Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK
Authors for correspondence (e-mail: vm237{at}mole.bio.cam.ac.uk and ama11{at}cus.cam.ac.uk)
Accepted 7 April 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Dorsal closure, Armadillo, Wingless, Planar cell polarity (PCP), Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At the end of germband retraction, the cells of the amnioserosa undergo
cell shape changes through a reduction of their apical surface
(Harden et al., 2002). The net
result of these changes is a pulling force exerted by the amnioserosa on the
epidermis, which appears to be the main force driving DC during these early
stages (Harden et al., 2002
;
Hutson et al., 2003
;
Kiehart et al., 2000
).
Simultaneously, the epidermal cells elongate in the dorsoventral direction,
first the dorsal-most epidermal (DME) cells and then the cells positioned more
ventrally. There is no cell division during DC and these cell shape changes
account for the increase of the epidermal surface, which eventually enables
the epidermis to enclose the embryo (Foe,
1989
; Young et al.,
1993
). In a second step, the two edges of the epidermis meet at
the ends of the embryo and initiate a zippering process powered by filopodia
and lamellipodia that protrude from the actino-myosin cable
(Jacinto et al., 2000
;
Kaltschmidt et al., 2002
).
These filopodia are thought to contribute to the interactions between the two
edges of the epidermis both by facilitating adherens junction establishment
and contributing to a correct segment-matching of the two sides of the
epidermis (Jacinto et al.,
2000
). The interactions between the two sides of the epidermis
proceed from both the anterior and the posterior edges in a double zipper-like
fashion until the continuity of the epidermis is achieved. At the same time,
the amnioserosa ingresses, dislodges itself from the epidermis and undergoes
apoptosis (Hartenstein,
1993
).
Genetic screens, which are mainly based on surveying defects in the
patterning of the dorsal epidermis, have identified a large number of genes
required for DC. These can be grouped into two main categories: genes involved
in signalling pathways, e.g. JNK, Wnt and Dpp pathways, and genes coding for
components of cellular architecture or its regulation, e.g. cytoskeletal
components like Myosin or proteins associated with the adherens junctions like
Canoe/L-afadin or Abl (Harden,
2002). Although early studies focused on large defects in the
patterning process, more recent ones have targeted cellular activities
(Harden et al., 2002
). These
studies have revealed a number of specialisations of the DME cells and in
particular a progressive polarisation of their membrane components in the
plane of the epithelium (Kaltschmidt et
al., 2002
). At the end of germband retraction, the DME cells and
the more ventrally located epidermal cells exhibit an isotropic shape. At the
onset of DC, the microtubules of the DME cells bundle in the dorsoventral (DV)
direction, which defines an axis along which these cells begin to elongate
(Fig. 1A').
Simultaneously, Flamingo (Fmi), an atypical cadherin
(Chae et al., 1999
;
Usui et al., 1999
), localises
to the membrane of the DME cells, except at their leading edge (LE,
Fig. 1A). A similar
localisation pattern is also adopted by other membrane associated proteins
such as Discs-large (Dlg) and Fasciclin 3 (Fas3; also known as Fas III). As
the process of polarisation progresses, actin begins to accumulate at the LE
and concentrates in actin nucleating centres (ANCs)
(Kaltschmidt et al., 2002
),
while Myosin accumulates at the LE forming a `beads on a string' pattern
(Young et al., 1993
) (V.M.,
unpublished; Fig. 1A'').
By the end of that first phase, the DME cells are thus elongated in the DV
direction and characterised by a polarised localisation of both cell membrane
associated proteins and cytoskeletal components. At present, the function of
this polarisation is unclear. It might contribute to the correct actin
dynamics because in wingless and dishevelled mutants, in
which the polarisation is affected, actin dynamics are defective.
|
PCP manifests itself in the asymmetric organisation in the plane of the epithelium of membrane associated proteins, including Fmi, and cytoskeletal components, such as microtubules. These act as a scaffold for the directional bundling of actin at the distal vertex of each cell and the formation of the hairs. At first sight, the polarisation of the DME cells during dorsal closure can be seen as a similar process with polarised actin dynamics instead of actin bundling. A similarity between the polarisation of the wing hairs and the DME cells of the embryo is also suggested by the requirement for dishevelled in both processes.
