Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
* Author for correspondence (e-mail: paul.hamel{at}utoronto.ca)
Accepted 26 October 2001
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Summary |
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Key words: Pax3, Wnt-signaling, Dishevelled, Focal adhesion, JNK, Frizzled
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Introduction |
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Tissue morphogenesis is characterized by the coordinated effect of several
different cell behaviors including changes to cell shape, position, adhesion,
proliferation and death (Atchley and Hall,
1991; Gumbiner,
1996
). Patterning of the vertebrate neural tube, for example,
involves a coordinated set of cell behaviors known as convergent extension,
which results in the simultaneous narrowing of the tissue in one dimension and
its lengthening in a perpendicular dimension
(Colas and Schoenwolf, 2001
).
Characterization of Drosophila and vertebrate morphogenetic mutants
has revealed the common use of conserved signaling cascades such as the
c-Jun-N-terminal kinase/stress activated protein kinase (JNK/SAPK) pathway
during morphogenesis (Noselli,
1998
; Noselli and Agnes,
1999
; Sokol,
2000
). JNK signaling pathways have been specifically implicated in
processes such as dorsal closure, thorax closure and in planar tissue/cell
polarity (Martin-Blanco et al.,
2000
; Noselli,
1998
; Zeitlinger and Bohmann,
1999
). Additionally, a signaling cascade involving a Wnt receptor,
Frizzled, and a downstream effector, Dishevelled, is implicated in the
regulation of morphogenesis in Drosophila and is also coupled to JNK
activation in tissue polarization (Sokol,
2000
). This Frizzled-mediated signaling cascade, which regulates
planar cell polarity (PCP) in Drosophila, appears homologous to a
Wnt-signaling cascade recently shown to regulate convergent extension
movements in vertebrates (Heisenberg et
al., 2000
; Wallingford et al.,
2000
).
Numerous studies have suggested a role for Pax genes in the regulation of
Wnt signaling (Dahl et al.,
1997; Uusitalo et al.,
1999
). One such study has revealed that the combined activity of
Pax3 and Pax7 is required for normal Wnt4 expression in the neural tube during
murine development (Mansouri and Gruss,
1998
). Analogous to the consequences of mutant Pax genes,
mutations to Wnt and to JNK genes in mice also result in
morphological defects in specific tissues. For example, as for Pax3
mutant mice, mutations to distinct Wnt genes result in neural tube patterning
defects (Uusitalo et al.,
1999
). Thus, on the basis of these and other data, we hypothesized
that Pax proteins regulate one or more of the aforementioned cell behaviors
through modulation of signaling cascades controlling morphogenetic cell
behavior.
Using an in vitro approach, we recently showed that ectopic Pax3 expression
induces cell aggregation and a phenotypic mesenchymal-to-epithelial transition
in vitro (Wiggan et al.,
2002). This study also confirmed c-met as a direct transcriptional
target of Pax3 and showed the ability of Pax3-induced cell aggregates to
dissociate in response to hepatocyte growth factor/scatter factor (HGF/SF). In
the present study we examined the cellular mechanisms by which Pax3 induced
cell aggregation of Saos-2 cells. We show that ectopic Pax3 expression in
Saos-2 cells induced cell behaviors that resulted in morphogenetic cell
movements similar to those used by cells during vertebrate gastrulation and
neurulation. These Pax3-induced morphogenetic cell movements are associated
with the induction of a signaling cascade resulting in activation of JNK and
in altered subcellular localization of Dishevelled, Frizzled and activated
JNK. Our results showing the localization of Dishevelled and Frizzled to the
actin cytoskeleton and their redistribution in response to Pax3 expression
indicate a novel mechanism by which Pax3 may regulate pattern formation during
development.
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Materials and Methods |
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Cell culture
Saos-2 osteosarcoma cells were grown in DMEM supplemented with 10% fetal
calf serum (Sigma, Oakville, ON, Canada). For infections, typically
1x105 cells were plated on 35 mm plates (Nunc) the day before
infection. The following day, cells were infected at a multiplicity of
infection (m.o.i.) of 10 in a total of 2 ml of normal media. Additionally, for
time-lapse analyses, cultures were overlaid with liquid paraffin.
Transfections were done using standard calcium phosphate procedures.
