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First published online 1 February 2006
doi: 10.1242/dev.02243
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Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 20 80 16, India
Author for correspondence (e-mail:
pradips{at}iitk.ac.in)
Accepted 9 December 2005
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wg, Wnt signaling, Cell-cell adhesion, DE-Cadherin (Shg), Fat, Growth regulation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Imaginal discs, the primordia of the adult appendages of Drosophila,
offer elegant models with which to investigate the cellular mechanisms that
determine the developmental outcome of the signaling pathways regulating organ
growth and pattern (Day and Lawrence,
2000). Evolutionarily conserved Wg/Wnt signaling pathway
(Logan and Nusse, 2004;
Peifer and Polakis, 2000)
plays a crucial role in the patterning of Drosophila wing
(Martinez Arias, 2003), and
its role in the regulation of its growth is now well established
(Giraldez and Cohen, 2003;
Johnston and Sanders, 2003;
Neumann and Cohen, 1996). The
wing imaginal disc epithelium is also postulated to develop a gradient of cell
affinities (Ripoll et al.,
1988) and perturbations in Wg signaling affect its cell-cell
adhesion (Blair, 1994;
Chen and Struhl, 1999;
Hoffmans and Basler, 2004).
Besides growth and pattern, Wg signaling may thus regulate cell-cell adhesion
in wing imaginal disc epithelium.
The Wg/Wnt ligand signals through its receptor Frizzled (Fz), the
seven-pass trans-membrane receptor that acts through Dishevelled (Dsh) to
antagonize the activity of GSK3/Shaggy, a component of the ß-catenin/Arm
degradation complex, thereby leading to stabilization of ß-catenin/Arm in
the cytoplasm. ß-Catenin/Arm then translocates to the nucleus where it
interacts with the transcription factor TCF/Lef1, known as Pangolin
(previously DTcf) in Drosophila, and activates nuclear targets (for a
review, see Logan and Nusse,
2004). ß-Catenin/Arm is also a component of the epithelial
cell-cell adhesion complex at adherens junction (AJ). DE-Cadherin (DE-Cad; Shg
– FlyBase), the Drosophila homolog of vertebrate E-Cadherin,
assembles the AJ complex, as a sub-apical ring around the neck of the
epithelial cells and mediates cell-cell adhesion by Ca2+ dependent
homophilic interactions (Oda et al.,
1994). Cytoplasmic domain of DE/E-Cad binds to ß-catenin/Arm
which in turn associates with the actin cytoskeleton through 
-catenin
(Gumbiner, 2005;
Nelson and Nusse, 2004).
Stabilization of the cytoplasmic pool of ß-catenin/Arm in response to Wg
signaling may influence cell-cell adhesion by facilitating its binding to
DE-Cad at AJs (Gumbiner, 2000;
Nelson and Nusse, 2004). In
mammalian cultured cells, for example, Wnt signaling upregulates the levels of
junctional E-Cad (Hinck et al.,
1994). However, enhanced junctional recruitment of
ß-catenin/Arm may not be the primary mode of Wg-mediated regulation of
cell-cell adhesion as the levels of Cadherin expression, rather than of
ß-catenin, appear to be the rate-limiting factor for AJ complex formation
(Gumbiner, 2000). Wg signaling
may affect cell-cell adhesion by regulating DE/E-Cad, which itself is a target
of Wg. Transcriptional regulation of DE-Cad by Wg signaling enhances
cell-cell adhesion in Drosophila cell lines
(Yanagawa et al., 1997). By
contrast, in mammalian hair follicles
(Jamora et al., 2003) or in
mouse brain (Shimamura et al.,
1994), Wnt signaling represses E-Cad transcription. More
strikingly, during zebrafish gastrulation, Wnt11 modulates E-Cad-mediated cell
cohesion by regulating its endocytosis by Rab5c
(Ulrich et al., 2005). Wnt/Wg
signaling thus modulates cell-cell adhesion by regulating DE/E-Cad in a
variety of ways.
The Drosophila tumor suppressor Fat (Ft)
(Bryant et al., 1988) also
regulates epithelial cell-cell adhesion in imaginal discs. Ft is an enormous
transmembrane protein with 34 extracellular Cadherin repeats
(Mahoney et al., 1991) and a
prototype of a class of Cadherins called `Fat-like Cadherins'
(Tanoue and Takeichi, 2005).
