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First published online 3 August 2006
doi: 10.1242/dev.02468
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Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY, USA.
* Author for correspondence (e-mail: marek.mlodzik{at}mssm.edu)
Accepted 1 June 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Cadherins, Cell motility
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Klein and Mlodzik, 2005;
Mlodzik, 2002;
Shulman et al., 1998;
Veeman et al., 2003). Although
genetic screens have identified many signaling molecules that participate in
PCP signaling, the relationship between signaling events and changes in
cytoskeletal organization and/or cellular adhesion remains unclear.
The read-out of PCP signaling is often the organization of cytoskeletal
elements or orientation of the mitotic spindle
(Adler, 2002;
Bellaiche et al., 2001;
Klein and Mlodzik, 2005;
Shulman et al., 1998). PCP is
also evident in neural sensory epithelia, most prominently in the
Drosophila eye (Mlodzik,
1999; Strutt and Strutt,
1999) and mammalian inner ear
(Dabdoub et al., 2003;
Montcouquiol et al., 2003). In
the Drosophila eye, PCP establishment includes transcriptional events
linked to photoreceptor cell fate specification and complex cellular movements
that cause ommatidial precursors to undergo a 90° rotation towards the
midline of the developing eye field
(Mlodzik, 1999;
Strutt and Strutt, 1999).
Drosophila eyes are composed of 
800 ommatidia, each
consisting of eight photoreceptor cells, and 12 accessory cells. Ommatidial
assembly begins posterior to an organizing center, the morphogenetic furrow
(MF), an indentation that forms at the posterior of the eye disc and moves
across the developing disc. Photoreceptor precursors are assembled and
specified behind the MF in a stereotyped manner, with R8 being the first,
followed by pairwise recruitment of R2/R5, R3/R4 and R1/R6, and finally R7
(Wolff and Ready, 1993).
Correct R3/R4 specification is crucial for PCP establishment. Initially,
this photoreceptor pair is symmetrical. Then, the cell located closer to the
dorsoventral (D/V) midline, the equator, receives a higher level of Fz signal,
which specifies it as R3, and causes a transcriptional upregulation of
Delta in the same cell. Delta then activates Notch signaling in the
neighboring cell of the pair, specifying it as R4
(Cooper and Bray, 1999;
Fanto and Mlodzik, 1999;
Tomlinson and Struhl, 1999).
As a result of distinct R3/R4 specification, ommatidial preclusters lose their
symmetry and adopt two opposing mirror-image chiral forms in the ventral and
dorsal eye hemispheres (Mlodzik,
1999; Strutt and Strutt,
1999).
Following R3/R4 specification, ommatidial preclusters rotate towards the
equator (Mlodzik, 1999;
Strutt and Strutt, 1999;
Wolff and Ready, 1993). Each
cluster rotates as a unit; this process is highly coordinated as clusters of
the same maturity rotate simultaneously. At its end, ommatidia have rotated
90° away from their original position. R3/R4 specification is a
prerequisite for the correct direction of rotation, resulting in dorsal and
ventral ommatidia rotating in opposite directions
(Mlodzik, 1999;
Strutt and Strutt, 1999). Both
the establishment of asymmetry within nascent ommatidial clusters and the
subsequent rotation event lead to the final ommatidial arrangement, reflected
in the mirror-image symmetry across the equator
(Fig. 1).
How ommatidial rotation initiates is unknown. The signal to stop rotation
at 90° is also unknown, but likely involves EGF-receptor (Egfr) and Notch
(N) signaling (Brown and Freeman,
2003; Chou and Chien,
2002; Gaengel and Mlodzik,
2003; Strutt and Strutt,
2003). Fz/PCP signaling may regulate rotation through effects on
cytoskeletal elements and cell-adhesion molecules. This is supported by the
observation that rok (Drosophila Rho-associated kinase), an
effector of the RhoA GTPase that has been placed genetically downstream of Fz
has a rotation-specific eye phenotype
(Winter et al., 2001).