In Drosophila both PCP and `canonical' Wingless signalling require Dishevelled, but there is no solid evidence for an involvement of Wingless in other than the `canonical' pathway. The observation that during DC the polarity of the DME cells is abnormal in wingless and dishevelled mutants has raised the possibility that Wingless is involved in establishing PCP in these cells. Here, we test the possibility of a role for the PCP pathway and Wingless in the polarisation of the DME cells. Our results reveal that Armadillo-mediated Wingless signalling is necessary for the correct polarisation of the DME cells during embryogenesis, while the PCP pathways seems dispensable.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunostaining
Embryos were fixed and stained as described previously
(Kaltschmidt et al., 2002). We
used the following primary antibodies (and concentration): rabbit antisera
against Discs-large (1/200; a gift from P. J. Bryant), mouse monoclonal
against Flamingo (1/10; from T. Uemura)
(Usui et al., 1999
), mouse
antibodies against Fas3 [1/50; Developmental Studies Hybridoma Bank (DSHB),
University of Iowa; developed by C. Goodman], mouse antibodies against
ßTubulin (1/100; DSHB; developed by M. Klymkowsky), rabbit antibodies
against Myosin (1/500; gift from R. Karess), mouse antibodies against Wingless
(1/250; DHSB; developed by S. M. Cohen), rabbit antibodies against
ß-galactosidase (1/10,000; from Cappel), mouse antibodies against
Engrailed (1/100; DSHB; developed by C. Goodman). The following conjugated
secondary antibodies were used at 1/200: Cy5-conjugated antibodies from
Jackson ImmunoResearch; Alexa488- and Alexa568-conjugated antibodies from
Molecular Probes and biotin-conjugated antibodies from Vector Laboratories.
With biotin-conjugated secondary antibodies we used the Elite ABC kit (Vector)
before staining with diaminobenzidene.
Fluorescently labelled embryos were mounted in Vectashield (Vector) and examined under confocal microscope (BioRad). Diaminobenzidene stained embryos were mounted in DPX (Fischer) and examined using an Axioplan2 microscope (Zeiss) and a KY-F55B camera (JVC).
RNA in situ hybridisation
We fixed embryos using standard protocols in 4% formaldehyde. In situ
hybridisation was carried out using a digoxygenin-labelled DNA dpp
probe using standard methods (Lecourtois
and Schweisguth, 1995). After staining, the embryos were washed
several times in PBTw (PBS, 0.1% Tween 20), dehydrated in 70% ethanol,
rehydrated in PBT-BSA (PBS, 0.1% Triton, 1% BSA) and blocked for 1 hour at
room temperature. They were incubated overnight at 4°C with rabbit
anti-ß-galactosidase antibody and anti-rabbit Alexa-488-conjugated
secondary antibody was added as described above. Embryos were hand sorted
under a MZFLIII GFP-scope (Leica) and mounted in Spurr embedding medium
(Fullam).
Cuticle preparation
Two-hundred and fifty embryos were collected from an overday egg collection
and aged 48 hours at 25°C. The unfertilised embryos were removed and
deducted from the count and the remaining embryos were mounted in acetic
acid/Hoyers 50/50. The slide was cooked overnight and the different phenotypes
counted.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The `canonical' Wingless pathway is required for dorsal closure
The involvement of both wingless and dishevelled in the
polarisation of the DME cells and the similarity of this process in the planar
polarisation of epithelial cells (Eaton,
2003) led us to enquire which of the two Wingless signalling
pathways, the `canonical' or the PCP pathway, is involved in this process. We
tested this by attempting to rescue wg mutant embryos with effector
elements of Wg signalling pathways. In these experiments we made use of the
Gal4 targeted expression system (Brand and
Perrimon, 1993
) to express different signalling molecules in the
wg mutant background.
We first examined the ability of different levels of Wingless signalling to rescue wg embryos. To do this, we used an ubiquitous Gal4 driver, daughterless-Gal4 (daGal4) to express either Wingless (wg>da>Wg) or an activated form of Armadillo (Armact, wg>da>Armact). We assumed that if the `canonical' Wingless pathway is involved in DC, overexpression of both Wg and Armact should lead to some rescue of DC. However, if the `canonical' pathway is not involved but the function of Wg during DC involves the PCP pathway, one should observe some rescue only with the overexpression of Wg. As an internal control of the experiment, we monitored the rescue of two features of the `canonical' pathway. One with a low requirement for Wingless signalling, the presence of naked cuticle on the ventral side of the embryo, and one with a higher requirement for Wingless, the expression of Engrailed (En) in stripes in the ectoderm. Both overexpression of Wg and Armact allowed rescue of those two features (Fig. 2), validating our experimental conditions.
|
Therefore both Wg and Armact are able to rescue the DC defects of a wg embryo to a similar extent. These experiments indicate that the `canonical' Wingless pathway is required for the correct behaviour of the DME cells during DC.
Spatial requirement for the Wingless `canonical' signalling pathway
During germband elongation and until the beginning of retraction,
wg is expressed in the epidermis of the embryo in a two-cell-wide
stripe in each segment. During stage 11, expression in the lateral epidermis
is lost and a new pattern emerges with a ventral stripe and a dorsal patch,
just below the DME cells, per segment. There is never wg expression
in the amnioserosa cells. Wingless protein expression pattern reflects the
dynamics of the RNA expression (Gonzalez
et al., 1991) (L. E. Owen, PhD Thesis, University of Cambridge,
1994).