In the absence of Ft, imaginal discs display overproliferation and altered
cell-cell adhesion (Agrawal et al.,
1995; Fanto et al.,
2003; Garoia et al.,
2000; Mahoney et al.,
1991; Shashidhara et al.,
1999). Ft is localized in the apicolateral membrane, above the
sub-apical AJ (Ma et al.,
2003) and displays Ca2+-dependent heterophilic binding
(Matakatsu and Blair, 2004)
with another Fat-like Cadherin, Dachsous (Ds)
(Clark et al., 1995). Ft also
regulates cell-cell adhesion both in cell culture assays
(Matakatsu and Blair, 2004)
and in vivo (Ma et al., 2003;
Strutt and Strutt, 2002), and
is also known to be involved in an intercellular signaling pathway where its
partners ds and four-jointed (fj) modulate its
activity during PD patterning (Cho and
Irvine, 2004; Rodriguez,
2004; Strutt et al.,
2004) and cell-cell adhesion
(Strutt and Strutt, 2002).
Finally, the Fj-Ft-Ds intercellular signaling is also implicated in the
regulation of planar cell polarity (PCP), the orientation of epithelial cells
orthogonal to their apicobasal axis (Casal
et al., 2002; Ma et al.,
2003; Rawls et al.,
2002; Strutt and Strutt,
2002; Yang et al.,
2002). Ft, therefore, links tissue growth with cell-cell adhesion
and PCP. However, the genetic pathways and cellular mechanisms of Ft-dependent
regulation of growth and cell-cell adhesion have yet to be uncovered (for a
review, see Tanoue and Takeichi,
2005).
Several indirect observations suggest convergence of Ft and Wg signaling in
the regulation of wing development. For example, ft genetically
interacts with Wg signaling (Greaves et
al., 1999), while both Ft (Cho
and Irvine, 2004) and its partner Ds
(Rodriguez, 2004) regulate Wg
expression/signaling in the proximal wing. Ft and Wg signaling may thus
coordinate tissue growth with cell-cell adhesion during wing development.
Here, we show that Wg signaling positively regulates expression of
DE-Cad and sets up a PD gradient of cell-cell adhesion and cell shape
in the distal wing imaginal disc epithelium. Ft, by contrast, represses
DE-Cad and regulates cell-cell adhesion and cell shape in distal
wing, although it does not co-localize with DE-Cad. Ft also intersects Wg
signaling by downregulating the cytoplasmic pool of ß-catenin/Arm,
downstream of its ligand Wg. Finally, Ft and Wg signaling exert opposing
regulations on Distalless (Dll), Vestigial (Vg)
(Neumann and Cohen, 1997;
Zecca et al., 1996) and DE-Cad
expression, thereby linking tissue growth with cell-cell adhesion during
Drosophila wing development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
ft422 (Rawls et al.,
2002), UAS-armS10, UAS-armS2
(Pai et al., 1997),
UAS-dsh (Neumann and
Cohen, 1996), UAS-DE-Cad
(Greaves et al., 1999),
UAS-tcf
N (van de Wetering
et al., 1997), UAS-ft
(Matakatsu and Blair, 2004),
UAS-GPI::DFz2 (Cadigan et al.,
1998), N23-Gal4
(Prasad et al., 2003),
vg-Gal4 (Simmonds et al.,
1995). Q-vg-lacZ (Kim
et al., 1996), BE-vg-lacZ
(Williams et al., 1994) and
fzH15fz2C1
(Chen and Struhl, 1999). Other
stocks are described in FlyBase.
Loss-of-function clones were generated by flp/FRT system
(Xu and Rubin, 1993) and
gain-of-function clones were generated using flip-out technique
(Ito et al., 1997). Ectopic
expression was induced by UAS/Gal4 system
(Brand and Perrimon, 1993). All
fly stocks were grown at 24±1°C. For experiments in the
vg1 genetic background, cultures were maintained at
22-23°C for a consistent phenotype. vg1 is a strong
hypomorphic allele (Williams et al.,
1990). We noticed that at 25°C or above
vg1 discs show a mild expression of Wg in the DV boundary.