Finally, based on genetic evidence, initiation of the second rotation step
(45° to 90°) is regulated by nemo, a MAP kinase
(Choi and Benzer, 1994). A link
of nemo, Egfr or N-activity to Fz signaling or rok-mediated
events has not been established. Rotation still takes place in Fz/PCP mutants,
albeit at random and mutations in rotation-specific genes do not affect the
R3/R4 fate decision and ommatidial chirality. It thus appears likely that
distinct signaling inputs are required downstream of Fz/PCP to regulate the
mechanistic aspects of ommatidial rotation.
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;
Tepass et al., 2000;
Yeaman et al., 1999).
Classical cadherins are single pass transmembrane glycoproteins that mediate
cell-cell adhesion through their Ca-dependent extracellular domains, which are
composed of `cadherin repeats'. Their cytoplasmic tails interact with the
cytoskeleton by binding to catenins, which then recruit other cytoskeletal or
signaling proteins (Perez-Moreno et al.,
2003). The cadherin-catenin complex, which forms adherens
junctions in vertebrates and invertebrates, is dynamically regulated to
participate in cell adhesion, sorting and signaling.
Drosophila has three cadherins that are functionally related to
vertebrate classical cadherins: DE-cadherin (DE-cad/shotgun),
expressed in almost all epithelia; and two DN-cadherins (DN-cad1/cadN
and DN-cad2/cadN2), encoded by adjacent genes and predominantly
expressed in mesodermal and neural tissues
(Iwai et al., 1997;
Tepass et al., 1996;
Uemura et al., 1996;
Prakash et al., 2005).
Vertebrate E- and N-cadherins often show complementary expression patterns.
Similarly, Drosophila DE-cad is early expressed ubiquitously and
later is downregulated in newly forming mesoderm and neural tissues, and
replaced with DN-cad. This `cadherin switch' from an epithelial cadherin,
maintaining cells in an epithelial layer, to a N-cad that promotes cell
migration is a required step during development
(Hazan et al., 2004). As
cadherins are required to maintain tissue integrity, it is difficult to
demonstrate an instructive role in tissue remodeling. However, there is
evidence that, in addition to N-cad, E-cad also promotes cell movement
(Montero et al., 2005;
Niewiadomska et al.,
1999).
Studies with Egfr signaling in ommatidial rotation suggest that DE-cad is
involved in this process. In particular, decreasing the dose of endogenous
DE-cad enhances the rotation specific phenotype of Egfr-signaling components
(Brown and Freeman, 2003;
Gaengel and Mlodzik, 2003),
suggesting that DE-cad is required during rotation. Here, we demonstrate that
shotgun (shg/DE-cad) promotes ommatidial rotation. By
contrast, the DN-cadherins appear to restrict rotation, most probably by
preventing ommatidia from rotating too fast. Our data indicate that the
balance between DE-cad and DN-cad is crucial for the precision required to
achieve correct ommatidial rotation. Consistent with this conclusion is the
largely complementary subcellular accumulation of DE-cad and DN-cad1 in
developing ommatidial preclusters. The dynamic cell-cell adhesion mediated by
the two cadherins regulates the extent of motility in several developmental
contexts and disease states.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For
UAS-DN-cad1, two independent lines were tested with medium to high
expression levels (T. Uemura, personal communication). All were driven with
the UAS/Gal-4 system (Brand and Perrimon,
1993). sevGal4 drives expression in R3/R4, R1/R6 and R7
precursors (gift from K. Basler). The flip-out gain-of-function clones of
UAS-DN-cad and UAS-DE-cadDN were generated with hs-FLP,
actin>CD2>Gal4 (Pignoni and
Zipursky, 1997) and marked with UAS-GFP. Interaction
crosses were grown at 25°C and eyes from female flies examined. The
transgenes that modified sev>DE-cadDN (UAS-EcadWT,
UAS-EcadEx, UAS-Ecad
B, UAS-ArmS2) showed no
phenotype with sevGal4 by themselves.
For loss-of-function clonal analyses, shgP34-1 (gift
from U. Tepass) was recombined onto the w-; FRT42
chromosome and clones were induced with eyFLP (marked with
ubiGFP) (Newsome et al.,
2000). Minute (M) clones [giving mutant tissue a growth advantage;
Garcia-Bellido et al. (Garcia-Bellido et
al., 1976)] were induced with eyFLP; FRT42, ubiGFP, M.
For rescue experiments, eyFLP; shgP34-1, FRT42, ubiGFP;
sev>DEcadWT genotype was analyzed.
