(Mello et al., 1994
). All of
the known genes that are involved in POP-1 asymmetry in the early embryo are
expressed maternally, as are the components of the Notch pathway. Thus, it may
be difficult to eliminate translation of maternally provided mRNAs by
laser-irradiating the early embryonic cells.
The AB4 cells contact one or more of the P1
descendants MS, E, C and P3. We have shown that each of these
P1 descendants can induce high/low POP-1 polarity in AB
descendants, similar to the ability of P2 to induce high/low POP-1
polarity in the daughters of EMS. Indeed, at least two of the P1
descendants, E and C, appear to be fully equivalent to P2 in their
ability to polarize EMS. C and P2 require MOM-2/Wnt for signaling,
however the MS cell appears to provide an additional, or alternative, high/low
signal. We presume this unidentified signal is the source of POP-1 asymmetry
in the AB4 daughters in mom-2 mutants. The C.
elegans genome can encode five Wnt proteins, raising the possibility that
other Wnts contribute to high/low signaling during the first few embryonic
cell divisions (Ruvkin and Hobert,
1998).
The AB4 spindles, in contrast to the AB2 spindles,
align with the signaling cells P2, MS, E or C in cell culture
experiments. This difference might result from the synthesis of new regulators
in the AB4 cells, or from the degradation of inhibitors present in
the AB2 cells. In normal development each AB4 division
results in an anterior/posterior pair of daughters. However, all of these
divisions are oblique, and some are nearly transverse, with respect to the a/p
axis of the egg (see Fig.
5A,C). The alignment of the AB4 spindles with
posterior-localized signaling cells such as E provides a partial explanation
for the a/p pattern of high/low POP-1 polarity at the AB8 stage. In
addition, the ability of MS to align the ABal spindle may be crucial for the
normal pattern of Notch-mediated interactions. MS can interact with the ABal
daughters through a Notch-like signaling pathway, and the alignment of the
ABal spindle by MS ensures that only one daughter undergoes this interaction
(Mello et al., 1994;
Hutter and Schnabel, 1994
).
Detailed cell-killing experiments on early C. elegans embryos have
revealed unexpected and surprisingly complex networks of positive and negative
interactions that are involved in the specification of muscle fates and that
are presumably integrated for proper development
(Schnabel, 1994;
Schnabel, 1995
). Although the
molecular basis of muscle specification is not yet understood, our present
study shows that the embryo contains multiple cells with the potential to
influence POP-1 polarity. In our cell culture experiments, signaling from E
predominated over signaling from MS when an AB4 cell simultaneously
contacted both E and MS. Similarly, we propose that signaling from C
predominates over signaling from MS in polarizing the division of ABar. The
polarity of POP-1 at the ABar division is reversed in mom-2 mutants,
presumably because of the absence of a signal from C and the persistence of a
non-MOM-2/Wnt signal from MS. An analogous example was found in a previous
analysis of the C. elegans Wnt family member, EGL-20, which functions
in the asymmetric division of a larval epidermal cell called V5. In the
absence of EGL-20/Wnt, the normal asymmetry of the V5 division is reversed by
signaling from a neighboring cell (Whangbo
et al., 2000
).
POP-1 asymmetry after the AB8 stage
By the AB16 stage, the descendants of both AB and EMS have an
ability to generate POP-1 asymmetry that appears to be independent of prior
contact with signaling cells that express MOM-2/Wnt. This conclusion is based
on experiments in which cells were sequentially isolated in culture, but is
supported by the observation that intact mom-2;mom-1 mutant embryos
show robust POP-1 asymmetry between sister pairs at the AB16 and
later stages. Although sequentially isolated AB16 cells can divide
with POP-1 asymmetry, contact with other AB descendants can determine POP-1
polarity. These interactions induce low/high POP-1 polarity, in contrast to
the high/low polarity induced by MOM-2/Wnt signals. Because adjacent
AB16 cells divide with mirror image POP-1 polarity, we assume that
each cell can both signal and respond to signaling. Thus, a single, isolated
AB16 cell may generate POP-1 asymmetry through self-signaling.
MOM-5/Frizzled is essential for POP-1 asymmetry in isolated cells that have
not been exposed to MOM-2/Wnt signaling. Therefore MOM-5/Frizzled may be a
component of the signaling pathway that generates low/high POP-1 polarity
independent of MOM-2/Wnt. Drosophila Frizzled is an essential
component of the planar cell polarity pathway, however the role of Wnt
proteins has not been determined (Lawrence
et al., 2002). It will be of interest to determine whether other
genes involved in Drosophila planar cell polarity have functions in
low/high signaling in C. elegans. MOM-4/MAPKKK and proteins such as
LIT-1/Nemo and WRM-1/Beta-catenin are essential for POP-1 asymmetry in AB
descendants, and thus appear to be core components of the asymmetry-generating
machinery (this study) (Kaletta et al.,
1997
; Lin et al.,
1998
; Meneghini et al.,
1999
; Rocheleau et al.,
1997
; Rocheleau et al.,
1999
).