At a lower temperature, however, Wg expression in the DV boundary was
completely abolished. Unless specified, to generate clones, larval cultures of
the desired genotypes were given heat shock (37°C) for 1 hour during 48-72
hours interval after egg laying. For experiments with Nts
mutants, larval culture was grown at 18°C. Culture was shifted to 30°C
for 48 hours prior to dissection. Adult wings were mounted in DPX and images
were acquired under Leica DMRA.
Antibodies
The antibodies used were anti-ß-galactosidase (Sigma), anti-Arm,
anti-Wg (DSHB), anti-DE-Cad (Oda et al.,
1994), anti-Dll (Panganiban et
al., 1995) and anti-Vg
(Williams et al., 1993).
Secondary antibodies (Alexa 488, Alexa 555, Alexa 633) were from Molecular
Probes.
For staining of Actin filaments, fixed imaginal discs were incubated in a 1:1000 dilution of PBS/Phalloidin-Rhodamine (Sigma) for 15 minutes and washed in PBS. Stained discs were mounted in Vectashield mounting medium with DAPI (Vector Labs). Images were acquired under Leica-SP2 Confocal microscope and processed using Leica Confocal software and Adobe Photoshop.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Neumann and Cohen, 1996). The
lacZ reporter of the quadrant enhancer of vestigial
(vg), Q-vg-lacZ (Kim et
al., 1996) marks the entire distal wing [i.e. the presumptive wing
blade (pouch) (Fig. 1A)].
In optical sections of the imaginal disc epithelium, AJs are visualized in
the XY (Fig. 1B) or XZ planes
(Fig. 1C) based on
immunolocalization of DE-Cad and ß-catenin/Arm besides binding with
fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm
(Collins and Treisman, 2000;
Jiang and Struhl, 1998;
Prasad et al., 2003) and
DE-Cad display characteristic upregulation across the DV boundary along the PD
axis of wing imaginal disc (Fig.
1D-D''). Optical sections along the XY plane reveal
higher levels of DE-Cad localization and narrower apical circumferences in the
AJs of cells flanking the DV boundary when compared with those of the more
proximally located cells (Fig.
1E) (see Fristrom and
Fristrom, 1993). Optical sections along the XZ plane further
confirmed upregulation of DE-Cad (Fig.
1F) and F-actin (Fig.
1F') in the AJs of cells flanking the DV boundary (blue in
Fig. 1F''). Thus,
along the PD axis of the wing disc, cell shapes and DE-Cad levels are
graded.
Wg signaling regulates DE-Cad levels and cell shapes along the PD axis of wing imaginal disc
We further sought to test if the PD gradient of cell shape and DE-Cad
levels are linked to Wg signaling. Somatic clones displaying constitutive Wg
signaling [induced by overexpression of Dsh
(Yanagawa et al., 1997) or of
a degradation resistant variant of ß-catenin/Arm, ArmS10
(Pai et al., 1997) (not shown
here)] induced cell-autonomous upregulation in the levels of DE-Cad and apical
cell constrictions (Fig.
2A,A' and inset in Fig.
2A). Somatic clones expressing secreted Wg, however, are expected
to induce non-cell-autonomous effects. Indeed, these clones induced non-cell
autonomous and graded upregulation in the levels of DE-Cad in the AJs
(Fig, 2B,B') and changes
in apical cell shapes (Fig.
2C,C'). In the presumptive hinge region
(Fig. 2D,D'), Wg
overexpression produced a more striking pattern of non-cell autonomous changes
in cell shapes: cells neighboring the Wg-expressing cells appeared to organize
as whorls around the former and displayed epithelial misfolding
(Fig. 2D,D').
Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which
compromises Wg signaling (Cadigan et al.,
1998), obliterated the characteristic PD gradient in the levels of
DE-Cad (Fig. 2E,E') and
F-actin in the AJs (Fig.
2F-G'). Finally, loss of Wg expression in the DV boundary of
wing imaginal disc of Nts mutants grown at a restricted
temperature (Fig. 2H)
(Diaz-Benjumea and Cohen,
1995) also abolished the PD gradient of DE-Cad
(Fig. 2I) and apical cell
shapes (Fig. 2I', see
Fig. 1D'',E). To
further test if apical cell constrictions are linked to elevated levels of
DE-Cad in AJs (Fig. 1E,
2A-D'), we expressed
DE-Cad in somatic clones. These clones were apically constricted (not
shown here), consistent with the role of DE-Cad/E-Cad in remodeling cell shape
and tissue architecture (Pilot and Lecuit,
2005). These results thus link Wg signaling to the PD gradient in
the levels of DE-Cad and apical cell shapes in the wing imaginal discs.
|
).