Mutant alleles and UAS lines: shgP34-1, shgR6,
shgIg29 (from U. Tepass), DE-cadWT (UAS-DECH and
UAS-DEFL), DE-cadDN (dCR3h 7-1), DE-cad-dEx (UAS-dEx), DE-cadDN (UAS-dCR4h,
UAS-dCR4, UAS-dCPc3, UAS-mNcGSP) (see Oda
and Tsukita, 1999); UAS-DE-cadß
(Pacquelet et al., 2003),
ubi-DE-cad-GFP (from F. Schoeck), UAS-DN-cad, NcadM19
(Iwai et al., 1997),
UAS-RhoAIR (Billuart et al.,
2001), Ncad14
(Prakash et al., 2005),
UAS-DE-cadwt (Lee et al.,
2003), stbm6, and dgo, stbm double mutant
(Das et al., 2004).
shg2, mys1, UAS-ArmS2, armYD35,
armXM19, wgCX4, spi259, EgfrElpB1,
pntD88, fzR52, dsh1, fmiE59,
dgo380, nmoP, dRok2, scaBP1,
rhoA72R and UAS-GFP are as described in FlyBase
(http://flybase.bio.Indiana.edu).
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-tubulin (Sigma),
rhodamine-phalloidin (Molecular Probes); anti-ß-Gal (Cappel, Promega),
rat anti-Elav, mouse anti-Armadillo (N27A1), rat anti-DN-cad (Dn-Ex#8) and
mouse anti-Flamingo from Hybridoma Bank. Secondary antibodies coupled to
fluorochromes were from Jackson Laboratories. Imaginal discs were stained as
described (Fanto et al.,
2000), mounted in 80% glycerol/2% N-propyl-gallate and viewed with
a Zeiss 510 confocal laser-scanning microscope. Eye sections were prepared as
described (Tomlinson et al.,
1987). For genetic interactions, eyes were sectioned near
equatorial region and only ommatidia with correct photoreceptor number were
scored; a rotation defect was defined as being either less than 80° or
more than 110° (compared to 90° in wild type). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DE-Cadherin is positively required for ommatidial rotation
To further examine the role of DE-cad in rotation, we used the UAS/Gal4
system (Brand and Perrimon,
1993) to analyze the consequences of eye specific expression of
different forms of the DE-cad protein (Oda
and Tsukita, 1999) (see Materials and methods). In addition to
full-length protein, transgenic lines with deletions of either the entire
extracellular, intracellular or deletions in cadherin (CAD) repeats were
tested (see Materials and methods). Expression of full-length DE-cad under the
control of sevGal4 (expressed in the R3/R4 pair at the time of
rotation initiation; see Materials and methods) had no effect at moderate
levels, and caused epithelial defects at high levels (not shown). By contrast,
a sevGal4 driven dominant-negative construct [DE-cadDN, with
an internal deletion of two CAD repeats;
(Oda and Tsukita, 1999)]
resulted in specific rotation defects (sev>DE-cadDN;
Fig. 3A). Importantly, as
photoreceptor loss and patterning defects were minimal and PCP associated
chirality defects were not observed, this suggested that DE-cad specifically
affects ommatidial rotation. This phenotype is comparable with rok or
nemo (Choi and Benzer,
1994; Winter et al.,
2001), which also specifically affect rotation but not the R3/R4
fate decision. We confirmed that DE-cadDN indeed acts as a dominant
negative, as removing one copy of endogenous DE-cad/shg
enhanced the sev>DE-cadDN phenotype
(Fig. 3B). Moreover,
coexpression of DE-cadWT (moderate levels) with
DE-cadDN (under sevGal4 control) rescued the rotation
defects (Fig. 3C).
A striking feature of all rotation defects associated with the DE-cad loss-of-function phenotypes (either in hypomorphic allele, shgR6, shgP34-1, or DE-cadDN combinations) is that the majority of ommatidia rotate less than 90°, displaying variable levels of under-rotation or lacking rotation completely (Fig. 2A-E; Fig. 3A,B; Table 1). The severity of the under-rotation correlates with allelic strength (see Fig. S1 in the supplementary material). The above data indicate that DE-cad/shg acts as a positive regulator of ommatidial rotation.
