MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Author for correspondence (e-mail:
MF1{at}mrc-lmb.cam.ac.uk)
Accepted 30 December 2003
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SUMMARY |
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Key words: EGF receptor, Denticle belt fusion, spitz group, Rhomboid, Epidermis, Intramembrane proteolysis, Apoptosis, Cuticle, Pointed
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Introduction |
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The Drosophila embryonic ventral epidermis has served as a
tractable tissue for the genetic analysis of patterning, and remains one of
the rare instances where the developmental programs used to pattern fields of
cells have been studied from early through to late stages
(Alexandre et al., 1999;
DiNardo et al., 1994
;
Hatini and DiNardo, 2001
;
O'Keefe et al., 1997
;
Payre et al., 1999
;
Szüts et al., 1997
).
Here, two cell types are specified: epidermal cells that secrete short, thick
hair-like structures called denticles (used by the larvae for traction), and
smooth-cuticle cells, which secrete a protective cuticle that lacks denticles.
Denticles occur in belts composed of several rows of denticle-secreting cells
in the anterior half of each parasegment, which alternate with smooth cuticle
regions that make up the posterior of each parasegment. This segmental pattern
is repeated along the anterior-posterior axis (see
Fig. 1A). Although deceptively
simple, this pattern is quite intricate and precise; each of the six rows of
denticle-secreting cells in the abdominal parasegments produce denticles of
distinct polarity and morphology, while many denticle belts have
segment-specific characteristics
(Szüts et al., 1997
;
Wiellette and McGinnis,
1999
).
|
The `spitz group' genes including rhomboid-1, Star and
spitz that initiate EGFR signalling were originally identified and
grouped according to their defects in cuticle patterning
(Mayer and Nusslein-Volhard,
1988; Nusslein-Volhard et al.,
1984
). Mechanistic analyses have established that Spitz is the
primary ligand of EGFR signalling during embryogenesis, but is produced in all
cells in an inert transmembrane form
(Rutledge et al., 1992
).
Signalling is activated when and where it is needed by the membrane proteins
Star and Rhomboid-1 (reviewed by Shilo,
2003
). Star is an export factor required for Spitz exit from the
ER (Lee et al., 2001
;
Tsruya et al., 2002
), while
Rhomboid-1 is the protease responsible for Spitz activation
(Urban and Freeman, 2003
;
Urban et al., 2001
).
Rhomboid-1 is expressed in three rows of cells in the future denticle regions
(Alexandre et al., 1999
;
Sanson et al., 1999
), which
constitute the site of Spitz processing during ventral epidermal patterning
and induce the denticle cell fate in these and neighbouring cells.
The cuticle phenotype of several spitz group genes indicates that
the role of EGFR signalling in epidermal patterning is more complex. In
addition to the predictable defects in denticle specification, mutation of
many spitz group genes also results in denticle belt fusions
(Mayer and Nusslein-Volhard,
1988; Nusslein-Volhard et al.,
1984
). This is perhaps the most striking and distinguishing
cuticle phenotype of the spitz group genes, and results in the
variable fusion of adjacent denticle belts in their central regions at the
expense of a region that is normally smooth cuticle
(Mayer and Nusslein-Volhard,
1988
) (see Fig.
1A). Since EGFR signalling is believed to be involved only in
specifying denticle fate and to have no role in smooth-cuticle cells
(O'Keefe et al., 1997
;
Payre et al., 1999
;
Szüts et al., 1997
), it
is unclear why this EGFR signalling defect causes a phenotype in
smooth-cuticle regions.
We have investigated the additional roles of EGFR signalling during epidermal development by studying the denticle fusion phenotype. Although high levels of EGFR signalling specify the denticle fate, lower levels of signalling are required in smooth-cuticle cells for survival. Reduction of EGFR signalling in spitz group mutant embryos causes smooth-cuticle cells to die, resulting in fusions of adjacent denticle belts. Rhomboid-3/Roughoid, but not Rhomboid-2 or -4, and the soluble ligand Vein cooperate with canonical spitz group genes in stimulating this survival signalling. These analyses now specifically demonstrate an unrecognized survival function for EGFR signalling during epidermal patterning, and illustrate one way in which different rhomboid proteases are deployed to fulfil the requirements of EGFR signalling during development.