Indeed, somatic clones displaying gain of Wg signaling owing to Dsh
(Fig. 2J) or ArmS10
(Fig. 2K) misexpression sorted
out from their neighbors and displayed smooth clone borders, akin to those
misexpressing DE-Cad (Fig. 2L).
Wg signaling may alter cell-cell adhesion by enhancing recruitment of
ß-catenin/Arm to the AJs (Hinck et
al., 1994) and/or by its transcriptional input
(Nelson and Nusse, 2004). In
many cell types, for example, expression of cadherins rather than the levels
of catenins appears to be the rate-limiting step of Catenin-Cadherin complex
formation at AJs and cell-cell adhesion
(Gumbiner, 2000). Wild type
ß-catenin/Arm (ArmS2), when overexpressed, does not transduce
Wg signaling (Pai et al.,
1997). Somatic clones overexpressing ArmS2 display
`wiggly' clone borders (Fig.
2M), unlike those expressing Dsh
(Fig. 2J) or ArmS10
(Fig. 2K). Thus, expression of
ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling,
fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc
epithelium is therefore likely to be regulated by transcriptional input from
Wg signaling.
Wg signaling regulates expression of DE-Cad
To test if canonical Wg signaling regulates DE-Cad expression, we
examined the response of its lacZ reporter, DE-Cad-lacZ.
Cells receiving high threshold of Wg signaling in the wing imaginal discs, as
in those flanking the DV boundary, displayed higher levels of
DE-Cad-lacZ reporter activity when compared with those further away
from the source of Wg expression (Fig.
3A,A'). Furthermore, somatic clones expressing
ArmS10 (Fig.
3B,B') or Dsh (not shown here) displayed cell-autonomous
activation of the DE-Cad-lacZ. Finally, clones expressing the
secreted Wg induced non-cell-autonomous activation of DE-Cad-lacZ:
i.e. in cells within (Fig.
3C,C') and surrounding the clones (arrows in
Fig. 3C,C'). Together
(Figs 1,
2,
3), these results suggest that
regulation of DE-Cad by the long-range activity of the Wg morphogen
(Neumann and Cohen, 1997;
Zecca et al., 1996) sets up
the PD gradient of cell-cell adhesion and cell shape in the distal wing.
Ft negatively regulates DE-Cad expression in the PD axis
Somatic clones lacking Ft (ft-/ft-), marked
by loss of GFP (arrows in Fig.
4A), displayed overgrowth and altered cell-cell adhesion with
characteristic circular and smooth clone borders, unlike the `wiggly' borders
of their wild type (ft+/ft+) twins that are
marked by brighter GFP (arrowheads in Fig.
4A). Furthermore, cells lacking Ft displayed upregulation of
DE-Cad in their AJs (Fig.
4B-B'') and DE-Cad-lacZ
(Fig. 4C,C'). By
contrast, when we overexpressed Ft, levels of both DE-Cad
(Fig. 4D,E') or
DE-Cad-lacZ (not shown here) were downregulated. Besides, following
overexpression of Ft in the posterior wing compartment, cells flanking the DV
boundary (arrowheads, Fig.
4E,E') displayed wider apical circumferences when compared
with those of the anterior wing compartment. These results suggest that Ft
regulates DE-Cad expression, cell-cell adhesion and apical cell
shapes in the distal wing.
Ft negatively regulates wing growth and pattern in the PD axis
Dynamic regulations of Wg expression controls both PD wing growth and
pattern (Diaz-Benjumea and Cohen,
1995; Klein and Arias,
1998; Neumann and Cohen,
1996; Rodriguez et al.,
2002). Ft (Cho and Irvine,
2004) and its partner Ds
(Rodriguez, 2004) both
regulate proximal wing growth and pattern by modulating Wg
expression/signaling. Furthermore, opposing regulation of DE-Cad by
Ft and Wg signaling (Figs 2,
3) suggests that these two
pathways might intersect during distal wing development. We sought to examine
the role of Ft in the regulation of two distal wing markers, namely Dll
(Cohen et al., 1989;
Diaz-Benjumea and Cohen, 1995)
and Vg or its pouch specific enhancer Q-vg-lacZ
(Kim et al., 1996;
Williams et al., 1994).