Dynamic expression of DE-cad and DN-cad during ommatidial rotation
We next analyzed the expression pattern of DE-cad during ommatidial
rotation. E-cad is expressed uniformly in epithelia close to the apical cell
surface, establishing adherens-junctions through its homophilic cell-adhesion
behavior and its intracellular interactions with the cytosolic catenins
(Perez-Moreno et al., 2003).
Similarly, in the eye disc epithelium, prior to photoreceptor recruitment and
adjacent to the furrow (MF), DE-cad is equally distributed as an apicolateral
membrane `ring' in all cells (Fig.
4A-A', left of MF). Posterior to MF, DE-cad expression is
patterned (Fig. 4A,A',
right of MF). As photoreceptor precursors are recruited into preclusters,
DE-cad becomes enriched in membranes bordering R8, and the R2/R5 pair
(Fig. 4A,A', see
Fig. 4D,D'' for detail),
and interestingly it is detected at much lower levels at the membranes between
the R3/R4 pair, as well as membranes between precluster cells and surrounding
undifferentiated cells (Fig.
4A,A',4D,D'').
DE-cad remained upregulated at all membranes between precluster cells, except
the R3/R4 border, throughout the process of rotation and precluster assembly
(Fig. 4A).
The Drosophila ß-catenin protein Armadillo (Arm) colocalizes
with DE-cad through its interaction with cytoplasmic DE-cad sequences
(Tepass et al., 2000).
Interestingly, Arm is still enriched at the R3/R4 border where DE-cad
expression is low (Fig. 4A,
arrows; Fig. 4D,D'),
suggesting that Arm interacts with another cadherin. We, thus, analyzed
DN-cad1 expression and observed that its distribution within the precluster is
largely complementary to DE-cad; DN-cad1 is enriched at R3/R4 cell border
membranes (Fig.
4B,C,4E-E'').
DN-cad1 is not detected ahead of and close to the MF
(Fig. 4C). Later, DN-cad1 is
also detected in axonal projections of all photoreceptors as these extend out
of the retinal layer (Lee et al.,
2001; Prakash et al.,
2005). At the apical R3/R4 border, DN-cad1 colocalizes with the
atypical cadherin Flamingo [Fmi; a core PCP-signaling and R3/R4 specification
factor (Das et al., 2002)]
(Fig. 4E).
To gain insight as to how the DE-cad and DN-cad1 complementary accumulations are generated within the precluster, we analyzed their localization in loss-of-function and overexpression clones of each other (see Materials and methods). First, we determined the contribution of DE-cad and DN-cad1 to apicolateral Arm localization. DE-cad levels strongly affect Arm levels (Fig. 4G), suggesting that the bulk of endogenous apicolaterally localized Arm is anchored through DE-cad, the exception being the R3/R4 cell border membranes. By contrast, decrease in DN-cad1 and 2 levels (Fig. 4F) has no effect on DE-cad (not shown) or Arm, except at the R3/R4 border, where Arm levels are reduced (Fig. 4F,F'). The DN-cad1 expression pattern is unchanged in DE-cad/shg (shgP34-1) loss-of-function clones (Fig. 4G,H). Arm is significantly decreased in such clones, but still accumulates apicolaterally between the precluster cells, where DN-cad is present (Fig. 4G). Clones overexpressing DE-cadDN do not affect endogenous DN-cad1 or Fmi (not shown). However, an increase in DN-cad1 expression lowers DE-cad levels (Fig. 4I,I'), without affecting Fmi localization (Fig. 4I,I''; the same is observed anterior to MF, not shown). Both DN-cad1 and DE-cadDN overexpression causes an increase in membrane associated Arm (Fig. 5E; not shown). These data indicate that there is largely mutually exclusive localization of DE-cad and DN-cad1 in wild type. The regulation of this pattern might be in part mediated through a repressive effect of DN-cad on DE-cad, as observed in the gain-of-function DN-cad clones (Fig. 4I). However, it is more complex, as in DN-cad mutant clones DE-cad is not upregulated.
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).
Although single loss-of-function analysis did not show a retinal phenotype,
mosaic eyes of mutants lacking both genes
[DN-cad14 deletes
DN-cad1 and DN-cad2
(Prakash et al., 2005)]
revealed retinal phenotypes, including misrotated ommatidia, symmetrical
clusters and loss of photoreceptor cells
(Fig. 5A, and not
shown).