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Materials and methods |
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|
|
RNA interference
Achieving a robust phenocopy using RNA interference
(Kennerdell and Carthew, 1998;
Misquitta and Paterson, 1999
)
was dependent on maximising penetrance while reducing nonspecific embryo
lethality. High concentrations of dsRNA were essential to maximise penetrance.
RNA was synthesised in 50 µl in vitro transcription reactions (using
Promega's Ribomax method) with 5 µg of linearized pBluescript containing
rhomboid genes as templates. Transcription was allowed to continue
for 4 hours at 37°C, which resulted in the production of
100 µg of
RNA for each strand. 1U DNaseI per µg template was added and incubated at
37°C for 1 hour, and the RNA was purified using RNeasy (Qiagen). The yield
and integrity of the RNA was examined by formaldehyde denaturing agarose gel
electrophoresis. Equivalent quantities of each strand were mixed, boiled for 5
minutes and allowed to cool to room temperature overnight. The dsRNA was
precipitated with sodium acetate/ethanol, and resuspended in 0.1x PBS at
1-2 µg/µl prior to use.
The variable that had the most significant effect on increasing embryo survival was injecting the embryos through the chorion. This enhanced survival since the embryos are much heartier in this state, and the chorion also keeps them from leaking after injection. Survival also relied on using uncrowded, well fed, young flies as older flies laid fewer eggs with significantly decreased hatching rates (with a concomitant increase in the number of unfertilised eggs). Embryos were washed off plates and aligned along the length of the slide while wet. The chorions of embryos that were dried for 5 minutes in silica gel containers became immobilized onto the slide surface (no glue required). Embryos were covered with Voltalefs 10S oil, which quickly rendered the chorion transparent, allowing embryo staging. Embryos were injected laterally, not posteriorly, as this was found to increase survival by 5-10%.
The injected embryos were incubated on slides at room temperature in a level humidified chamber. Hatching rate was assessed by counting the number of unhatched embryos after 2 days, with both positive (lethal gene) and negative (buffer) controls included in each set of injections. Overall, a typical hatching rate of 80% was achieved with injecting buffer, which was very consistent (approx. ±5%). Unhatched embryos were mounted for cuticle analysis.
Embryo stainings
RNA expression patterns of wg-gal4 and prd-gal4 (driving
rhomboid-2 and -4 as probe targets, respectively) were
visualized using digoxigenin-labelled antisense RNA probes prepared from 1-2
µg linearized DNA templates using Boehringer Mannheim reagents. Probes were
fragmented in 40 mM NaHCO3, 60 mM Na2CO3 pH
10.2 for 135 minutes, and hybridisation and detection were performed according
to standard protocols.
Embryos were stained with anti-Engrailed (4D9) and anti-GFP using standard protocols, and mounted in Vectashield. TUNEL labelling was performed after antibody detection by permeabilizing embryos in 0.5% Triton X-100, 0.1 M sodium citrate for 30 minutes at 70°C, rinsing in PBS + 0.5% Triton X-100, and incubating with TUNEL reaction components (Roche) at 37°C for 2.5 hours. Confocal images were collected using a MRC Radiance 2001 confocal microscope.
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Results |
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The lower frequency of denticle belt fusions in rhomboid-1 null mutants, and the variability and overall low number of fusions per embryo in all spitz group mutants suggested that other EGFR signalling components might also be involved in epidermal patterning. We therefore investigated the role of other possible rhomboids and EGFR ligands in this process.
Rhomboid-1 and -3 cooperate in suppressing denticle-belt fusions
Since Spitz, Star and Rhomboid are all obligate components of the EGFR
signal activation pathway (Lee et al.,
2001; Mayer and
Nusslein-Volhard, 1988
; Tsruya
et al., 2002
; Urban and
Freeman, 2003
), the lower penetrance of denticle belt fusions in
rhomboid-1 mutant embryos suggested that another rhomboid protease
might be acting with Rhomboid-1 in epidermal patterning. Previous biochemical
analysis suggested that at least three other rhomboid proteases could
substitute for Rhomboid-1 (Urban and
Freeman, 2002
; Urban et al.,
2002
). We therefore examined the physiological role of these
proteases in embryogenesis using RNA interference
(Fire et al., 1998
;
Kennerdell and Carthew, 1998
;
Misquitta and Paterson,
1999
).