In wild type wing imaginal discs, Dll
(Fig. 5A) and Vg (data not
shown) display a characteristic PD gradient in their expression
(Neumann and Cohen, 1997;
Zecca et al., 1996). In
ft mutant wing imaginal discs, expressions of both Dll
(Fig. 5B) and
Q-vg-lacZ (not shown here) are upregulated and expanded along the PD
axis. By contrast, a strong overexpression of Ft in the posterior compartment
of wing imaginal disc resulted in downregulation of Dll
(Fig. 5C,C'; larvae grown
at 26-27°C; also compare with Fig.
8H,H') and Vg (data not shown). Furthermore, in mosaic wing
imaginal disc, Q-vg-lacZ displayed cell autonomous
upregulation in cells lacking Ft throughout the PD axis
(Fig. 5D,D'). Plot of the
intensity of Q-vg-lacZ expression in the mosaic discs revealed an
upregulated and flatter gradient of Q-vg-lacZ activity in
cells lacking Ft when compared with the wild-type
(ft+/ft-) domains in the mosaic wing pouch
(Fig. 5E). Ft thus negatively
regulates Dll and Vg expression suggesting its role in distal wing
development. Furthermore, overexpression of Ft resulted in truncated growth
along the PD axis (Fig.
5F,G).
|
|
). We
sought to further confirm genetic interaction of ft with Wg signaling
by reducing the dosage of ft (ft-/ft+)
in genetic backgrounds where Wg signaling is altered. High threshold of Wg
signaling is essential for margin bristle formation
(Couso et al., 1994;
Neumann and Cohen, 1997).
Overexpression of ArmS10 along the DV boundary induced ectopic wing
margin bristles (22-40 bristles, n=25)
(Fig. 6A). This phenotype was
enhanced by a reduction in the dose of ft (45-87 bristles,
n=25) (Fig. 6B).
Similarly, the ectopic wing margin bristle phenotype induced by overexpression
of the Wg receptor Fz2 was also enhanced by a reduction in the dosage of
ft (not shown here). Conversely, compromising Wg signaling by
overexpression of dTCFN (van de
Wetering et al., 1997) abolished the wing margin bristles
(Fig. 6C, n>100),
while overexpression of Dfz2-GPI (Cadigan
et al., 1998) induced notching of wing margin (not shown here).
These, phenotypes were partially suppressed by a reduction in the ft
gene dose (dTCFN: Fig.
6D, n>100; Dfz2-GPI: not shown here).
These results suggest that a reduction in Ft activity enhances Wg
signaling. To further test the consequences of complete loss of Ft on Wg
signaling, we examined the levels of ß-catenin/Arm in somatic clones
lacking Ft. Upregulation of ß-catenin/Arm signifies stabilization of its
cytoplasmic pool, a characteristic of cells receiving Wg signaling
(Peifer et al., 1994). In the
wing imaginal discs, for example, cells flanking the DV boundary show higher
levels of cytoplasmic ß-catenin/Arm
(Fig. 1D') in response to
Wg signaling (Collins and Treisman,
2000; Jiang and Struhl,
1998; Prasad et al.,
2003). Somatic clones lacking Ft displayed upregulated levels of
ß-catenin/Arm (Fig.
6E-F'). Conversely, overexpression of Ft
(en-Gal4xUAS-Ft) downregulated ß-catenin/Arm levels
(Fig. 6G,H). Taken together
with the earlier studies of genetic interactions
(Greaves et al., 1999), these
results show that Ft modulates Wg signaling, presumably by altering
ß-catenin/Arm levels.
|
).
Furthermore, loss of Ft in somatic clones upregulated Wg in the proximal but
not in the distal wing (Fig.
7C,C'). Wg expressions in the distal
(Diaz-Benjumea and Cohen,
1995) and proximal wing domains are maintained by different
mechanisms (by a positive auto-regulatory loop in the latter)
(Rodriguez et al., 2002). In
accordance with this interpretation, we could further show that constitutive
gain of Wg signaling upregulates expression of its ligand, Wg in the proximal
but not in the distal wing (data not shown). These results suggest that
upregulation of Wg expression in the proximal wing of ft mutants
could be due to a positive feedback from enhanced Wg signaling: for example,
by upregulation in the levels of ß-catenin/Arm (see
Fig. 6).