To gain better insight into the role of DN-cad in rotation, we analyzed
N-cad14 clones in 3rd instar
eye discs, during ommatidal rotation and prior to terminal photoreceptor
differentiation [also affected in the
N-cad14 mutant
(Prakash et al., 2005)].
Interestingly, the disc phenotype of
N-cad14 mutant clusters is the
opposite of DE-cad/shg mutant clones, with the majority of mutant
ommatidial clusters reaching a 90° angle prematurely as compared with
wild-type neighbors (Fig. 5B;
Table 1). Therefore, DN-cad
function appears to provide the correct rate of movement.
We next tested the effects of DN-cad1 overexpression, driven with sevGal4 (sev>DN-cad). The sev>DN-cad genotype showed rotation defects reflected in ommatidia having rotated to a random degree (Fig. 5F). Random rotation angles are also observed in 3rd instar sev>DN-cad eye discs (Fig. 5C), consistent with the loss-of-function analysis and the suggestion that the DN-cadherins function in controlling the rate of rotation.
DE-cad affects rotation through its interactions with cytoskeletal elements
To gain further insight into cadherin requirements in ommatidial rotation,
we used the dose-sensitive sev>DE-cadDN and sev>DN-cad
genotypes for dominant genetic interaction studies. This approach is often
used in Drosophila to identify members of a signaling pathway or
interacting factors involved in the regulation of a particular developmental
or morphological process (e.g. Boutros et
al., 1998; St Johnston,
2002). Our initial analysis of sev>DE-cadDN indicated
that the phenotype is dose sensitive and modified by decreasing endogenous
DE-cad levels (Fig. 3B).
We first tested mechanistic aspects of DE-cad-regulated rotation. The
sev>DE-cadDN phenotype is enhanced by dose reduction of an
arm null allele (armYD35;
Fig. 3D), but not an
arm allele that maintains its cell architecture function
(armXM19, only defective for Wg-signaling;
Fig. 3E). This suggests that
the cytoskeletal anchoring function of DE-cad, via Arm/ß-catenin, is
crucial for ommatidial rotation, and is supported by several observations.
First, the enhancement is, in addition to rotation, in defects associated with
ommatidial architecture and adhesion (Fig.
3D; e.g. causing gaps between ommatidia; also
Fig. 5G). Second, unlike
full-length DE-cad that rescues the rotation defects of
sev>DE-cadDN (Fig.
3C), co-expression of sev>DE-cadDN with
DE-cadß, which lacks Arm interaction sequences, does not
rescue the defects (Table 2).
Third, DE-cad lacking the extracellular Cad-repeats (DE-caddEx) does
not rescue sev>DE-cadDN when co-expressed
(Table 2). Taken together,
these data suggest that the cell adhesion and cytoskeletal anchoring aspects
of DE-cad are crucial in ommatidial rotation.
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14;
Table 2). The
sev>DN-cad phenotype is enhanced both by DE-cad/shg dose
reduction and by co-expression of DE-cadWT (not shown). The
enhancement of sev>DE-cadDN under-rotation by co-expression of
DN-cad may be due to competition for Arm (as suggested from DN-cad
gain-of-function clones; Fig.
5E,E'). However, the sev>DN-cad rotation
phenotype is not enhanced by arm reduction
(Fig. 5G) and co-expression of
UAS-ArmS2 (full-length) enhances both the rotation defects
and defects in epithelial integrity of sev>DN-cad
(Fig. 5H). Taken together,
these observations suggest that a proper balance between DE-cad, DN-cads, and
Arm levels is crucial for the precise 90° ommatidial rotation.
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;
Klein and Mlodzik, 2005;
Mlodzik, 1999;
Strutt and Strutt, 1999).
Egfr-signaling is also required for the process, although its specific
regulatory input remains unclear (Brown and
Freeman, 2003; Gaengel and
Mlodzik, 2003; Strutt and
Strutt, 2003). We thus tested for genetic interactions of
components of these pathways with sev>DE-cadDN.