Microinjection of rhomboid-1 dsRNA resulted in dose-dependent embryonic
lethality with all embryos recapitulating the characteristic
rhomboid-1 epidermal phenotype, including missing row one denticles,
reversal of row four denticle polarity, and denticle belt fusions
(Fig. 2A). Although this effect
was very reproducible, it required high concentrations of dsRNA, possibly
because rhomboid proteases are such efficient enzymes
(Urban et al., 2001;
Urban et al., 2002
). However,
injection of dsRNA corresponding to rhomboid-2, -3 and -4
had no detectable effect on survival rates or phenotypes of the injected
embryos (Fig. 2B). This is
consistent with genetic analysis of rhomboid-2 and -3 since
their null mutants have been recently isolated and do not display embryonic
phenotypes (Schulz et al.,
2002
; Wasserman et al.,
2000
).
|
Although rhomboid-1 and -3 are within 80 kb of each other
on chromosome 3L and as such are too close to be practically recombined to
produce a double mutant, we had previously determined that one
rhomboid-1 mutation (7M43) was originally generated on a chromosome
that contained a rhomboid-3 mutation
(Wasserman et al., 2000).
Analysis of these rhomboid-3 rhomboid-1 double mutant embryos
revealed that the frequency of fusions was more than double compared to
rhomboid-1 alone (Fig.
1A), confirming the RNAi analysis. Although this rhomboid-3
rhomboid-1 double mutation did not fully recapitulate the severity of the
Star and spitz mutants, this rhomboid-3 allele
(roughoid1) causes a reduction rather then a loss of
Rhomboid-3 activity (Wasserman et al.,
2000
). It should be noted that the fusion phenotype of this widely
used rhomboid-1 stock (7M43) has been attributed to Rhomboid-1 alone
as the significance of the roughoid mutation was unknown
(Mayer and Nusslein-Volhard,
1988
).
The only aspect of embryonic development for which we detected cooperation between Rhomboid-3 and Rhomboid-1 was in the formation of denticle belt fusions. In all other contexts examined, including ventral narrowing, which is diagnostic of a defect in ventrolateral specification (Fig. 3A,B), and other aspects of denticle determination (Fig. 3C), the rhomboid-1 mutation alone was fully penetrant.
|
To distinguish between these possibilities, we analysed the effect of
removing Vein, a soluble neuregulin-like protein that is the only EGFR ligand
thought to be independent of Rhomboid-1 and Star
(Schnepp et al., 1996). Since
spitz; vein double mutants are too severely affected for analysis of
denticle patterning (Schnepp et al.,
1996
), we generated a rhomboid-1 vein double mutant
(because rhomboid-1 mutants produced weaker denticle belt fusion
phenotypes). Under these conditions about one third of rhomboid-1
vein mutant embryos could be analysed for denticle phenotypes, the rest
being too severely affected (Fig.
4C).
Intriguingly, although the proportion of embryos displaying at least one
fusion was not significantly increased compared to rhomboid-1 alone,
these embryos displayed an increase in the frequency of multiple fusions per
embryo (Fig. 4D). This
suggested that the lack of complete fusions in spitz group mutant
embryos is due to EGFR stimulation by Vein. Indeed, Vein is known to be
expressed in the ventral epidermis at the time epidermal fates are being
specified (Schnepp et al.,
1996), although it has not previously been described to have a
role in epidermal patterning.
Since the denticle belt fusion phenotype caused by EGFR loss could be accounted for by removing multiple ligands or their activators, this analysis indicates that ligand-independent EGFR activation may not occur physiologically, at least not during epidermal patterning.
A new requirement for EGFR signalling in smooth-cuticle cells
EGFR signalling is known to stimulate cells to adopt the denticle fate,
while Wingless signalling antagonises EGFR signalling, allowing cells to adopt
the smooth cuticle fate (O'Keefe et al.,
1997; Payre et al.,
1999
; Sanson et al.,
1999
; Szüts et al.,
1997
). Contrary to these established roles, the denticle belt
fusion phenotype in spitz group mutants was manifest in smooth
cuticle domains, where Wingless signalling is high and there is no known
function for EGFR signalling. To test whether EGFR signalling is indeed
specifically required in these cells, we blocked EGFR signalling by expressing
a dominant negative form of the receptor
(Freeman, 1996
) in all five
rows of smooth cuticle cells of alternating parasegments using the
prd-gal4 driver (Moline et al.,
1999
; Yoffe et al.,
1995
), or in one or two of the five posterior rows of smooth
cuticle cells in each parasegment using the wg-gal4 driver
(Pfeiffer et al., 2000
). The
expression patterns of these drivers have been characterized previously, and
are shown for reference in Fig.