Wg ligand-independent signaling by Ft
Ft is proposed to regulate growth in the entire wing by a broad based
mechanism (Cho and Irvine,
2004). As Wg synthesis is not upregulated in the DV boundary of
overgrown ft mutant wing imaginal discs
(Fig. 7) (see also
Cho and Irvine, 2004),
regulation of distal wing development by Ft could be independent of the
activity of the Wg ligand. We sought to examine this possibility.
During wing development, Wg and Vg maintain their mutual expression in the
DV boundary (Prasad et al.,
2003). In vg1 wing imaginal discs, synthesis
of the Wg ligand in the DV boundary is abolished (arrowhead in
Fig. 8A), besides those of
Q-vg-lacZ (Fig. 8A)
(Prasad et al., 2003) or Dll
(Fig. 8E). These distal wing
markers are activated in vg1 wing imaginal discs under
conditions that circumvent the loss of the endogenous Wg ligand activity: for
example, by expressing the Wg ligand from a transgene
(Fig. 8B) or by overexpression
of Dsh (not shown here). Expression of Q-vg-lacZ or Dll in the
vg1 wing imaginal disc thus signifies activation of the Wg
signaling pathway, independent of the activity of endogenous Wg ligand.
Loss of Ft in vg1 discs in somatic clones showed cell-autonomous activation of Q-vg-lacZ; expression of Wg in these clones, however, remained extinguished (Fig. 8C,C'). In ftfd vg1 double mutant discs, Q-vg-lacZ (Fig. 8D') and Dll (Fig. 8F) were activated, while Wg in the distal wing remained extinguished (arrow, Fig. 8D-F). Levels of expression of Q-vg-lacZ (Fig. 8D') and Dll (Fig. 8F) appeared milder in ftfd vg1 double mutants than in their respective wild-type counterparts (see Fig. 1A and Fig. 8H'). Furthermore, between these two markers, Q-vg-lacZ (Fig. 8D') displayed stronger level of derepression than that of Dll (Fig. 8F) in ftfd vg1 double mutant discs.
We carried out additional tests in genetic backgrounds that
abolished/undermined the activities of the Wg ligand in the distal wing. Notch
(N) regulates Wg expression in the DV boundary
(Diaz-Benjumea and Cohen,
1995). At a restrictive temperature, Wg expression in the DV
boundary (Fig. 8G,G') of
Nts mutant wing imaginal disc is extinguished
(Fig. 2F); in addition,
expression of Vg or its boundary and quadrant enhancers is also silenced in
Nts mutants (not shown here). Nts/Y;
ftfd/ft422 double mutant larvae grown at
restrictive temperature, however, showed distal wing growth and activation of
Q-vg-lacZ (Fig.
8G') but not that of Wg (arrow,
Fig. 8G). In wild-type wing
disc, expression of Dll is seen to be nearly symmetrical on either side of the
AP boundary (Fig. 8H,H')
and flanking the DV boundary (Fig.
5A). Expression of the GPI-DFz2 in the wild-type discs
downregulates Wg ligand activity (Cadigan
et al., 1998) and reduced the level of expression of Dll
(Fig. 8I,I'). Expression
of Vg was also repressed following expression of GPI-DFz2 (data not shown). In
the ftfd mutant wing discs, however, expression of
GPI-Dfz2 largely failed to repress Dll
(Fig. 8J,J') or Vg (data
not shown). Taken together, these results show that Ft intersects Wg signaling
downstream of its ligand and upstream of the cytoplasmic stabilization of
ß-catenin/Arm.
|
|
|
;
Chen and Struhl, 1999). In the
complete absence of Fz activity (Fz null), which is abolished when both
fz and fz2 are mutated, cells of the wing pouch fail to grow
and survive (Fig. 8K)
(Chen and Struhl, 1999).
However, when induced late during larval development, these Fz-null mutant
clones displayed modest survival but failed to express Dll
(Chen and Struhl, 1999).
Activation of the downstream components of Wg signaling, however, rescues the
Fz-null embryos from growth and pattern defects
(Chen and Struhl, 1999). We
argued that if Ft signaling were Fz independent, then Fz-null clones would
survive when induced in ft mutant discs. However, Fz null clones
induced in ft mutant discs during early larval stages failed to
survive (Fig. 8K,L). However,
Fz-null clones induced 24 hours before dissection survived but appeared
undergrown by comparison with their wild-type twins and also failed to
activate Dll expression (Fig.