Previous studies identified shg/DE-cad alleles as
enhancers of rotation defects caused by reduction in Egfr signaling
(Brown and Freeman, 2003;
Gaengel and Mlodzik, 2003) (a
decrease in DE-cad enhances the randomness of rotation in Egfr-signaling
mutants). Our data show that reduced Egfr-signaling levels (e.g. reduced gene
dose of the Egfr-ligand Spitz) specifically enhanced the under-rotation of
sev>DE-cadDN (Table
2), suggesting that Egfr-signaling promotes rotation by positively
regulating DE-cad function. This is supported by the observation that the
gain-of-function EgfrElp allele appears to suppress
sev>DE-cadDN under-rotation (not shown; owing to the dominant
effects of EgfrElp on photoreceptor numbers and survival
this effect cannot be directly compared with the other interactions). The
enhancement of sev>DE-cadDN by pointed
(Table 2; pnt, a
common nuclear effector of RTK/MAPK pathways) suggests that the entire
RTK/MAPK cascade is functioning in this context (see Discussion).
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We next wished to determine whether DE-cad/shg during rotation is required in a specific cell or cells within the precluster, and in particular tested for R3/R4 requirement. Mosaic analysis of shgP34-1 clones (in discs) suggested that mosaic ommatidia with both R3 and R4 wild type do not have a higher probability of rotating normally. Largely, the overall number of wild type R-cells correlates with correct rotation: the more wild-type cells are present per cluster, the higher is the probability of normal rotation (see Tables and Fig. S1 in the supplementary material). Furthermore, we have also observed fully wild-type preclusters directly adjacent to mutant interommatidial cells that failed to rotate properly (Fig. 2C-D''; orange arrowheads), suggesting that DE-cad/shg function is required not only in R-cells but also in adjacent interommatidial cells. Nevertheless, expression of sev>DEcadWT (expressed in R3/R4 at the onset of rotation) increased the number of clusters that initiated rotation in shgP34-1 clones (Fig. 2E,E'; Table 1), suggesting that sufficient levels of DE-cad in R3/R4 are crucial. Taken together, these data suggest that in rotation DE-cad/shg is required in more cells than the R3/R4 pair, including cells that are adjacent but outside the precluster. Moreover, the genetic interactions suggest that parallel input from PCP and Egfr-signaling regulates DE-cad activity during rotation.
RhoA might link PCP signaling to DE-cad function in rotation
Classical cadherins have multiple links to the cytoskeleton, primarily
through 
- and ß-catenin
(Perez-Moreno et al., 2003).
Reduction of the Fz/PCP effector RhoA enhanced the sev>DEcadDN
phenotype. Mutants in rok, a downstream effector of RhoA, have a
rotation-specific loss-of-function eye phenotype, and RhoA has been linked to
the regulation of DE-cadherin apical localization and clustering at the
membrane (Bloor and Kiehart,
2002; Magie et al.,
2002; Yap and Kovacs,
2003).
Our genetic interactions have revealed that decreasing endogenous RhoA
levels enhanced the rotation and adhesion defects of sev>DEcadDN
(Table 2; and not shown). To
minimize the RhoA-associated defects that are not linked to rotation [e.g.
general cellular architecture as RhoA null clones are cell lethal
(Strutt et al., 1997)], we
expressed UAS-RhoAIR [lowering endogenous RhoA
levels through RNA interference (Billuart
et al., 2001)] in the R3/R4 precursors. Co-expression of
sev>DEcadDN with UAS-RhoAIR causes a specific
enhancement of the sev>DE-cadDN under-rotation phenotype
(Fig. 6A,B;
Table 2). Whereas a general
decrease in endogenous RhoA affects cell adhesion (in addition to rotation),
the specific RhoA RNAi knock-down in a subset of R-cell precursors at the
onset of rotation only enhances ommatidial under-rotation
(Table 2; and not shown).
Clones of hypomorphic RhoA alleles display an irregular DE-cad
localization in eye discs, with malformed preclusters and a general reduction
of DE-cad levels outside the precluster
(Fig. 6C,C') (see also
Bloor and Kiehart, 2002;
Magie et al., 2002;
Yap and Kovacs, 2003). Similar
results were obtained with dRok2 clones (not shown). These
data show that in addition to the proposed role for RhoA in DE-cad
localization and maintenance of tissue integrity, the dynamic rearrangements
of DE-cad and DN-cad during rotation require RhoA function. Fz/PCP-signaling
thus probably regulates DE-cad function and/or localization through RhoA-Rok.