The experiments described above support the suggestion that a localised source of Wingless may not be required for DC. However, there might be a differential requirement for Wg signalling between the amnioserosa and the epidermis for DC to proceed. We previously showed that when activated in both tissues Wg signalling rescues DC and epidermis defects. We have now tested the requirement for Wg signalling in the amnioserosa by overexpressing Wingless or Armact in the amnioserosa of wg- embryos and assessing its effects on DC and the epidermis.
When Wingless is expressed using the amnioserosa specific driver,
332.3-Gal4 (ASGal4) (Wodarz et al.,
1995), Wingless can be seen to be expressed and secreted by the
amnioserosa cells from the stage 11 onwards. In these embryos, Wingless can be
detected in the cytoplasm of the amnioserosa cells but not in the epidermis.
At stage 12 [mid-way through retraction of the germband, stages as in
Hartenstein (Hartenstein,
1993
)], some Wingless can be observed in the head epidermis up to
5-6 cells away from the amnioserosa. At the onset of DC, late stage 12 and
stage 13, some Wingless can be detected in the form of dots over the DME cells
and the lateral epidermis up to five cells away from the amnioserosa
(Fig. 3D). This pattern
persists until the end of closure. Although at stages late 12 and 13 the dots
are on the apical side of the epidermis, later on they are located on the
basal side of the epidermis (data not shown).
|
Although we observe Wingless protein over the epidermis, the rescue of the wg phenotype is restricted to the DME cells. This raises the possibility that the rescue is due to an interaction between the epidermis and the amnioserosa rather than to a direct effect of Wingless on the epidermal cells. To circumvent this, we overexpressed Armact in the amnioserosa (wg>AS> Armact). As Arm is an intracellular protein, any observed effect is likely to be cell autonomous and thus associated with activation of the Wingless pathway within the amnioserosa. Under these conditions, neither the DME cell shape, nor the localisation of Fmi, the organisation of the microtubule bundles nor the localisation of the Myosin were significantly rescued when compared with similar staining in wg embryos (compare Fig. 1B, Fig. 3E and Fig. 1B'', Fig. 3F). Activation of the `canonical' Wingless pathway within the amnioserosa cells is thus not sufficient to rescue the cellular aspects of DC.
These results show that the polarisation and cytoskeletal reorganisations of the DME cells require the activity of the `canonical' Wingless signalling pathway within the epidermis. In addition, they indicate that different cellular events respond to different thresholds of Wingless signalling and that activity, and elongation, of the DME cells does not trigger elongation in more ventrally located cells.
Ubiquitous activation of the Dpp pathway does not rescue the polarity defects of wingless mutant embryos
The activity of the DME cells during dorsal closure requires the activation
of the JNK pathway and the expression of two target genes, puckered
(Glise et al., 1995;
Riesgo-Escovar et al., 1996
)
and decapentaplegic (dpp)
(Glise and Noselli, 1997
;
Harden, 2002
;
Hou et al., 1997
;
Riesgo-Escovar and Hafen,
1997
; Zeitlinger et al.,
1997
). McEwen et al. (McEwen
et al., 2000
) reported that wg is required for
dpp expression in the DME cells. As the `canonical' Wingless pathway
leads to the activation of gene expression, we investigated its contribution
to dpp expression. Reciprocally, we investigated if all the effects
of the `canonical' Wingless pathway are mediated by Dpp.
In wild-type embryos, dpp shows a complex and dynamic pattern of expression. During retraction of the germband, dpp expression is restricted to a single row of cells corresponding to the DME cells. As DC begins, the level of dpp expression in the DME cells decreases (Fig. 4A) and during the zippering phase dpp transcripts cannot be detected anymore. In wg embryos, dpp is expressed in the DME cells at the onset of DC but its levels, as detected by in situ hybridisation, are decreased compare with wild type (Fig. 4B). Nevertheless, the dynamic of dpp expression seems to be conserved, and, as in wild-type embryos, dpp expression disappears during the zippering process. In wg mutant embryos expressing high levels of either Wg or Armact with daGal4, the levels of dpp are restored to wild type levels (Fig. 4C,D). Interestingly, regardless of the ubiquitous overexpression of either Wg or Armact throughout the epidermis, the pattern of dpp expression during DC is preserved, suggesting that there is only a subpopulation of cells that are competent to express dpp. However, amnioserosa-specific expression of Wingless, but not of Armact, in wg mutants results in a rescue of dpp expression in the DME cells (Fig. 4E,F). This confirms that the effects that we observe are due to Wingless signalling directly to the DME cells and not to secondary signalling across cell types.
|
Altogether, these results challenge the proposal that in wild-type embryos the elongation of the ventral epidermal cells is induced by Dpp secreted from the DME cells and suggest that if Dpp contributes to the elongation of the epidermal cells, it requires an additional input from Wingless signalling.