Immunofluorescence staining and microscopy
For methanol/acetone fixation, cells grown on glass coverslips were fixed
for 10 minutes in 50% methanol/50% acetone at room temperature. Coverslips
were rinsed in PBS and cells were then exposed to a blocking solution of 3%
bovine serum albumin (BSA) in PBS for 30 minutes. This fixation procedure
efficiently extracted all the green fluorescent protein (GFP), which is
co-expressed in both Ad-Pax3flag and Ad-ß-gal adenoviruses,
thus enabling dual staining of cells. GFP signals after methanol/acetone
fixation were equivalent to those obtained for control staining with
FITC-conjugated secondary antibodies alone. For paraformaldehyde fixation
cells were fixed in freshly prepared 4% paraformaldehyde for 10 minutes at
room temperature followed by exposure to a solution of 0.2% Triton X-100 and
3% bovine serum albumin (BSA) in PBS for 30 minutes for permeabilization and
to block nonspecific binding. For paraformaldehyde-cytoskeletal fixation,
cells were fixed in 4% paraformaldehyde in the presence of 0.2% Triton X-100
for 10 minutes at room temperature. This fixation procedure was suitable for
dual labeling of cytoskeletal components as it resulted in efficient
extraction of all cytoplasmic GFP but maintained variable levels of nuclear
GFP. After fixation, cells were incubated for 60 minutes at room temperature
with the appropriate primary antibodies, diluted in 3% BSA in PBS. Cells were
then rinsed briefly 4-5 times with PBS and subsequently incubated sequentially
with the appropriate Texas Red- or FITC-conjugated secondary antibodies in 3%
BSA in PBS for 50 minutes. After four washes with PBS, coverslips were then
mounted with vinol mountant. Fluorescent images were captured either on a
Zeiss axiophot microscope equipped with a CCD camera or on a Zeiss LSM
confocal microscope. Phase-contrast images were obtained with a Nikon inverted
microscope onto Kodak Plus-X pan 125ASA film. Images were processed using
Adobe Photoshop software.
Time-lapse videomicroscopy, cell-tracking analysis, protrusion
analysis
Time-lapse analyses were carried out in a chamber kept at 37°C and 5%
CO2. Time-lapse recordings were made at 1/240 of real time to a
Panasonic time-lapse video recorder with a Hitachi CCD camera connected to an
Olympus phase-contrast microscope. Images were captured with either a
10x or 20x objective. Videos were digitized and processed using a
Matrox frame grabber and Adobe Premier 5.0 video-processing software. Cell
tracings and tracking of centroid positions were carried out using NIH Image
software. Analyses of cell protrusive behavior was carried out with NIH image
software as described (Elul et al.,
1997); 4-10 cells per treatment were analyzed. Cell speed was
computed by tracking nuclear positions of at least 25 cells per treatment from
two to three videos at 10 or 30 minute intervals, over a 10 hour period using
Image-1 software.
Western immunoblots, immunoprecipitation, cell fractionation
Cell fractionation and western blotting were carried out as previously
described (Wiggan et al.,
2002). Immunoprecipitations were performed by incubating 200 µg
of soluble or 100 µg of insoluble extracts with the indicated primary
antibodies and protein G-Sepharose beads (Pharmacia) for 2 hours at 4°C.
Following this incubation beads were washed five times with lysis buffer,
followed by SDS-PAGE and western blotting. The antibodies used for
immunoprecipitations and western blotting were the same as those used for cell
staining. Quantification of bands was done with NIH image software.
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Results |
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To gain more insight into the cellular basis of these distinct Pax3-induced cell rearrangements, time-lapse video microscopy analyses of Ad-ß-gal- or Ad-Pax3flag-infected cultures was performed. Analyses of these recordings revealed a number of distinct cell behaviors contributing to Pax3-induced cell aggregation and rearrangements. For example, between 36 and 72 hours postinfection, Ad-ß-gal-infected cells began to exhibit contact inhibition of cell movement (Fig. 2A). By contrast, Ad-Pax3flag-infected cells appeared more motile and displayed both directed and random cell movements (Fig. 2B). Analysis of cell speed indicated the Ad-Pax3flag-infected cells had an average speed of 7.86±2.7 µm/hour, which was 2.5-times greater (P<0.0001) than that of control infected cells (3.05±1.1 µm/hour). In Ad-Pax3flag-infected cells, cell movement was not restricted to individual cells, as cells comprising aggregates also moved cohesively as a group (data not shown).