8M,M', n=10). These results suggest that Ft
intersects Wg signaling either upstream or parallel to its receptors, Fz/Fz2,
reminiscent of its interaction with the non-canonical Wg signaling pathway for
the regulation of PCP (Ma et al.,
2003; Strutt and Strutt,
2002; Yang et al.,
2002). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Linking
developmental regulation of Cadherins to signaling pathways, implicated in the
control of organ growth and pattern, would ensure that cells within a regional
specialization do not migrate and expand in inappropriate places
(Gumbiner, 2005;
Vleminckx and Kemler, 1999).
Here, we have shown that apical cell shapes and cell-cell adhesion are both
graded along the PD axis of the developing wing imaginal discs. Furthermore,
readout of Wg signaling along the PD axis regulates expression of
DE-Cad. As DE-Cad levels affect cell-cell adhesion
(Fig. 2) (see
Dahmann and Basler, 2000), our
results suggest that by regulating DE-Cad expression, Wg signaling
integrates cell-cell adhesion with tissue growth and pattern. Regulation of
DE-Cad expression could be a prevalent mechanism for coordination of
the emerging pattern in an organ primordium with the spatial control of its
cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells
flanking the stripe of cells, along the AP boundary (Figs
1 and
3) which express the morphogen
Decapentaplegic (Dpp) (Lecuit et al.,
1996; Nellen et al.,
1996); misregulation of Dpp signaling also affects DE-Cad
expression (M.J. and P.S., unpublished). The Ft tumor suppressor, by contrast,
negatively regulates DE-Cad expression in the distal wing. This may
also explain the inverse correlation between the levels of DE-Cad in AJs and
the activity of Ft (Fig. 4). Thus, besides its heterophilic binding with Ds
(Matakatsu and Blair, 2004;
Strutt and Strutt, 2002), Ft
controls cell-cell adhesions at AJs by regulating DE-Cad
expression.
|
). E-Cad has
been shown to mark the sites of actin assembly on cell surface
(Kovacs et al., 2002).
Cadherin complexes regulate cytoskeletal networks and cell polarity
(Harris and Peifer, 2004),
while disruption of AJ associated components affect asymmetric cell division
(Lu et al., 2001). Fat1, a
mammalian homolog of Drosophila Ft, modulates actin dynamics
(Moeller et al., 2004;
Tanoue and Takeichi, 2004).
Interestingly, Ft also regulates orientated cell division (OCD) in imaginal
epithelium, which is mirrored by orientation of the spindles of the dividing
cells; OCD may also regulate organ shape along the PD axis
(Baena-Lopez et al., 2005).
Misregulation of DE-Cad may thus affect the cytoskeleton and produce
OCD phenotype in ft mutant discs.
Intersection of Ft and Wg signaling and the regulation of distal wing growth and pattern
In both loss- and gain-of-function (Figs
5,
8 and data not shown) assays,
we could show that Ft downregulates Dll and Vg/Q-vg-lacZ
(Fig. 5, data not shown) in the
distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained
to be the direct targets of Wg, all available evidence
(Klein and Arias, 1999;
Neumann and Cohen, 1997;
Prasad et al., 2003;
Zecca et al., 1996) so far
suggests that these targets positively respond to Wg signaling
(Schweizer et al., 2003).
These results also show that Ft and Wg signaling intersect and control distal
wing growth and pattern, presumably through their opposing regulation of a
common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling,
Dpp signaling also regulates Q-vg-lacZ
(Kim et al., 1996); however,
its long-range target, Omb (Lecuit et al.,
1996; Nellen et al.,
1996) is not upregulated in ft mutant clones (not shown
here) suggesting that regulation of distal wing targets by Ft is mediated by
its intersection with Wg signaling.