Overexpression of Dishevelled in wg mutant embryos is not sufficient to rescue polarity defects
To analyse the possibility of a contribution of the PCP pathway in DC, we
tested the ability of Dsh to rescue the wg mutant phenotype. In these
experiments, we overexpressed Dsh with the ubiquitous driver daGal4
(wg>da>Dsh). We first assessed the ability of Dsh to rescue the
naked cuticle and the expression of En in wg
embryos. Although overexpression of either Wg or Armact restored
naked cuticle and exhibited a dominant effect, the overexpression of Dsh
exhibits a partial rescue and only restores some naked cuticle (compare
Fig. 5B',C',
Fig. 2). A similar effect is
observed with regard to the expression of En, which is fully rescued by
ubiquitous expression of Wg or Armact, but only weakly rescued by
the ubiquitous overexpression of Dsh (compare
Fig. 5B'',C'',
Fig. 2). Thus, overexpression
of Dsh in a wg embryo partly rescues the
`canonical' pathway, as assessed by the naked cuticle and En expression.
|
|
|
DIX and the DEP domains of Dishevelled have different contributions to DC
Dsh contains three highly conserved domains, the DIX, PDZ and DEP domains
(for a review, see Wharton,
2003). The DEP domain mediates interaction of Dsh with the cell
cortex and is required for PCP but not `canonical' Wg signalling
(Axelrod, 2001
;
Axelrod et al., 1998
;
Rothbacher et al., 2000
),
while the DIX domain is required for the `canonical' Wg signalling but seems
dispensable for PCP (Axelrod,
2001
; Penton et al.,
2002
). To investigate further an involvement of the PCP pathway in
the activities of the DME cells during DC, we repeated the rescue experiments
of wg embryos using truncated forms of Dsh deleted
for either the DEP (Dsh
DEP) or the DIX (Dsh
DIX) domain. Although
overexpression of Dsh
DEP leads to the partial rescue of naked cuticle
(compare Fig.
5B',D') and of En expression (compare
Fig. 5B'',D''), no
naked cuticle nor rescue of En expression are observed in
wg>da>Dsh
DIX embryos
(Fig. 5E',E''). We
thus confirm that Dsh
DEP is able to signal within the `canonical' Wg
pathway but not Dsh
DIX.
We then tested the ability of either protein to rescue DC in
wg embryos. wg>da>DshDEP
embryos are longer than wg mutants and their dorsal cuticle is
improved as no hole is observed and only occasional warts can be seen
(Fig. 6C'). The DME cells
are oriented in the DV direction and most of them show a slight elongation in
the DV direction when the zippering process has started. Simultaneously, Fmi
is observed at the membrane and accumulates at the level of the ANCs
(Fig. 6C''). Although no
clear elongation of DME or ventral epidermal cells is observed, DC process is
improved as two zippers, at the anterior and posterior ends of the embryo, are
initiated, whereas only the posterior one is observed in
wg embryos (not shown). By contrast,
wg>da>Dsh
DIX embryos have a shorter cuticle than
wg mutants and show a more severe puckering and hole on the dorsal
side (compare Fig. 6A,D with
Fig. 6A',D').
Furthermore, neither the shape nor the polarisation of DME cells is improved
in these embryos (Fig.
6D'').
Thus, although DshDEP can rescue partially the DC defects of
wg mutants, ubiquitous overexpression of Dsh
DIX does not
rescue any of the observed features confirming the requirement for the Wg
`canonical' pathway during DC.
The activity of Dsh is not required for dorsal closure when the Wg `canonical' pathway is constitutively activated
Although overexpression of DshDEP in wg mutant embryos
leads to a rescue comparable with the one observed with overexpression of Dsh,
one cannot exclude the possibility that the PCP pathway plays a role in this
process. Dsh function is not affected in wg embryos
and the endogenous Dsh protein is potent to mediate PCP. Thus, it is possible
that in many of our experiments some of the observed rescue is due to an
activity of the endogenous Dsh induced by interactions of the overexpressed
forms with other regulatory proteins. To rule out this possibility, we
generated dsh mutant embryos in which the `canonical' Wg signalling
pathway is constitutively activated through a loss of function of sgg
(sgg,dshGLC).