The fact that we do not observe gross mislocalization of DE-cad in mutant
clones of primary PCP genes (e.g. fmi or stbm; not shown)
suggests that effects of RhoA on DE-cad localization/activity in rotation are
subtle, influenced by several inputs and not affecting tissue integrity.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
We propose that such conserved molecular mechanisms drive cell movement
processes in general during development and disease (see below).
DE-cadherin and cell movement
Although the role for DE-cad in tissues undergoing rearrangements during
development is established (Tepass et al.,
2000; Perez-Moreno et al.,
2003), a direct role for DE-cad in cell and tissue movement has
been more difficult to study in vivo owing to its essential role in
maintenance of epithelial integrity. Analysis of adult eye phenotypes of a
homozygous viable shg/DE-cad allele and a dominant-negative DE-cad
construct (DE-cadDN), expressed in the R3/R4 and later R1/R6, R7
precursors, indicate that DE-cad is required throughout the rotation process.
The ability of ommatidia to complete the precise 90° rotation directly
depends on DE-cad activity. Both the extracellular domain, responsible for
cell-cell adhesion, and the intracellular domain, linking DE-cad to the actin
cytoskeleton, are required for rotation. DE-cad associates with the actin
cytoskeleton primarily through interactions with Arm/ß-catenin. Although
ß-catenin has a dual role in cell adhesion and Wg signaling [which can be
separated (Orsulic and Peifer,
1996; Sanson et al.,
1996)], our data indicate that during ommatidial rotation
ß-catenin acts through its role in cell adhesion.
Ommatidial rotation represents the final step in establishing PCP during
eye development. The direction of rotation depends on proper R3/R4 cell fate
specification, which is determined by PCP signaling. The Egfr pathway and
input by rotation-specific genes, e.g. nemo, are thought to function
in parallel to Fz-PCP signaling. We observed an enhancement of the
sev>DE-cadDN rotation defects by dose reduction in core regulatory
PCP genes dgo and stbm
(Table 2; see Fig. S1 in the
supplementary material; ommatidial under-rotation and the number of ommatidia
that did not initiate rotation in
sev>DE-cadDN/dgo-/+, stbm-/+ was
comparable with the enhancement of sev>DE-cadDN by heterozygosity
of a shg null allele). The localization of PCP protein complexes at
the level of adherens junctions (Djiane et
al., 2005; Wu et al.,
2004) is consistent with the idea that PCP factors can influence
DE-cad function. The mechanism of this regulation remains unclear. The
RhoA-RNAi transgene, which was expressed only in R3/R4 precursors
during the initiation of ommatidial rotation, enhanced
sev>DE-cadDN associated under-rotation defects. Although a RhoA
requirement in multiple cellular processes makes it difficult to dissect its
specific role in rotation, the specificity of the phenotype (enhanced
under-rotation in sev>DEcad/RhoAIR) suggests a role for
RhoA in the regulation of cadherin-mediated cell movement.
Although Egfr signaling appears to be required for the precise 90°
rotation (Brown and Freeman,
2003; Gaengel and Mlodzik,
2003; Strutt and Strutt,
2003), its role in the process -promoting motility or antagonizing
it -has remained unclear. Our genetic data suggest that Egfr signaling acts
positively to promote rotation, as a reduction in Egfr signaling enhances the
sev>DE-cadDN under-rotation phenotype
(Table 2,
Fig. 7). This may reflect a
positive role for Egfr signaling in the regulation of DE-cad activity or
turnover at the membrane, as suggested from human tumor cell lines
(Lu et al., 2003). Affecting
the function of endocytic pathway components can also have an effect on
ommatidial rotation (Weber et al.,
2003). This might be mediated by Egfr signaling, as is thought to
be the case in human cancer cells, leading to recycling and redistribution of
E-cad at the plasma membrane (Naora and
Montell, 2005).
|
Distinct requirements for DN-cad and DE-cad in regulating the rotation process
Drosophila DN-cadherins, which are encoded by the adjacent
cadN and cadN2 genes, are the main cadherins expressed in
the nervous system. In developing photoreceptors they participate in axon
guidance, and in pupal eye discs they mediate terminal patterning of the
retina [through specific expression in cone cells
(Hayashi and Carthew, 2004;
Iwai et al., 1997;
Lee et al., 2001;
Prakash et al., 2005)]. During
PCP establishment, DN-cad1 is concentrated at the border between R3/R4
precursors, in a pattern largely complementary to DE-cad
(Fig. 4B,C,E). This suggested a
possible combinatorial role for DE-cad and DN-cad in rotation, with DN-cad
either providing a structural role in rotating clusters, or participating in
signaling cascades that regulate cell movement. Analysis of DN-cad
mutant clones in discs during rotation demonstrated a specific function, as
many mutant clusters have completed rotation well before wild-type clusters of
the same stage (Fig.
5B-B''). These data indicate that DN-cadherins function to
slow down rotation, serving an opposing function to DE-cad.
The balance and complementary distribution of DE-cad and DN-cad appear crucial for correct rotation to occur. Mild overexpression of DN-cad1 in R3/R4 (sev>DN-cad) is sufficient to interfere with the process, possibly by affecting DE-cad levels. Consistently, DN-cad1 overexpression enhances sev>DE-cadDN induced under-rotation and overexpression clones of DN-cad1 cause a decrease in endogenous DE-cad levels (Fig. 4I). Alternatively, the negative effect of DN-cad on DE-cad might be through competition for ß-catenin, as sev>DE-cadDN is partially rescued by UAS-ArmS2 (full length; not shown), although as sev>DN-cad is not enhanced by arm dose reduction this appears less likely. Interestingly, sev>DN-cad is enhanced by co-expression of full-length DE-cad and full-length Arm. These phenotypes resemble those of a strong sev>DN-cad line (not shown), suggesting that DN-cad is stabilized by increased levels of available Arm, and also that co-overexpression of two cadherins may interfere with optimal turnover rate at the membrane.
|
). This is consistent with our data, as overexpression
of DN-cad induces apparent accumulation of endogenous Arm in the cell, largely
at the cell membrane.
Parallels between ommatidial rotation and cell migration during development and disease
Previous work has identified some signaling input into ommatidial rotation,
e.g. PCP and Egfr signaling. Here, we begin to address mechanistic aspects of
rotation, defining the involvement of cell adhesion molecules that are linked
to cytoskeletal elements.
Ommatidial rotation shares similarities with border cell migration (BCM)
during Drosophila oogenesis
(Niewiadomska et al., 1999).
In both cases, DE-cad is upregulated in the cell cluster as the respective
cells are being recruited from surrounding epithelial cells, and as migration
is initiated, DE-cad is downregulated at the membrane between the migrating
cells and surrounding `substrate' cells. Egfr signaling is required positively
for both processes (Duchek and Rorth,
2001) (this work). Thus, although the regulatory role for Egfr in
rotation is complex, our data suggest that both Egfr and PCP components
promote DE-cadherin mediated rotation. Other RTKs may act redundantly in
promoting rotation (I.M. and M.M., unpublished), as they do in BCM
(Duchek et al., 2001).
Although the role of DN-cad in BCM remains unclear, existing data suggest that
it may serve an inhibitory function there. DN-cad1 is detected in all
epithelial follicular cells from which border cells are recruited but is lost
after mid-oogenesis when BCM initiates
(Tanentzapf et al., 2000). Our
data suggest that DN-cad1 is initially present, albeit reduced, in border
cells, but becomes undetectable during BCM (I.M. and M.M., unpublished).
A positive role for DE-cad in migration is not limited to
Drosophila. A recent study addressing zebrafish gastrulation finds a
similar role for E-cad during cell migration
(Montero et al., 2005).
Furthermore, BCM (Naora and Montell,
2005) and ommatidial rotation (this work) share similarities with
the progression of ovarian carcinoma
(Naora and Montell, 2005).
Ovarian cancer cells tend to cluster and metastasize as cell aggregates that
show upregulation in E-cad expression. The proposed role for RTK signaling in
the three processes (BCM, ommatidial rotation and ovarian carcinoma
metastasis) suggests an evolutionarily conserved mechanism that can switch the
function of E- and N-cad between promoting and inhibiting cell movement.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/17/3283/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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