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Pax3flag also induced cell behavior where columns of aggregated cells formed from initially loosely associated and apparently randomly arranged cells. Fig. 3 illustrates the dynamic changes to cell size and shape. Specifically, cells were observed to episodically lengthen and contract over time (compare outline of cells numbered 2 and 3 in Fig. 3A and B). Cell tracings from images captured at selected intervals beginning at 30 hours postinfection revealed that cells underwent rearrangement mediated by both cell migration and cell intercalation. For example, in the 10 hour interval depicted in Fig. 3, cells labeled 2 and 3 in Fig. 3A had migrated upwards to and intercalated between cells 1 and 5 (Fig. 3B). By 83 hours postinfection, through a combination of further reduction in cell size as well as changes in cell shape, increased cell-cell adhesion and cell intercalation, the initially loosely and randomly positioned cells became rearranged into two parallel columns of closely associated cells (data not shown). Taken together, these analyses revealed that cell migration, cell intercalation and alterations to cell shape, cell size and intercellular adhesion properties contributed to the Pax3-induced cell aggregation. These cell behaviors described for the formation of the aggregate illustrated in Fig. 3 contributed to the formation of all Pax3-induced aggregates.
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Pax3-induced cell rearrangements beginning around 48 hours postinfection
was associated with an increased presence of lamellipodia, filopodia and
microspike-like protrusions (Fig.
4) (Wiggan et al.,
2002). Analyses of time-lapse recordings revealed, however, that
at 24 hours postinfection, control infected and Pax3-infected cells extended
protrusions episodically at similar rates. For protrusions greater than 5
µm in length, control cells extended an average of 8±5 protrusions
per cell per hour, whereas an average of 11±3 protrusions per cell per
hour in Pax3-infected cells was observed. At this time point for both control
and Pax3-infected cells, these protrusions were unstable; up to 60% of
protrusions were withdrawn within 5 minutes of extension and 100% of all
protrusions withdrew within 25 minutes of extension. Between 36 and 48 hours
postinfection there was a significant reduction in the number of protrusions
extended by control cells that were greater than 5 µm in length (average of
1±1 protrusion per cell per hour). These cells, however, extended and
withdrew many smaller unstable protrusions. By contrast, as cells formed
aggregates at 48 hours post-Ad-Pax3flag infection, cells in
aggregates extended an average of 4±1 (P=0.001) protrusions
per cell per hour. More significantly, these protrusions were considerably
more stable than those of control infected cells, with 54% of protrusions in
aggregating Pax3-expressing cells persisting for periods up to or greater than
50 minutes. This is in contrast to control infected cells, where there were no
protrusions that persisted for 50 minutes or greater. Thus, Pax3-induced cell
aggregation is associated with the extension of larger and more persistent
protrusions relative to control cells.
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The appearance of these persistent protrusions in Pax3-expressing cells was also evident by examining the arrangement of actin in Ad-ß-gal vs. Ad-Pax3flag-infected cells (Fig. 4A). As illustrated, at three days postinfection, multiple large actin-containing protrusions in control cells were infrequent, whereas multiple large protrusions were evident in condensed cells expressing Pax3. The episodic nature of these protrusions in Pax3-expressing cells is illustrated in Fig. 4D-F. Persistent protrusions in Pax3-expressing cells, some of which extended over 100 µm in length, were often very elaborate and resembled growth cones (Fig. 4B). Furthermore, during the aggregation process, persistent filopodia-like protrusions from individual cells appeared to `seek out' and to be directed to neighboring cells, often over extended distances (Fig. 4C, for example). These persistent protrusions were involved in behavior not observed in control cells. Specifically, at 72 to 96 hours postinfection, extension of a filopodia-like protrusion from one aggregate to another typically preceded the frequently observed fusion of neighboring aggregates. So, for example, Fig. 4D-J illustrates the type of cell behavior involved in the fusion of neighboring aggregates and in the recruitment of free cells into aggregates. Following the extension of a filopodia-like protrusion from one cell to another (Fig. 4E), the cell making the protrusion was often observed to extend to the newly contacted cell, creating a bridge between aggregates or to a neighboring cell (Fig. 4F,H-I). Furthermore, cell division, cell migration and cell intercalation appeared to be coordinated during aggregation such that bridges and the columnar arrangement of aggregates were maintained (Fig. 4G,J).