5A.
Strikingly, removal of EGFR signalling in only smooth cuticle cells using
prd-gal4 resulted in strong denticle belt fusions in essentially all
paired domain denticle belts (Fig.
5B). Although wg-gal4 expresses in only one or two rows
of smooth cuticle cells (Pfeiffer et al.,
2000), expressing dominant-negative EGFR (DN-EGFR) in these cells
also resulted in partial denticle belt fusions. We examined the physiological
significance and specificity of the fusions caused by reducing EGFR signalling
in smooth-cuticle cells by testing genetic interactions between spitz
group mutant embryos and perturbing EGFR signalling using these transgenes.
The fusions in rhomboid-3 rhomboid-1 double mutant embryos were
completely rescued in paired domains by the expression of activated
forms of EGFR (TorD-EGFR) or Ras (RasV12)
(Fig. 5C). Conversely, reducing
EGFR signalling by expressing a weak line of DN-EGFR or dominant negative
forms of Ras (RasN17) or Raf (DN-Raf) all enhanced the fusions of
rhomboid-3 rhomboid-1 double mutant embryos
(Fig. 5C). Note that these
transgenes are weak and did not result in phenotypes when expressed by
themselves in wild-type embryos using prd-gal4. These observations
strongly indicate that smooth-cuticle cells, which are receiving the Wingless
signal to antagonise the denticle-inducing effects of EGFR signalling, are
nevertheless specifically dependent on EGFR signalling for their normal
development.
Interestingly, cells near the midline appear particularly sensitive to reduced EGFR signalling since denticle belt fusions of spitz group mutant embryos have an hourglass shape and vary in thickness at the point of fusion (Fig. 5D). But from this observation it was not clear whether all ventral cells have some requirement for EGFR signalling. We addressed this further by expressing the strong DN-EGFR transgene along the entire width of the smooth-cuticle parasegment using prd-gal4 (see Fig. 5A and Fig. 6B for expression pattern). This resulted in fusion of no more than the central two thirds of each denticle belt (Fig. 5D), suggesting that only the ventral epidermal cells, but not ventrolateral cells, are sensitive to reduced EGFR signalling.
|
To distinguish between these alternatives, we marked future smooth-cuticle
cells expressing DN-EGFR in paired domains by co-expressing GFP, and
analysed the fate of these cells at different stages of embryogenesis.
Importantly, expression of DN-EGFR results in strongly penetrant fusions in
paired domains, and this is the only cuticle phenotype of these
embryos (Fig. 6G). No defects
could be observed in paired domains of stage 10/11 embryos expressing
DN-EGFR compared to those expressing only GFP
(Fig. 6B). Since epidermal
cells do not proliferate significantly after the initial series of three
mitoses following syncitial development, with the final division occurring
around stage 10 when ventrolateral fates are being specified
(Bodmer et al., 1989;
Campos-Ortega and Hartenstein,
1997
), this observation indicated that reduced proliferation is
not the cause of the fusion phenotype.
Conversely, expressing DN-EGFR in future smooth-cuticle cells resulted in dramatically increased apoptosis in paired domains as visualized by TUNEL labelling (Fig. 6C,D). Elevated levels of apoptosis were first evident at stages 10/11, became strong at stage 12 (Fig. 6C), and persisted in regions expressing DN-EGFR in and around the midline during stages 13-14 (Fig. 6D). Strikingly, following these late stages of apoptosis, denticle belt fusions first became evident as curvatures of Engrailed-expressing cell stripes in ventrolateral regions of paired domains and the absence of Engrailed-marked cells in the midline, around stage 15 before any epidermal differentiation occurs (Fig. 6E,F). Note that since Engrailed marks parasegment boundaries rather than epidermal cell fate, curvature of Engrailed cell stripes directly confirms that the fusion phenotype results from pulling of denticle cells into smooth regions rather than fate change of smooth cells into denticle cells. Collectively, these observations indicate that denticle belt fusions result from apoptosis of future smooth-cuticle cells as a consequence of reduced EGFR signalling during stages 13 and 14, resulting in fusion of adjacent denticle belt regions at stage 15.