In agreement with a role for the `canonical' Wg pathway in DC, the cuticle of the sgg,dshGLC embryos is improved compared with that of dsh embryos. The embryos exhibit a severe defect in germband retraction but no dorsal hole is observed and the dorsal cuticle appears severely puckered (compare Fig. 7A,C with Fig. 7A',C'). Furthermore, although in dsh mutants the DME cells do not elongate in the DV direction and show a `dotty' cytoplasmic localisation of Fmi (Fig. 7A''), in embryos derived from sgg,dshGLC females DME cells are elongated in the DV direction and Fmi localises to the cell membrane as it does in the wild type (Fig. 7C''). These observations show that the function of Dsh is dispensable for the organisation of the DME cells when the `canonical' Wg pathway is activated. They also indicate that the rescue of wg mutants by Armadillo is due to an activation of the `canonical' Wg signalling pathway without a major contribution of the PCP pathway.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcriptional control of the polarisation of the DME cells and DC by the ß-catenin-dependent Wg pathway
The polarisation of the DME cells occurs in the plane of the epithelium and
can be seen as a manifestation of the phenomenon of planar cell polarity
(PCP). As a specific branch of Wg signalling has been implicated in PCP and
there is evidence for an interaction between Dishevelled and JNK signalling
during dorsal closure (Boutros et al.,
1998), we tested whether there is a role for this mode of Wg
signalling in the process of DC. Our results clearly show that the `canonical'
Wg signalling pathway that leads to activation of Armadillo and of the
transcription of target genes is necessary and sufficient to restore the
polarity of the DME cells and to promote a normal process of dorsal closure in
a wg mutant embryo. Surprisingly, we find that the PCP pathway does
not appear to play a major role in DC or the polarisation of the DME cells as
activation of the `canonical' pathway in the absence of dsh activity
rescues the polarity and function of the DME cells. This conclusion is
supported by the observation that although a moiety of Dishevelled that
promotes Armadillo signalling is capable of rescuing the defects of
wg mutants, a moiety that promotes JNK signalling and PCP does not.
Altogether, these results indicate that the polarisation and activity of the
DME cells during dorsal closure requires Armadillo/ß-catenin-dependent Wg
signalling. Furthermore, this requirement is restricted to the epidermis as
activation of Wg signalling in the amnioserosa has no effect on the
epidermis.
The polarisation of the DME cells and subsequent dynamics of actin at the
LE can be construed as the development of the leading edge of a motile cell
and to a certain extent is akin to an epidermal/mesenchymal transition (EMT),
as one of the features of this process is the reorganisation of the actin
cytoskeleton and the acquisition of motility by the cells. In this regard, it
is interesting to note that ß-catenin-dependent Wnt signalling has been
implicated in EMT both in normal and cancerous cells
(Martinez Arias, 2001;
Muller et al., 2002
) and that
therefore there are precedents for the involvement of the
ß-catenin-mediated transcriptional regulation in the development of actin
dynamics. However, the targets of the Wnt pathway mediating this process are
not known.
Dpp is not the central target of the ß-catenin-dependent Wingless signalling
It has been suggested that the Drosophila BMP homologue Dpp is a
central effector of dorsal closure
(Affolter et al., 1994;
Glise and Noselli, 1997
;
Hou et al., 1997
;
Zeitlinger et al., 1997
).
Embryos mutant for dpp signalling exhibit defects in dorsal closure.
dpp is expressed in the DME cells and has been proposed to act as a
long range signal for the elongation of the more ventral cells
(Glise and Noselli, 1997
;
Hou et al., 1997
;
Riesgo-Escovar and Hafen,
1997
). Here, we show that Wingless is required for the correct
maintenance of dpp expression in the DME cells, although in our
experiments the input is less significant than has been reported before
(McEwen et al., 2000
).
Altogether, these observations suggest that some of the activity of Wingless
during DC is mediated by Dpp. Indeed, when we ubiquitously activated the Dpp
pathway by the means of an activated form of its receptor Tkv, we observe some
rescue of the polarity of the DME cells. However, although in this case the
DME cells orient themselves in the DV direction and Fmi localises as it does
in wild type, neither the DME nor the ventral epidermal cells elongate, and
the DC process is not substantially improved. This contrasts with the full
rescue of both the polarisation of DME cells and the DC process following
ubiquitous activation of the ß-catenin-dependent Wg pathway. Thus, if Dpp
contributes to DC, it is not as the only target of Wg signalling.
Expression of Wingless from the amnioserosa in wg mutants induces
high and continuous levels of dpp in the DME cells together with some
rescue of the polarity of the DME cells but without any effect on the
elongation of these or the more ventral cells. This rescue is very similar to
the one observed with ubiquitous expression of the activated Tkv. These
results indicate that Dpp does not act as a long-range signal for the
elongation of the more ventral epidermal cells as rescue of Dpp expression in
the DME cells or activation of Dpp signalling throughout the epidermis in
wg mutants does not lead to the elongation of the more ventral cells.
A similar conclusion had been suggested from the observation that epidermal
cells initially elongate in the absence of Dpp signalling but resume their
polygonal shape soon after (Ricos et al.,
1999; Zeitlinger et al.,
1997
). However, an alternative explanation for our observations is
that the elongation of the ventral epidermal cells requires inputs from both
Dpp and Wingless signalling.