Together, all of the aforementioned cell behaviors culminated in the formation of highly extended three-dimensional structures four to seven days following Ad-Pax3flag infection (Fig. 1F). These Pax3-induced structures ultimately began to dissociate 10 to 12 days postinfection, cells eventually reverting to an appearance indistinguishable from uninfected cells (data not shown). Reversion coincided with the loss or significant reductions in the level of Pax3 expression, determined by western analysis. This reversion is consistent with specific levels of Pax3 expression being required to initiate and to maintain the observed morphological changes.
Pax3 induces a Wnt/PCP signaling cascade
The cell rearrangements and many of the cell behaviors induced by Pax3 in
Saos-2 cells were reminiscent of morphogenetic cell movements observed during
gastrulation and neurulation known as convergent extension
(Jacobson, 1981;
Keller et al., 1985
). During
convergent extension, where a tissue narrows along one axis while elongating
along a perpendicular axis, both polarized cell protrusive activity as well as
cell intercalation occurs (Elul et al.,
1997
; Shih and Keller,
1992
). We observed that ectopic Pax3 expression induced
convergence-like behavior in Saos-2 cells with groups of cells converging
along a single axis to form columns. This convergence-like behavior was
associated with episodic and directed cell protrusive activity, as well as
cell intercalative behavior. Given the specific morphological changes of
Pax3-expressing Saos-2 cells, we hypothesized that Pax3 may induce activation
of a Wnt/PCP-signaling cascade. To test this hypothesis we determined whether
factors in the Wnt-signaling pathway known to mediate convergent extension and
PCP would behave in a characteristic manner indicative of activation of this
signaling cascade. Thus, we first examined whether Dishevelled is recruited to
the plasma membrane, as occurs during Frizzled-dependent PCP signaling
(Axelrod, 2001
;
Axelrod et al., 1998
).
We examined the subcellular distribution of Dishevelled by indirect
immunofluoresence confocal microscopy, using an antibody that recognizes all
three known human Dishevelled proteins
(Fig. 5). The staining pattern
of Dishevelled in both uninfected and Ad-ß-gal-infected Saos-2 cells was
identical. In both instances, punctate vesicular cytoplasmic staining was
observed. The staining pattern in these cells also suggested that Dishevelled
was associated with the cytoskeleton, as has been described previously
(Torres and Nelson, 2000).
Indeed, dual staining of control infected Soas-2 cells with Dishevelled and
phalloidin revealed extensive overlap between Dishevelled and F-actin signals,
as well as prominent Dishevelled localization along F-actin stress fibers. In
contrast to control infected cells, in Ad-Pax3flag-infected cells
between 48 to 72 hours postinfection, there was significant accumulation of
Dishevelled at the cell cortex, at the tips of filopodia-like protrusions, at
cell-cell junctions and peripherally around the nucleus. In
Ad-Pax3flag-infected cells, Dishevelled also colocalized with
F-actin primarily with actin bundles at the cell cortex and at cell-cell
junctions. Distinct from its association with the actin cytoskeleton,
Dishevelled did not show any appreciable overlap with microtubules in the
presence or absence of Pax3, as determined by dual staining with
ß-tubulin antibodies (data not shown). Thus, Pax3 induces altered
subcellular distribution of Dishevelled, including strong membrane
localization, consistent with the potential activation of a Wnt/PCP signaling
cascade.
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The subcellular distribution of endogenous Frizzleds, which act upstream of
Dishevelled in the PCP signaling cascade, has not been previously described in
mammalian cells. Using commercially available antibodies to Frizzled 2, which
also detect Frizzled 1 due to the high degree of amino acid similarity between
Frizzled 1 and Frizzled 2, we examined the subcellular localization of
Frizzled in Saos-2 cells (Figs
6,
7). Immunofluorescence confocal
microscopy of methanol/acetone-fixed uninfected, Ad-ßgal- and
Ad-Pax3flag-infected cells revealed strong membrane staining at
sites of lamellipodia and filopodia protrusions, strong membrane staining at
sites resembling focal adhesions and diffuse punctate cytoplasmic staining
(Fig. 6A-C, respectively).