In support of this model, elevated levels of apoptosis were also prominent
in the midline regions of stage 13/14 spitz null embryos, and less so
earlier in stages 10-12 (Fig.
7A,B). Removing the three main apoptosis-activating genes using
the H99 deletion (Foley and
Cooley, 1998; White et al.,
1994
) also partly rescued denticle belt fusions in rhomboid-3
rhomboid-1 double mutant embryos (Fig.
7C). The absence of any other recognizable smooth cuticle
phenotypes in these mutant embryos partially blocked for apoptosis suggests
that EGFR signalling does not have any additional roles in smooth cuticle
patterning (Fig. 7D). These
analyses indicate that EGFR signalling provides an important survival function
in the developing ventral epidermis: ventral smooth-cuticle cells, which are
receiving Wingless signalling to antagonise the effect of EGFR signalling on
the denticle fate, are nevertheless dependent on EGFR signalling for
survival.
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Discussion |
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This direct phenotypic analysis is also supported by several independent
genetic observations. EGFR signalling is not required for survival in future
smooth-cuticle cells early, when the ventrolateral fates are being specified
(stage 10/11) since removing Rhomboid-1 expression at only this stage using
the single-minded mutation never results in denticle belt fusions
(Mayer and Nusslein-Volhard,
1988). Defects at this early stage also cause ventral narrowing in
spitz group genes (Mayer and
Nusslein-Volhard, 1988
), and since rhomboid-3 does not
enhance this phenotype, this suggests that it cooperates with
rhomboid-1 only later in development. Vein acts independently of
spitz group genes to suppress denticle belt fusions, and this cannot
occur at stage 10/11 since at this early stage Vein expression is dependent on
EGFR signalling through a positive feedback loop
(Golembo et al., 1999
;
Wessells et al., 1999
).
Finally, the fusion phenotype itself suggests that it forms late since
denticle cells are being pulled into smooth cuticle regions and, as such,
their denticle fate must have already been determined and cannot be altered by
receiving signals from these smooth domains.
Thus, two thresholds with different outcomes exist for EGFR signalling in
patterning the ventral epidermis (Fig.
8). The level of EGFR signalling that a cell receives is
presumably dependent on its distance from the Spitz-processing cells;
activated MAPK staining indicates that these rows of cells receive high levels
of EGFR signalling (Payre et al.,
1999). High levels of EGFR signalling are required to induce the
denticle fate, while lower levels that reach smooth-cuticle cells are
sufficient to suppress apoptosis. All ventral epidermal cells therefore
require EGFR signalling, but the exact level, together with antagonism of
shavenbaby transcription by Wingless signalling, determines the
biological outcome. Importantly, these functions may be separate, as Wingless
signalling is known to antagonise shavenbaby transcription to repress
the denticle fate, but may not repress EGFR signalling itself in
smooth-cuticle cells: activated MAPK staining suggests that some
smooth-cuticle cells in the midline may also receive higher levels of EGFR
signalling (see Payre et al.,
1999
).
These results indicate that cells only require EGFR signalling for their
survival when they are starting to differentiate. A similar pattern was also
observed in the developing eye imaginal disc where removing the EGFR resulted
in cell death only once the morphogenetic furrow had passed
(Domínguez et al.,
1998). These observations raise the intriguing possibility that
establishing a requirement for survival signals may be inherent in the
differentiation program itself, perhaps for protecting against developmental
errors. However, the observation that the requirement for survival signalling
is restricted to the central region of the ventral epidermis implies that
either this requirement is not ubiquitous, or that another signal is also
involved.