Our results show that Ft negatively regulates Wg signaling. Loss or gain of
Ft induces a telltale sign of perturbations in Wg signaling, namely, changes
in the cellular pool of ß-catenin/Arm
(Collins and Treisman, 2000;
Jiang and Struhl, 1998;
Peifer et al., 1994;
Prasad et al., 2003),
consistent with its role as a suppressor of Wg signaling in genetic tests
(Fig. 6). Our results further
reveal intersection of Ft with Wg signaling downstream of the Wg ligand
(Fig. 8), while with respect to
its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2
(Fig. 8). It is interesting to
note here that the role of Ft in PCP regulation has also been suggested to be
either parallel to or upstream of the Fz receptor
(Ma et al., 2003;
Strutt and Strutt, 2002;
Yang et al., 2002). We also
note that Ft co-localizes with neither Fz
(Ma et al., 2003) nor Fz2 (not
shown here) and does not mediate their subcellular localization (not shown
here), thereby suggesting that Ft interacts with Fz indirectly. Unraveling the
genetic and molecular basis of this interaction may explain how Ft straddles
both the canonical (growth and cell-cell adhesion) and non-canonical (PCP)
(Veeman et al., 2003) Wnt
signaling pathways.
One of the remarkable aspects of development of an organ primordium is that
a stereotypic PCP is achieved even while it passes through dynamic changes in
its size and shape (Day and Lawrence,
2000; Eaton,
2003). The fact that changing organ sizes/shapes does not alter
PCP suggests an in-built mechanism to regulate constancy of PCP during animal
development (Eaton, 2003). A
link between PCP and growth through the activity of Ft has been speculated, as
it regulates both (Lawrence,
2004). Intersection of Ft and the canonical Wg signaling seen here
might provide a mechanism to coordinate PCP and organ growth.
Wg signaling and regulation of wing growth by the tumor suppressor Ft
Drosophila wing growth is under dynamic spatial and temporal
regulation by Wg signaling (Giraldez and
Cohen, 2003; Johnston and
Sanders, 2003; Martinez Arias,
2003; Neumann and Cohen,
1996). Furthermore, different thresholds of Wg signaling impact
cell proliferation in their characteristic ways and activate distinct sets of
PD markers. Although at a very high threshold, Wg signaling inhibits cell
proliferation (Johnston and Sanders,
2003; Neumann and Cohen,
1996), at a modest threshold it has been shown to stimulate growth
(Giraldez and Cohen, 2003;
Neumann and Cohen, 1996). We
note that loss of Ft fail to activate Wg targets that demand a high threshold
of its signaling, e.g. Ac (not shown here), which is required for wing margin
specific bristle development (Couso et
al., 1994; Neumann and Cohen,
1997). Conversely, overexpression of Ft also did not lead to loss
of margin bristles (Fig. 5),
suggesting that it is not a strong repressor of Wg signaling either. The
short-range Wg target, fz3-lacZ
(Sato et al., 1999;
Sivasankaran et al., 2000),
which responds to a high threshold of Wg signaling, is also not upregulated by
loss of Ft (not shown here). Dll responds to a higher threshold of Wg
signaling than that required for Vg/Q-vg
(Neumann and Cohen, 1997;
Zecca et al., 1996). Dll and
Vg display modest and strong upregulation (Figs
5 and
8), respectively, following
loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to
only modest thresholds, consistent with the growth-promoting effect of the
latter (Neumann and Cohen,
1996).
Over-proliferation in ft mutant imaginal discs is induced by
perturbation of as yet unidentified disc-intrinsic mechanisms that determine
their characteristic final sizes (Bryant et
al., 1988; Cho and Irvine,
2004). The imaginal discs of ft mutants continue to grow
and the extent of their over-proliferation appears to be constrained only by
the developmental time available during the extended periods of their larval
life (Bryant et al., 1988). By
contrast, growth in wild-type imaginal discs is determinate, which ceases
after they attain their predetermined sizes even under conditions of unlimited
developmental time; for example, on transplantation into wild-type adult host
abdomen that can sustain development
(Bryant, 1975;
Bryant et al., 1988).
ft mutant imaginal discs thus acquire unlimited proliferative
potential, akin to immortalization, a crucial step during tumorigenesis
(Hanahan and Weinberg, 2000).
It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling,
a pathway implicated in cancers (Logan and
Nusse, 2004; Peifer and
Polakis, 2000). Several orthologs of Ft have been identified in
vertebrates with diverse functions (Tanoue
and Takeichi, 2005). It will thus be interesting to explore if
these orthologs of Ft in higher vertebrates also interact with Wnt signaling
and thereby behave as tumor suppressors.
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ACKNOWLEDGMENTS |
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Footnotes |
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