Identical staining patterns were observed in paraformaldehyde-fixed cells
(Fig. 6D,E), with the exception
that an accumulation of vesicular cytoplasmic staining in cells expressing
Pax3flag was more apparent. Like that observed for Dishevelled, the
staining pattern of Frizzled suggested it was associated with the
cytoskeleton. Dual staining of paraformaldehyde-fixed cells with Frizzled and
phalloidin revealed that in uninfected and control infected Saos-2 cells,
Frizzled colocalizes with F-actin along stress fibers, with a concentration of
Frizzled signals at the tips of stress fibers
(Fig. 6F-H, strong nuclear
Frizzled staining is an artifact of the paraformaldehyde fixation procedure
used). In Ad-Pax3flag-infected cells, there is also a dramatic
accumulation of Frizzled in cytoplasmic vesicles, corresponding to significant
reductions in the number of stress fibers and focal adhesions in these cells
(Fig. 6I-K;
Fig. 7G-I)
(Wiggan et al., 2002). In both
control infected and Ad-Pax3flag-infected cells, Frizzled also
showed near perfect overlap with F-actin at the leading edge of lamellipodia
and at the tips of filopodia (data not shown). Like Dishevelled, there was no
appreciable overlap of Frizzled staining with that of microtubules in the
presence or absence of Pax3 (data not shown). Thus, these results reveal the
localization of both Frizzled and Dishevelled to the actin cytoskeleton.
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The concentration of Frizzled at the tips of stress fibers predicted a
potential association with focal adhesions. We therefore examined whether
Frizzled colocalized with the focal adhesion proteins vinculin and paxillin.
In uninfected and control Ad-ß-gal-infected cells, Frizzled signals
overlapped almost perfectly with those of vinculin
(Fig. 7) and paxillin (data not
shown) at sites of focal adhesions. In Ad-Pax3flag-infected cells
there was a significant reduction of Frizzled at focal adhesion sites. Like
Frizzled, vinculin also exhibited vesicular cytoplasmic staining in
Pax3flag-expressing cells and there was considerable overlap
between both proteins at these vesicular structures
(Fig. 7G-I). The subcellular
distribution of Frizzled and its association with vinculin was pursued further
by biochemical fractionation and immunoprecipitation experiments
(Fig. 7J,K). Extracts of Saos-2
cells were partitioned into detergent-soluble and -insoluble fractions and
western blots probed with -Frizzled antibodies
(Fig. 7J). Frizzled products
were detected almost exclusively in the detergent-insoluble fraction,
supporting its association with the actin cytoskeleton. The Frizzled
antibodies detected two doublets in insoluble extracts; the upper band of the
first doublet migrated at 75 kDa, whereas the upper band of the second doublet
migrated at 65 kDa. The 65 kDa band is close to the predicted size of human
Frizzled 2 (predicted size 63 kDa), whereas the 75 kDa band is close the
predicted size of human Frizzled 1 (predicted size 72 kDa). The lower bands of
the two doublets may represent degradation products or, alternatively, they
may represent post-translational modified Frizzled products. As
Fig. 7K illustrates, both the
75 kDa and 65 kDa Frizzled products co-immunoprecipitated with vinculin from
detergent-insoluble extracts (lanes 7 and 8) but not from soluble extracts
(lanes 4 and 5), where very little of these proteins localize. Likewise,
antibodies directed against Dishevelled, which has recently been shown to
associate with other focal adhesion proteins
(Torres and Nelson, 2000
),
co-immunoprecipitated both the 75 and 65 kDa Frizzled products, as well as
vinculin from detergent-insoluble extracts (lane 9). Taken together, these
results show the association of both Frizzled and Dishevelled to the actin
cytoskeleton and complex formation of these proteins with focal adhesion
proteins. Our data also reveal that Pax3 expression culminates in the
induction of a signaling cascade, which alters the subcellular distribution of
both Frizzled and Dishevelled proteins.
Although PCP signaling in Drosophila results in recruitment of
Dishevelled to the cell membrane, canonical Wnt signaling has also been
reported to induce Dishevelled membrane localization
(Boutros et al., 2000).