Altogether, these observations indicate that Dpp is not the only effector of Wingless during DC and indicates that Wingless signalling via Armadillo controls genes that act either in parallel or together with those regulated by JNK and Dpp.
ß-catenin dependent Wg signalling plays a permissive role for the polarisation of the DME cells and dorsal closure
We have shown that Wingless is required in the epidermal cells but does not
act as a polarising signal, as ubiquitous activation of the pathway rescues
the defects of wg mutants. An important observation of our
experiments is that the DME cells of wg mutant embryos display a
polarity and an elongation at the very final stages of DC, suggesting that the
polarisation signal is received correctly by the DME cells but that in the
absence of Wingless signalling there is a delay either in its interpretation
or in its materialisation. This, together with the lack of importance of a
fixed source of Wingless for the polarisation of the DME cells
(Kaltschmidt et al., 2002),
suggests that Wingless makes the DME cells competent to interpret a
pre-existing polarisation signal. Such a permissive role of Wingless
signalling had been suggested by McEwen et al.
(McEwen et al., 2000
). It has,
furthermore, been emphasized in other transcriptional events
(Martinez Arias, 2003
).
In the case of DC, the permissive function of Wingless signalling translates itself in the correct coordination of the different events, i.e. the cells have to elongate at the right time and the activity of their cytoskeleton has to be properly linked to other events some of which are transcriptional. Failure to do this will result in defects in dorsal closure. These observations raise the question of the temporal requirements for Wg signalling during DC.
ASGal4 drives expression of Wingless from the elongation of the germband to
the end of DC. However, when driven by ASGal4, Wg can only be detected over
the epidermal cells during the first phase of DC. This is probably due to the
inability of Wingless to cross the deep fold existing between the AS and the
epidermis during germband retraction and the zippering process. The provision
of Wg from the amnioserosa rescues the defects of the DME cells of
wingless mutants but not those of the more ventral cells. Although
the DME cells, in contact with the AS, might have received Wg signal at the
very onset of the overexpression (around stage 9-10), the more ventral
epidermal cells seem to see the signal too late to elongate, suggesting that
Wg signalling is required before the beginning of DC for the cell shape and
polarity changes. A hint at the timing of Wnt requirement for DC is provided
by experiments using a temperature sensitive allele of wingless
(Bejsovec and Martinez Arias,
1991). Removal of wingless function between 4 and 4.5
hours after egg laying, i.e. at stages 9-10, affects the shape of the dorsal
cuticle in a way similar to DC defects. This suggests that the polarising
signal must occur very early, during germband elongation.
Establishment versus propagation. What is the function of the PCP pathway?
The notion of PCP has emerged from studies of the mechanism that determines
the orientation of the hairs in the cells of the wing of Drosophila
(Eaton, 2003). A number of
studies have revealed the existence of protein complexes that mediate this
orientation by becoming asymmetrically distributed between the proximal and
distal membranes of the epidermal cells. Thus, while Flamingo becomes
localised equally between the proximal and the distal sides of the cell
(Usui et al., 1999
), the
distal side of the cell accumulates a complex composed of Frizzled and
Dishevelled (Axelrod, 2001
;
Shimada et al., 2001
;
Strutt, 2001
) and the proximal
side accumulates a complex formed by Strabismus and Prickled
(Bastock et al., 2003
;
Tree et al., 2002
). Genetic
analysis of these complexes has led to the formulation of a model which
describes the propagation of the polarity from one cell to its neighbours
(reviewed by Eaton, 2003
), but
which says nothing about the origin of the polarity that is being propagated.
In this model, Dsh, like Strabismus, Prickled or Frizzled, is an essential
component of the mechanism that propagates the polarity.
The observation of polarised distributions of Fmi, Dsh and Fz in the DME
cells during dorsal closure has led us to suggest a link between the
polarisation of these cells and the process of PCP. However, we have not found
a requirement for elements of this pathway in dorsal closure. In particular,
the PCP function of Dsh is not required for the polarisation of the DME cells
and the polarised localisation of Fmi, which was quite unexpected considering
the interdependence of Dsh and Fmi for their asymmetric localisation in the
wing cells (Shimada et al.,
2001). This asymmetric distribution of Fmi is likely to play a
role in the polarised actin dynamics in response to the polarity signal.
Although this may appear surprising, it also invites a consideration of the
notion of PCP.
The PCP pathway has been defined in a context of propagation of a polarity but not of its initial definition. In fact none of the experiments performed in the wing of Drosophila address the origin of the polarity that is being propagated. In DC, however, the process that we observe in the asymmetric distribution of proteins in the DME cells reflects the establishment of a polarity and not its propagation. From this perspective, the lack of a requirement for the PCP branch of Wnt signalling might not be that surprising as PCP Wnt signalling might be related to propagation or coordination of a polarity signal that has been generated in a different manner. However, the requirement for the ß-catenin-dependent Wg pathway might be significant and indicate the requirement for a transcriptional event in the establishment of PCP. This observation might also apply to the wing.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Affolter, M., Nellen, D., Nussbaumer, U. and Basler, K.