EGFR survival signalling may be independent of Pointed
Pointed is an Ets domain-containing transcription factor that is
responsible for transducing most known instances of EGFR signalling. Although
it was previously clear that pointed mutant embryos rarely display
denticle belt fusions (Mayer and
Nusslein-Volhard, 1988), our analysis of a more recent null allele
that removes both P1 and P2 transcripts demonstrates that even complete loss
of pointed leads only to a very low frequency of denticle belt
fusions. This is also consistent with the milder effects of pointed
clones in the developing eye, and in particular the late onset of their
apoptosis (Yang and Baker,
2003
). These observations raise the possibility that EGFR-mediated
survival signalling in general occurs primarily at a non-transcriptional
level. Consistent with this model, EGFR signalling has been shown to reduce
Hid protein stability, thus directly inhibiting apoptosis
(Bergmann et al., 1998
;
Kurada and White, 1998
).
The role of the rhomboid gene family in embryogenesis
Rhomboid exists as a seven-member family in Drosophila, and at
least four of these are intramembrane serine proteases that can cleave all
Drosophila membrane-tethered EGFR ligands and specifically activate
EGFR signalling in vivo (Urban et al.,
2002). Although the precise role of the rhomboid protease family
in EGFR signalling and in other biological contexts has been unclear,
mutations have now been isolated for both Rhomboid-2 and -3
(Schulz et al., 2002
;
Wasserman et al., 2000
).
Genetic analysis with null alleles has revealed that both act as
tissue-specific activators of EGFR signalling much like Rhomboid-1. Rhomboid-2
is the only rhomboid known to be expressed early in gametogenesis
(Guichard et al., 2000
;
Schulz et al., 2002
), and is
involved in sending EGFR signals from the germline to the soma to guide its
encapsidation by somatic cells (Schulz et
al., 2002
). In this context, Rhomboid-2 appears to act alone.
Rhomboid-3 displays strong expression in the developing eye imaginal disc, and
is allelic to roughoid (Wasserman
et al., 2000
), one of the first Drosophila mutants
described. Rhomboid-3 is the dominant rhomboid protease during eye
development, but does not act alone: Rhomboid-3 cooperates with Rhomboid-1 in
the developing eye.
Despite the power of these genetic approaches, it should be noted that
rhomboid-1, -2 and -3 exist as a gene cluster on
chromosome 3L and, as such, combined mutations are difficult to generate by
recombination. Analysis of epidermal patterning using RNAi to overcome this
limitation is the first implication of a rhomboid homologue function in
embryogenesis. Interestingly, the rhomboid involved is Rhomboid-3, the
rhomboid that was previously thought to be eye-specific
(Wasserman et al., 2000).
However, unlike in the developing eye where Rhomboid-3 has the dominant role,
and removing Rhomboid-1 by itself has no effect
(Freeman et al., 1992
;
Wasserman et al., 2000
), the
exact opposite is true in embryogenesis: Rhomboid-1 is the main protease in
epidermal patterning while removing Rhomboid-3 alone did not result in
detectable defects. This analysis suggests that different rhomboid proteases
function predominantly to activate EGFR signalling in distinct tissues, but
often act cooperatively or with a degree of redundancy.
A reciprocal survival signalling mechanism and its conservation
The requirement for high levels of signalling for fate specification and
lower levels for viability in developing tissues may not be limited to the
EGFR pathway. Intriguingly, analysis of cell death in wingless mutant
embryos suggests that a reciprocal signalling function may also be required to
maintain cell viability in denticle regions of the ventral epidermis: in
conditions of reduced Wingless signalling, specifically during the stage of
epidermal fate specification (but not earlier), cells corresponding to two
denticle rows were observed to undergo apoptosis
(Pazdera et al., 1998).
Therefore, as with EGFR signalling, high levels of Wingless signalling induces
the smooth-cuticle cell fate, while lower levels may be required for survival
of a subset of denticle cells. Thus, the Wingless and EGFR signalling pathways
may act antagonistically in specifying cell fate, while having complementary
and reciprocal functions in maintaining cell viability in the developing
epidermis of Drosophila. These survival functions may be conserved
since EGFR signalling also has multiple roles in mammalian epidermal
development (Jost et al.,
2000
), including maintaining cell survival
(Rodeck et al., 1997
), while
some mammalian epidermal tumours are also specifically dependent on EGFR
signalling for cell survival (Sibilia et
al., 2000
). Wnt signalling has also been linked to maintaining
cell viability in certain developmental contexts
(Tepera et al., 2003
;
You et al., 2002
).
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Department of Genomic Sciences, University of Washington,
Seattle, WA 98195, USA
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