Pax3-induced Dishevelled localization to the plasma membrane did not appear to
invoke induction of canonical Wnt signaling, however, as we have shown the
absence of changes to the stability and total protein levels of ß-catenin
(Wiggan et al., 2002
). To
further examine whether canonical Wnt signaling played a role in Pax3-induced
Saos-2 cell aggregation, we generated stable cell lines expressing high levels
of either dominant-negative or constitutively active GSK3ß (data not
shown). Expression of either mutant GSK3ß proteins did not inhibit or
enhance the kinetics of cell rearrangement or aggregation induced by
Ad-Pax3flag (data not shown). These results further support the
notion that Pax3-induced redistribution of Dishevelled and Frizzled in Saos-2
cells does not entail activation of canonical Wnt signaling.
In Drosophila, Frizzled- and Dishevelled-mediated PCP signaling
results in JNK activation (Boutros et al.,
1998). In vertebrates, signaling by Dishevelled also activates JNK
(Li et al., 1999
;
Moriguchi et al., 1999
). We
therefore determined whether the Pax3-induced recruitment of Dishevelled to
the cell membrane and redistribution of Frizzled was associated with
activation of JNK. Using an anti-phospho-JNK-specific antibody, activated JNK
was detected at focal adhesions in Ad-ß-gal-infected cells
(Fig. 8A,C) and in small focal
complexes, in Ad-Pax3flag-infected cells
(Fig. 8B,D). In these cells,
diffuse cytoplasmic and nuclear staining was also observed. An obvious
increase in nuclear staining of phospho-JNK compared to Ad-ß-gal-infected
cells was evident 24 hours after Ad-Pax3flag infection (data not
shown). Strikingly, two days postinfection and later, there was an intense
juxtanuclear or perinuclear multi-vesicular accumulation of activated JNK in
Pax3-expressing cells (Fig.
8B,D). In Saos-2 cells transiently expressing HA-tagged JNK, Pax3
also induced juxtanuclear vesicular accumulation of this exogenously expressed
JNK as determined by anti-HA staining (Fig.
8D, inset) The juxtanuclear/perinuclear multi-vesicular staining
of activated JNK is characteristic for that of the Golgi
(Bershadsky and Futerman,
1994
). Our preliminary analyses have revealed that Pax3-induced
activated JNK colocalized partially with a number of Golgi markers such as
GM130 and ß-cop (O.W., unpublished). More extensive colocalization of
Pax3-induced juxtanuclear vesicular activated JNK was observed for markers
such as GS15 and Rab8 (O.W., unpublished), both factors involved in vesicular
traffic. Fig. 8E illustrates
further that Pax3 induced increased expression levels of JNK protein.
Specifically, by 48 hours postinfection, a 1.5 to 2-fold increase in total JNK
protein levels was detected in Ad-Pax3flag-infected cells relative
to control cells.
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Taken together, Pax3 induces a signaling cascade that results in activation of JNK and in the altered subcellular distribution of Dishevelled, Frizzled and activated JNK. This signaling cascade is associated with changes in cell morphology, increased cell motility and cell rearrangements, as well as altered arrangement of the cytoskeleton and cell adhesion properties. They further support the notion that Pax3 induces a PCP-or Wnt-signaling cascade that gives rise to morphological changes in Saos-2 cells in vitro, analogous to those observed during convergent extension cell movements in vivo.
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Discussion |
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Pax3 induced a distinctive set of cell behaviors during aggregation, where
convergence on a single axis resulted in the formation of columns of cells.
This convergence is associated with the formation of many persistent
lamellipodia and filopodia-like protrusions. Convergence resulted from cell
migration, cell intercalation and changes to cell shape and size. These
Pax3-induced cell movements are reminiscent of the convergent extension cell
movements that occur during Xenopus gastrulation and neurulation.
Particularly intriguing is the observation that, during neural tube closure in
Xenopus, the neuroepithelial cells that undergo cell intercalation
and convergent extension cell movements express Pax3
(Davidson and Keller, 1999).
Pax3-induced convergence behavior in vitro did not appear to be accompanied by
significant amounts of extension, as extension appeared to be countered by the
dramatic size reduction in Pax3-expressing cells. Our evidence that Pax3 can
induce convergence behavior in vitro suggests, however, that Pax3 may regulate
convergent extension movements of neuroepithelial cells in vivo. This role for
Pax3 is supported further by our data showing that Pax3 induces the same
Wnt-signaling cascade in vitro that plays a regulatory role in convergent
extension movements in vivo. Although the mechanism by which Pax3 induces this
Wnt/PCP-signaling cascade is not yet clear, a compelling possibility is that
Pax3 directly or indirectly induces expression of a Wnt or of some other
unknown PCP ligand. We are currently determining whether ectopic Pax3 induces
expression of endogenous Wnt proteins.