(1994). Multiple requirements for the receptor serine/threonine
kinase thick veins reveal novel functions of TGF beta homologs during
Drosophila embryogenesis. Development
120,3105
-3117.
Axelrod, J. D. (2001). Unipolar membrane
association of Dishevelled mediates Frizzled planar cell polarity signaling.
Genes Dev. 15,1182
-1187.
Axelrod, J. D., Miller, J. R., Shulman, J. M., Moon, R. T. and
Perrimon, N. (1998). Differential recruitment of
Dishevelled provides signaling specificity in the planar cell polarity and
Wingless signaling pathways. Genes Dev.
12,2610
-2622.
Bastock, R., Strutt, H. and Strutt, D. (2003).
Strabismus is asymmetrically localised and binds to Prickle and Dishevelled
during Drosophila planar polarity patterning.
Development 130,3007
-3014.
Bejsovec, A. and Martinez Arias, A. (1991). Roles of wingless in patterning the larval epidermis of Drosophila. Development 113,471 -485.[Abstract]
Boutros, M., Paricio, N., Strutt, D. I. and Mlodzik, M. (1998). Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94,109 -118.[Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Chae, J., Kim, M. J., Goo, J. H., Collier, S., Gubb, D.,
Charlton, J., Adler, P. N. and Park, W. J. (1999). The
Drosophila tissue polarity gene starry night encodes a member of the
protocadherin family. Development
126,5421
-5429.
Chou, T. B. and Perrimon, N. (1992). Use of a
yeast site-specific recombinase to produce female germline chimeras in
Drosophila. Genetics
131,643
-653.
Eaton, S. (1997). Planar polarization of Drosophila and vertebrate epithelia. Curr. Opin. Cell Biol. 9,860 -866.[CrossRef][Medline]
Eaton, S. (2003). Cell biology of planar polarity transmission in the Drosophila wing. Mech. Dev. 120,1257 -1264.[CrossRef][Medline]
Foe, V. E. (1989). Mitotic domains reveal early commitment of cells in Drosophila embryos. Development 107, 1-22.[Abstract]
Glise, B. and Noselli, S. (1997). Coupling of Jun amino-terminal kinase and Decapentaplegic signaling pathways in Drosophila morphogenesis. Genes Dev. 11,1738 -1747.[Abstract]
Glise, B., Bourbon, H. and Noselli, S. (1995). hemipterous encodes a novel Drosophila MAP kinase kinase, required for epithelial cell sheet movement. Cell 83,451 -461.[Medline]
Gonzalez, F., Swales, L., Bejsovec, A., Skaer, H. and Martinez Arias, A. (1991). Secretion and movement of wingless protein in the epidermis of the Drosophila embryo. Mech. Dev. 35, 43-54.[CrossRef][Medline]
Harden, N. (2002). Signaling pathways directing the movement and fusion of epithelial sheets: lessons from dorsal closure in Drosophila. Differentiation 70,181 -203.[CrossRef][Medline]
Harden, N., Ricos, M., Yee, K., Sanny, J., Langmann, C., Yu, H.,
Chia, W. and Lim, L. (2002). Drac1 and Crumbs participate in
amnioserosa morphogenesis during dorsal closure in Drosophila. J.
Cell Sci. 115,2119
-2129.
Hartenstein, V. (1993). Atlas of Drosophila Development. New York: Cold Spring Harbor Laboratory Press.
Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C. and Wilson, S. W. (2000). Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76-81.[CrossRef][Medline]
Hou, X. S., Goldstein, E. S. and Perrimon, N. (1997). Drosophila Jun relays the Jun amino-terminal kinase signal transduction pathway to the Decapentaplegic signal transduction pathway in regulating epithelial cell sheet movement. Genes Dev. 11,1728 -1737.[Abstract]
Hutson, M. S., Tokutake, Y., Chang, M. S., Bloor, J. W.,
Venakides, S., Kiehart, D. P. and Edwards, G. S.
(2003). Forces for morphogenesis investigated with laser
microsurgery and quantitative modeling. Science
300,145
-149.
Jacinto, A., Wood, W., Balayo, T., Turmaine, M., Martinez-Arias, A. and Martin, P. (2000). Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure. Curr Biol. 10,1420 -1426.[CrossRef][Medline]
Jacinto, A., Martinez-Arias, A. and Martin, P. (2001). Mechanisms of epithelial fusion and repair. Nat. Cell Biol. 3,E117 -E123.[CrossRef][Medline]
Kaltschmidt, J. A., Lawrence, N., Morel, V., Balayo, T., Fernandez, B. G., Pelissier, A., Jacinto, A. and Martinez Arias, A. (2002). Planar polarity and actin dynamics in the epidermis of Drosophila. Nat. Cell Biol. 25, 25.[CrossRef]
Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L.
and Montague, R. A. (2000). Multiple forces contribute
to cell sheet morphogenesis for dorsal closure in Drosophila. J.