The observation that POP-1 asymmetry is present in intact mom-5
mutant embryos, but not in mom-2;mom-5 double mutant embryos,
suggests that MOM-2/Wnt signaling can induce POP-1 asymmetry independent of
MOM-5/Frizzled. These results support the view from previous genetic studies
that MOM-2/Wnt and MOM-5/Frizzled have overlapping, but distinct roles in the
early embryo (Rocheleau et al.,
1997; Schlesinger et al.,
1999
). A survey of gene expression patterns in C. elegans
embryos has detected mRNAs corresponding to at least two additional
Frizzled-related proteins that are candidate receptors for MOM-2/Wnt (Y.
Kohara, personal communication;
http://nematode.lab.nig.ac.jp/).
However, our present study indicates that these Frizzleds cannot be
functionally redundant with MOM-5 for the POP-1 asymmetry shown by isolated,
cultured cells.
When two adjacent AB8 cells divide in culture they generate a line of two sister pairs with low/high-high/low POP-1 polarity. In an intact, normal embryo, similarly oriented divisions of adjacent AB8 cells would be expected to produce sister pairs with high/low-high/low POP-1 polarity. Thus, the behavior of the isolated cells does not reproduce the normal pattern of POP-1 polarity. Among the cell culture experiments described here, the only condition that resulted in high/low-high/low POP-1 polarity involved combining a low/high signaling cell (AB4) with a high/low signaling cell such as MS, E or C. Thus, it is possible that the normal pattern of POP-1 involves two distinct signaling pathways. We have shown that isolated AB descendants remain responsive to MOM-2/Wnt signaling until at least the AB16 stage, however we do not yet know whether Wnt signals persist in normal embryos at the AB16 and later stages.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Edgar, L. G. (1995). Blastomere culture and analysis. In Caenorhabditis Elegans: Modern Biological Analysis of an Organism. Vol. 48 (ed. H. F. Epstein and D. C. Shakes), pp. 303-321. San Diego: Academic Press.
Gendreau, S. B., Moskowitz, I. P., Terns, R. M. and Rothman, J. H. (1994). The potential to differentiate epidermis is unequally distributed in the AB lineage during early embryonic development in C. elegans. Dev. Biol. 166,770 -781.[CrossRef][Medline]
Goldstein, B. (1992). Induction of gut in Caenorhabditis elegans embryos. Nature 357,255 -257.[CrossRef][Medline]
Goldstein, B. (1993). Establishment of gut fate
in the E lineage of C. elegans: the roles of lineage-dependent
mechanisms and cell interactions. Development
118,1267
-1277.
Goldstein, B. (1995). An analysis of the
response to gut induction in the C. elegans embryo.
Development 121,1227
-1236.
Gotta, M. and Ahringer, J. (2001). Distinct roles for G-alpha and G-betagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos. Nat. Cell Biol. 3,297 -300.[CrossRef][Medline]
Herman, M. (2001). C. elegans
POP-1/TCF functions in a canonical Wnt pathway that controls cell
migration and in a non-canonical Wnt pathway that controls cell
polarity. Development
128,581
-590.
Hutter, H. and Schnabel, R. (1994).
glp-1 and inductions establishing embryonic axes in C. elegans.Development 120,2051
-2064.
Hutter, H. and Schnabel, R. (1995).
Specification of anterior-posterior differences within the AB lineage in the
C. elegans embryo: a polarising induction.
Development 121,1559
-1568.
Kadowaki, T., Wilder, E., Klingensmith, J., Zachary, K. and Perrimon, N. (1996). The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing. Genes Dev. 10,3116 -3128.[Abstract]
Kaletta, T., Schnabel, H. and Schnabel, R. (1997). Binary specification of the embryonic lineage in Caenorhabditis elegans. Nature 390,294 -298.[CrossRef][Medline]
Kawasaki, I., Shim, Y. H., Kirchner, J., Kaminker, J., Wood, W. B. and Strome, S. (1998). PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans.Cell 94,635 -645.[Medline]
Korswagen, H. C. (2002). Canonical and non-canonical Wnt signaling pathways in Caenorhabditis elegans: variations on a common signaling theme. BioEssays 24,801 -810.[CrossRef][Medline]
Lawrence, P. A., Casal, J. and Struhl, G.
(2002). Towards a model of the organisation of planar polarity
and pattern in the Drosophila abdomen.
Development 129,2749
-2760.
Lin, R., Hill, R. J. and Priess, J. R. (1998). POP-1 and anterior-posterior fate decisions in C. elegans embryos. Cell 92,229 -239.[Medline]
Lin, R., Thompson, S. and Priess, J. R. (1995). pop-1 encodes an HMG box protein required for the specification of a mesoderm precursor in early C. elegans embryos. Cell 83,599 -609.[Medline]
Maduro, M. F., Lin, R. and Rothman, J. H. (2002). Dynamics of a developmental switch: recursive intracellular and intranuclear redistribution of Caenorhabditis elegans POP-1 parallels Wnt-inhibited transcriptional repression. Dev. Biol. 248,128 -142.[CrossRef][Medline]
Mango, S. E., Thorpe, C. J., Martin, P. R., Chamberlain, S. H.
and Bowerman, B. (1994). Two maternal genes, apx-1
and pie-1, are required to distinguish the fates of equivalent
blastomeres in the early Caenorhabditis elegans embryo.
Development 120,2305
-2315.
Mello, C. C., Draper, B. W., Krause, M., Weintraub, H. and Priess, J. R. (1992). The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos. Cell 70,163 -176.[Medline]
Mello, C. C., Draper, B. W. and Priess, J. R. (1994). The maternal genes apx-1 and glp-1 and establishment of dorsal-ventral polarity in the early C. elegans embryo. Cell 77,95 -106.[Medline]
Meneghini, M. D., Ishitani, T., Carter, J. C., Hisamoto, N., Ninomiya-Tsuji, J., Thorpe, C. J., Hamill, D. R., Matsumoto, K. and Bowerman, B. (1999). MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans.Nature 399,793 -797.[CrossRef][Medline]
Moskowitz, I. P., Gendreau, S. B. and Rothman, J. H.
(1994). Combinatorial specification of blastomere identity by
glp-1-dependent cellular interactions in the nematode
Caenorhabditis elegans. Development
120,3325
-3338.
Nance, J. and Priess, J. R. (2002). Cell polarity and gastrulation in C. elegans. Development 129,387 -397.[Medline]
Neumann, C. J. and Cohen, S. M. (1997).
Long-range action of Wingless organizes the dorsal-ventral axis of the
Drosophila wing. Development
124,871
-880.
Rocheleau, C. E., Downs, W. D., Lin, R., Wittmann, C., Bei, Y., Cha, Y. H., Ali, M., Priess, J. R. and Mello, C. C. (1997). Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 90,707 -716.[CrossRef][Medline]
Rocheleau, C. E., Yasuda, J., Shin, T. H., Lin, R., Sawa, H., Okano, H., Priess, J. R., Davis, R. J. and Mello, C. C. (1999). WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.Cell 97,717 -726.[Medline]
Ruvkin, G. and Hobert, O. (1998). The taxonomy
of developmental control in Caenorhabditis elegans.Science 282,2033
-2041.
Schlesinger, A., Shelton, C. A., Maloof, J. N., Meneghini, M.
and Bowerman, B. (1999). Wnt pathway components orient a
mitotic spindle in the early Caenorhabditis elegans embryo without
requiring gene transcription in the responding cell. Genes
Dev. 13,2028
-2038.
Schnabel, R. (1994). Autonomy and nonautonomy in cell fate specification of muscle in the Caenorhabditis elegans embryo: a reciprocal induction. Science 263,1449 -1452.[Medline]
Schnabel, R. (1995). Duels without obvious
sense: counteracting inductions involved in body wall muscle development in
the Caenorhabditis elegans embryo.
Development 121,2219
-2232.
Schnabel, R., Hutter, H., Moerman, D. and Schnabel, H. (1997). Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of development and regional specification. Dev. Biol. 184,234 -265.[CrossRef][Medline]
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J. N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100,64 -119.[Medline]
Tabara, H., Grishok, A. and Mello, C. C.
(1998). RNAi in C. elegans: soaking in the genome
sequence. Science 282,430
-431.
Thorpe, C. J., Schlesinger, A., Carter, J. C. and Bowerman, B. (1997). Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm. Cell 90,695 -705.[CrossRef][Medline]
Way, J. C., Wang, L., Run, J.-Q. and Hung, M. (1994). Cell polarity and the mechanism of asymmetric cell division. BioEssays 16,925 -931.[Medline]
Whangbo, J., Harris, J. and Kenyon, C. (2000).
Multiple levels of regulation specify the polarity of an asymmetric cell
division in C. elegans. Development
127,4587
-4598.
Wittmann, C., Bossinger, O., Goldstein, B., Fleishmann, M.,
Kohler, R., Brunschwig, K., Tobler, H. and Muller, F. (1997).
The expression of the C. elegans labial-like Hox gene ceh-13
during early embryogenesis relies on cell fate and on anteroposterior cell
polarity. Development
124,4193
-4200.
Zecca, M., Basler, K. and Struhl, G. (1996). Direct and long-range action of a Wingless morphogen gradient. Cell 87,833 -844.[Medline]
Zwaal, R. R., Ahringer, J., van Luenen, H. G., Rushforth, A., Anderson, P. and Plasterk, R. H. (1996). G proteins are required for spatial orientation of early cell cleavages in C. elegans embryos. Cell 86,619 -629.[Medline]