Work by Heisenberg et al. (Heisenberg
et al., 2000) showed that Wnt signaling regulates convergent
extension movements. In both vertebrate convergent extension movements and
polarization of epithelial cells in Drosophila, signaling downstream
of Frizzled does not elicit the canonical Wnt/Wg signaling cascade
(Axelrod et al., 1998
;
Boutros et al., 1998
;
Tada and Smith, 2000
). Rather,
in both instances, signaling by Frizzled results in relocalization of
Dishevelled to the plasma membrane
(Axelrod, 2001
;
Axelrod et al., 1998
;
Wallingford et al., 2000
).
Mutations to Dishevelled protein that prevent its membrane localization
correlate with their ability to block both PCP signaling and convergent
extension movements (Axelrod,
2001
; Wallingford et al.,
2000
). Our results show that, in Saos-2 cells, Pax3 induces a
Wnt/PCP signaling cascade causing relocalization of Dishevelled to the plasma
membrane. The cortical staining pattern of Dishevelled in Pax3-xpressing
Saos-2 cells is highly indicative of activation of the PCP branch of the
Wnt-signaling cascade. Torres and Nelson
(Torres and Nelson, 2000
)
recently showed that Wnt1 induces actin cytoskeleton reorganization and
altered Dishevelled subcellular distribution in embryonic kidney metanephric
mesenchymal cells. Interestingly, although Dishevelled associates with the
actin cytoskeleton in these cells, Wnt1 stimulation did not cause cortical
Dishevelled accumulation, nor was activation of JNK apparent in these cells.
Thus, consistent with the work of Axelrod et al.
(Axelrod et al. 1998
;
Axelrod, 2001
), it appears that
recruitment of Dishevelled to the cell cortex is specific to PCP signaling.
Additionally, in Pax3-expressing Saos-2 cells there is no apparent activation
of the canonical Wnt-signaling cascade, as evidenced by a lack of altered
ß-catenin stability or the ability of GSK3ß mutants to alter the
cellular response to Pax3 expression. Rather, Dishevelled relocalization is
associated with significant activation and relocalization of JNK, further
supporting the notion that the Pax3-induced cell behavior in Saos-2 cells
relies on signaling cascades similar to those required for convergent
extension and PCP morphogenesis.
Our data also indicate that, in Saos-2 cells, Pax3-induced Wnt/PCP
signaling may play a role in regulating reorganization of the actin
cytoskeleton. In Saos-2 cells, Dishevelled localizes to the actin cytoskeleton
with prominent localization along stress fibers. Pax3 induces reorganization
of the actin cytoskeleton, causing a reduction in stress fibers and focal
adhesions and increased cortical actin bundles, as well as strong actin
polymerization at epithelial cell-cell junctions and around the nucleus. In
Pax3-expressing cells, Dishevelled colocalized with cortical actin bundles and
with actin at cell-cell junctions. Intriguingly, like Dishevelled, Frizzled
also localized to the actin cytoskeleton at sites of strong actin
polymerization such as at the leading edge of lamellipodia and at the tips of
filopodia. Thus, we hypothesize that Pax3-induced Wnt/PCP signaling may
regulate the formation and position of actin-rich structures such as
lamellipodia and filopodia. Dishevelled regulates the stability and
polarization of protrusive activity during Xenopus convergent
extension movements (Wallingford et al.,
2000). Our results indicate that Pax3-induced cell aggregation is
associated with increased persistent protrusive activity. Thus, Wnt/PCP
signaling may also regulate the turnover of actin structures that give rise to
lamelliform and filiform protrusions.
Formation of lamellipodia and filopodia occur in response to Rac and Cdc42,
respectively (Kozma et al.,
1995; Nobes and Hall,
1995
), which are also known mediators of JNK signaling
(Coso et al., 1995
). The
possibility that Rho family GTPases, such as Cdc42 or Rac, may be involved in
the JNK signaling cascade induced by Pax3 is intriguing. Genetic analysis in
Drosophila determined that Rho-family GTPases are upstream mediators
of the JNK signaling cascade in dorsal closure, planar tissue polarity and
thorax closure (Noselli, 1998
;
Noselli and Agnes, 1999
). It
is very likely, therefore, that Rho-family GTPases also participate in the JNK
signaling cascade induced by Pax3. Further studies using dominant negative
constructs of Rho-family GTPases will be required to address this question.
These ongoing studies are expected to define a more precise role for PCP
signaling in Pax3-expressing cells.
The localization of Frizzled, Dishevelled and activated JNK to focal
adhesions suggests that focal adhesion sites may be primary signaling centers
for Wnt/PCP signaling. In Pax3-expressing cells there was a reduction of
Frizzled at focal adhesions and an increase in its localization to cytoplasmic
vesicular structures. This vesicular accumulation may in part represent
internalization of Frizzled due to ligand binding. Pax3-induced Frizzled
vesicular accumulation also appears to be associated with a reduction of the
cytoskeletal structures with which it associates, such as stress fibers and
focal adhesions. Focal adhesion sites represent the major sites of
cell-extracellular matrix adhesion in cultured cells. These sites play an
important role in cell motility, and large focal adhesions have been reported
to retard cell motility (Sastry and
Burridge, 2000). Infection by Ad-Pax3flag in Saos-2
cells induced a significant increase in cell motility. Furthermore, cells
expressing Pax3 had considerably fewer focal adhesions that were generally
smaller in size relative to control infected cells. The localization of
Wnt/PCP signaling components to focal adhesions suggests that Wnt/PCP
signaling may regulate cell motility, possibly by regulating the dynamics of
focal adhesion assembly/disassembly. Indeed, many of the genetically
identified signaling cascade components that act downstream of Frizzled and
Dishevelled, such as the Rho-family GTPases and Rho-associated kinase, are
known regulators of focal adhesion assembly
(Sastry and Burridge, 2000
;
Winter et al., 2001
).
PCP signaling results in activation of JNK. Our studies revealed that Pax3
not only induced JNK activation but also induced increased expression levels
of JNK protein. Interestingly, the subcellular localization of Pax3-induced
activated JNK indicates that the major targets for JNK activity during Wnt/PCP
signaling are cytoplasmic rather than nuclear. Specifically, in Saos-2 cells,
Pax3-induced activated JNK localizes to a juxtanuclear multivesicular
structure. On the basis of preliminary colocalization experiments, this
structure appears to be a post-Golgi compartment. In this compartment,
activated JNK shows extensive colocalization with Rab8 and GS15. Rab8, a
member of the small GTPase family, and GS15, a SNARE protein, are thought have
roles in regulating vesicular traffic
(Peranen et al., 1996;
Xu et al., 1997
). Thus
juxtanuclear localized activated JNK may play a role in regulating membrane
traffic during PCP signaling. This possibility is particularly intriguing
given the recent report that a related stress-activated kinase, p38 MAPK,
associates with the small GTPase Rab5 to regulate endocytic traffic
(Cavalli et al., 2001
). The
localization of Frizzled, Dishevelled and activated JNK to focal adhesions
would suggest further that important Wnt/PCP JNK signaling targets are located
at focal adhesion sites.
It has become apparent that signaling cascades involving JNK activation
play crucial roles during embryonic morphogenesis in diverse organisms.
Genetic analyses in Drosophila revealed the importance of the JNK
signaling pathway in morphogenetic processes such as dorsal closure, planar
cell polarity and thorax closure
(Martin-Blanco et al., 2000;
Noselli and Agnes, 1999
;
Zeitlinger and Bohmann, 1999
).
For these processes, JNK signaling influences cytoskeletal architecture, cell
adhesiveness and cell polarity. We speculate that induction of a Wnt/PCP
signaling cascade resulting in JNK activation during vertebrate neural tube
development may be an important function of Pax3. Like Pax3 mutants, double
mutant JNK1 and JNK2 mice also have neural tube closure defects
(Kuan et al., 1999
;
Sabapathy et al., 1999
). A
role for Dishevelled in neural tube closure in Xenopus has also
recently been shown (Wallingford and
Harland, 2001
). We hypothesize that a conserved signaling cascade,
involved in the movement and fusion of sheets of epithelial cells in
Drosophila, has been recruited for vertebrate neural tube closure and
that this signaling cascade may be regulated by Pax genes. Analysis of JNK
activity in Pax3 mutant mice will be required to determine whether Pax3
influences JNK signaling in vivo.
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Acknowledgments |
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