Cell Biol. 149,471
-490.
Lawrence, N. and Morel, V. (2003). Dorsal closure and convergent extension: two polarised morphogenetic movements controlled by similar mechanisms? Mech. Dev. 120,1385 -1393.[CrossRef][Medline]
Lecourtois, M. and Schweisguth, F. (1995). The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev. 9,2598 -2608.[Abstract]
Martinez Arias, A. (2001). Epithelial mesenchymal interactions in cancer and development. Cell 105,425 -431.[CrossRef][Medline]
Martinez Arias, A. (2003). Wnts as morphogens? The view from the wing of Drosophila. Nat. Rev. Mol. Cell Biol. 4,321 -325.[CrossRef][Medline]
McEwen, D. G., Cox, R. T. and Peifer, M.
(2000). The canonical Wg and JNK signaling cascades collaborate
to promote both dorsal closure and ventral patterning.
Development 127,3607
-3617.
Mlodzik, M. (2002). Planar cell polarization: do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation? Trends Genet. 18,564 -571.[CrossRef][Medline]
Muller, T., Bain, G., Wang, X. and Papkoff, J. (2002). Regulation of epithelial cell migration and tumor formation by beta-catenin signaling. Exp. Cell Res. 280,119 -133.[CrossRef][Medline]
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85,357 -368.[Medline]
Penton, A., Wodarz, A. and Nusse, R. (2002). A
mutational analysis of dishevelled in Drosophila defines novel domains in the
dishevelled protein as well as novel suppressing alleles of axin.
Genetics 161,747
-762.
Ricos, M. G., Harden, N., Sem, K. P., Lim, L. and Chia, W.
(1999). Dcdc42 acts in TGF-beta signaling during Drosophila
morphogenesis: distinct roles for the Drac1/JNK and Dcdc42/TGF-beta cascades
in cytoskeletal regulation. J. Cell Sci.
112,1225
-1235.
Riesgo-Escovar, J. R. and Hafen, E. (1997). Drosophila Jun kinase regulates expression of decapentaplegic via the ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal closure. Genes Dev. 11,1717 -1727.[Abstract]
Riesgo-Escovar, J. R., Jenni, M., Fritz, A. and Hafen, E. (1996). The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye. Genes Dev. 10,2759 -2768.[Abstract]
Rothbacher, U., Laurent, M. N., Deardorff, M. A., Klein, P. S.,
Cho, K. W. and Fraser, S. E. (2000). Dishevelled
phosphorylation, subcellular localization and multimerization regulate its
role in early embryogenesis. EMBO J.
19,1010
-1022.
Shimada, Y., Usui, T., Yanagawa, S., Takeichi, M. and Uemura, T. (2001). Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarization. Curr. Biol. 11,859 -863.[CrossRef][Medline]
Strutt, D. I. (2001). Asymmetric localization of frizzled and the establishment of cell polarity in the Drosophila wing. Mol. Cell 7,367 -375.[Medline]
Tada, M. and Smith, J. C. (2000). Xwnt11 is a
target of Xenopus Brachyury: regulation of gastrulation movements via
Dishevelled, but not through the canonical Wnt pathway.
Development 127,2227
-2238.
Tree, D. R., Shulman, J. M., Rousset, R., Scott, M. P., Gubb, D. and Axelrod, J. D. (2002). Prickle mediates feedback amplification to generate asymmetric planar cell polarity signaling. Cell 109,371 -381.[Medline]
Usui, T., Shima, Y., Shimada, Y., Hirano, S., Burgess, R. W., Schwarz, T. L., Takeichi, M. and Uemura, T. (1999). Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell 98,585 -595.[Medline]
Veeman, M. T., Axelrod, J. D. and Moon, R. T. (2003). A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev. Cell 5, 367-377.[Medline]
Wharton, K. A., Jr (2003). Runnin' with the Dvl: proteins that associate with Dsh/Dvl and their significance to Wnt signal transduction. Dev. Biol. 253, 1-17.[CrossRef][Medline]
Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995). Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. Cell 82,67 -76.[Medline]
Young, P. E., Richman, A. M., Ketchum, A. S. and Kiehart, D. P. (1993). Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev. 7, 29-41.[Abstract]
Zeitlinger, J., Kockel, L., Peverali, F. A., Jackson, D. B.,
Mlodzik, M. and Bohmann, D. (1997). Defective dorsal
closure and loss of epidermal decapentaplegic expression in Drosophila fos
mutants. EMBO J. 16,7393
-7401.
Related articles in Development: