Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India 500 007
* Author for correspondence (e-mail: shashi{at}ccmb.res.in)
Accepted 3 July 2003
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SUMMARY |
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Key words: Drosophila, Wing, Peripodial membrane, Ultrabithorax, Egfr/Ras pathway, Notum
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Introduction |
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Surgical ablation experiments suggest that the wing disc DP is capable of
differentiating into wing blade without the PM, although the PM is required
for proper development of the wing margin
(Gibson and Schubiger, 2000).
Those experiments did not address the role of PM in patterning events, as the
PM was removed from late third instar larval discs. Subsequently, Gibson et
al. (Gibson et al., 2002
)
showed that survival of PM cells requires Decapentaplegic (Dpp) signaling from
the DP. More importantly, inhibition of Dpp signaling in the PM affected the
growth of the entire disc (Gibson et al.,
2002
), suggesting a crucial role for the PM in wing
development.
Although the PM has not been specifically studied for its role in wing
development, it has been observed that several wing-patterning genes are
expressed in PM cells. puckered (puc), a negative regulator
of the JNK pathway, and hemipterous (hep), which encodes
Drosophila JNK-Kinase, are expressed in cells at the medial edge of
the wing disc PM (Agnes et al.,
1999). The hep mutants show loss of Puc in the PM and,
perhaps as a consequence, are defective in thorax closure. In addition,
engrailed (en), decapentaplegic (dpp),
patched (ptc), combsgap, Capichua (Cic),
teashirt (tsh), Broad complex genes, E74A, DHR3,
hep and Ultrabithorax (Ubx) are expressed in the PM of
wing imaginal discs (White and Wilcox,
1985
; Brower, 1987
;
Boyd et al., 1991
;
Emery et al., 1994
;
Lam et al., 1997
;
Panin et al., 1997
;
Agnes et al., 1999
;
Svendsen et al., 2000
;
Roch et al., 2002
;
Wu and Cohen, 2002
). We have
made use of GAL4 drivers derived from some of these genes (particularly
Ubx-GAL4) to study the nature of interactions between the PM and DP,
and in lineage tracing experiments to study the possible lineage relationship
between the two.
Our results suggest the following.
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Materials and methods |
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en- and ptc-GAL4
(Brand and Perrimon, 1993),
ap- and pnr-GAL4
(Calleja et al., 1996
),
AgiR-GAL4 (Gibson et al.,
2002
), vg-GAL4
(Simmonds et al., 1995
), and
tsh-GAL4 [personal communication to FlyBase (Calleja, 1996.10.16)]
drivers have been previously reported. 426-GAL4, odd-GAL4 (an
insertion in odd-paired locus) and coro-GAL4 (insertion in
Drosophila homologue of coronin) drivers were identified in
the laboratory in a GAL4-enhancer trap screen. UAS lines used are UAS-Argos
[personal communication to FlyBase (Michelson, 1999.8.9)], UAS-P35
(Hay et al., 1994
), UAS-Ubx
(Castelli-Gair et al., 1994
),
UAS-Vg (Kim et al., 1996
) and
dominant-negative forms of Serrate (UAS-DN-Ser)
(Sun and Artavanis-Tsakonas,
1997
), Glued (UAS-DN-Glu)
(Allen et al., 1999
),
Drosophila Ras (UAS-DrasN17)
(Lee at al., 1996
), human RAS
(UAS-rasN17) (Lee at al.,
1996
), Raf (UAS-DN Raf3.1)
(Martin-Blanco et al., 1999
)
and DER (UAS-DN-DER) (Golembo et al.,
1996
).
Ubx-GAL4 driver was generated using previously reported
transposon-swapping strategy (Sepp and
Auld, 1999). Casares et al.
(Casares et al., 1997
) have
reported a Ubx-lacZ insertion (cytology: 89D6-9 and carried
ry+ marker), which reflects near-complete pattern of
Ubx expression. In wing imaginal discs, it is expressed only in PM.
We used ptc-GAL4 (cytology: 44D5-E1 and carried
w+ marker) as the donor GAL4-P element. Generation of
Ubx-GAL4 strain was confirmed by testing the strain for the absence
of lacZ by X-gal staining and for the presence of GAL4 by crossing to
UAS-GFP. Lineage-tracing technique is essentially as described by Weigmann and
Cohen (Weigmann and Cohen,
1999
). lacZ clones were generated by crossing
Actin5C>stop>lacZ to UAS-FLP;Ubx-GAL4 at 25°C. Third larval
instar wing discs were stained for lacZ with
anti-ß-galactosidase.
Histology
X-gal staining and immunohistochemical staining were essentially as
described by Ghysen and O'Kane (Ghysen and
O'Kane, 1989) and Patel et al.
(Patel et al., 1989
). The
lacZ reporter gene constructs used are lio-lacZ
(Bolwig et al., 1995
) and
aos-lacZ (Freeman et
al., 1992
). The primary antibodies used are anti-Arm
(Riggleman et al., 1990
); anti
ß-galactosidase (Sigma, St Louis, MO), anti-Ci
(Motzny and Holmgren, 1995
);
anti-Cut (Blochlinger et al.,
1993
); anti-En (Patel et al.,
1989
); anti-Ubx (White and
Wilcox, 1984
); and anti-Wg
(Brook and Cohen, 1996
).
Anti-Arm and anti-Wg antibodies were obtained from the Development Studies
Hybridoma Bank (University of Iowa, IA). Confocal microscopy was carried out
on Meridian Ultima. The adult appendages were processed for microscopy as
described before (Shashidhara et al.,
1999
).
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Results |
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Peripodial membrane and disc proper cells are clonally related
The anterior-most expression domain of Ubx-GAL4 is parasegment 4
(Fig. 2G). However, in the
third instar wing disc, it is expressed only in the PM
(Fig. 2F). We made use of this
dynamic expression pattern of Ubx-GAL4 to determine the lineage
relationship between PM and DP cells. We used a lineage-tagging method
described by Weigmann and Cohen (Weigmann
and Cohen, 1999) for this purpose. We generated
Act5C>lacZ-expressing clones by crossing UAS-flp;
Ubx-GAL4 to Act5C>stop>lacZ.
We examined a number of wing discs (n=42), some expressing
lacZ only in the PM (n=20) and others in both PM and the DP.
We did not observe any disc that expressed lacZ only in DP cells.
Because in wing discs that expressed lacZ in both PM and DP there was
no direct way of determining if lacZ-expressing cells correspond to a
single clone, we characterized the nature of clones in the following way. All
clones that expressed lacZ in both PM and DP were always large
(Fig. 3A-D), whereas the size
of PM-only clones varied from just two cells to more than 100 cells
(Fig. 3E-H). In all wing discs
that showed lacZ expression in both PM and DP cells, the ratio
between lacZ-expressing PM and DP cells remained at around 1:80,
similar to the ratio between PM to DP cells for the entire disc. Moreover, in
a wing disc, the number of clusters of lacZ-expressing PM cells was
always equal to or more than the number of clusters of
lacZ-expressing DP cells. These observations suggest that PM and DP
cells share a common lineage in the embryonic disc primordium and they become
separated later during development. The similar ratio of PM to DP cells in
clones and in the whole disc suggests that the proliferation pattern of PM
cells is similar to that of DP cells. Wing imaginal primordium divides every
10-12 hours during three larval instars before the wing disc everts and starts
differentiating (Cohen, 1993).
We observed large PM-only clones comprising >128 cells
(Fig. 3G,H). As such clones
would have undergone seven or eight cell divisions, they must have been
generated in the early first larval instar stage. Thus, it is likely that PM
and DP cells are separated before the onset or at the beginning of
proliferation phase of the disc primordium. Because we observed large PM-only
clones in virtually all parts of the wing disc, we also infer that
Ubx-GAL4 continues to be expressed in all PM-progenitor cells and is
switched-off in DP cells as soon as the lineages are separated.
|
Interestingly, in wing discs that showed lacZ expression in both PM and DP cells, the clones were always within the same subdivision of the wing disc. For example, for every cluster of lacZ-expressing DP cells in the notum there was a cluster of lacZ-expressing PM cells overlaying the notum (Fig. 3D). A similar correlation was observed for the wing pouch. This suggests that clonally related PM and DP cells maintain a spatial relationship, which may be indicative of a role for PM in wing patterning.
Ras is required for the viability of PM cells
Ras/Egfr signaling is required for the survival of notum cells. A
temperature sensitive allelic combination of Egfr
(Egfrtsla/Egfrf24) shows severe reduction of
notum (Wang et al., 2000) and
mitotic clones of Egfr and Ras mutations do not survive in
the notum (Zecca and Struhl,
2002
). We generated loss-of-function mitotic clones of
Ras to examine if it is required for the viability of PM cells. In
one set of experiments,
Ras/Ras clones were
marked with GFP and in the other set
Ras+/Ras+ cells were marked with GFP.
Consistent with the earlier reports, Ras clones
(induced at 48 to 72 hours AEL) were generally viable in the pouch region but
not in the notum (data not shown). We observed that in the PM too,
Ras/Ras clones were
associated with reduced cell viability
(Fig. 4A). Interestingly,
unlike in the DP (wherein Ras clones show
differential viability in the notum and the pouch),
Ras clones showed reduced viability in all parts of
the PM.
|
As en-GAL4 is expressed in both PM and DP cells
(Fig. 2D), it is possible that
the reported notum/hinge-to-wing transformation (caused by the overexpression
of DN-Raf or DN-Ras using en-GAL4 driver) is mediated through PM. A
parallel phenomenon has been demonstrated for eye discs. Overexpression of
Fringed (Fng) results in identical phenotypes when expressed using either a
PM-specific GAL4 (c311-GAL4) or a GAL4 driver (ey-GAL4),
which is expressed in both PM and DP
(Gibson and Schubiger,
2000).
Downregulation of the Egfr pathway in PM alone is sufficient to
induce fate transformations in the disc proper
We re-examined the role of the Egfr pathway in specifying notum/hinge fate.
We could not make use of Ras clones for this
purpose, as Ras clones induced at early stages of
development (coinciding with the time at which the Egfr pathway is required
for both survival and specification of the notum) were always lethal. Because
clones were induced during the rapid proliferation stage, they were generated
always in large numbers, invariably in both PM and DP. Furthermore, owing to
lethality associated with Ras clones in both PM and
the notum, it was often difficult to confirm if we had generated PM-only
clones, which made analysis of the effect of removal of Ras from PM
cells inconclusive.
To circumvent the problem of direct genetic analysis of Ras function in PM,
we used various GAL4 drivers and dominant-negative forms of DER, Ras and Raf
or wild-type Argos (Aos; a negative regulator of the Egfr pathway) to
downregulate the Egfr pathway in PM and/or DP cells. First, we overexpressed
these proteins using the PM-specific GAL4 driver Ubx-GAL4.
Overexpression of all the four proteins in the wing disc PM resulted in wing
duplication in the posterior compartment
(Fig. 5B-G). All the flies,
which showed distinct pattern duplication, also had partial loss of notum
and/or hinge (Fig. 5E). We also
observed several pharate adults, which died within the pupal case, with severe
loss of notum tissue. In such pharate adults, wing blade development was also
severely affected, probably owing to defective notum development (data not
shown). Ectopic expression of a dominant-negative form of the mammalian Ras
(UAS-rasN17) too showed the same phenotype when crossed to
Ubx-GAL4 (Fig. 5H).
Wing discs overexpressing DN-DER, DN-Ras, DN-Raf or Aos in the PM showed
altered morphology, particularly overgrowth in the posterior hinge and
mesonotum. When stained for Wg, they indicated a new pouch complete with the
DV boundary (Fig. 6A-C). Double
staining with Ci and Wg (Fig.
6D), and En and Wg (Fig.
6E) indicated that pattern duplication is associated with loss of
notum, confirming notum/hinge-to-wing transformations. When stained for Ubx
and Arm, transformed discs showed intact PM
(Fig. 6F-I), which rules out
loss of PM identity in the transformed wing discs. Interestingly,
Ras clones generated during larval stages do not
survive in the notum (Zecca and Struhl,
2002) or in the PM (Fig.
4A). The overexpression of DN-Ras, however, causes cell lethality
in the notum (Fig. 4E), but not
in the PM (Fig. 6F-I). This
observation suggests that notum cells are more sensitive to loss of Egfr
pathway than are PM cells.
|
|
We further examined the specificity of the observed phenotype using a
number of GAL4 drivers, which express either in the DP alone or in both DP and
the PM. They are 426-, pnr-, ap-(all express only in the
DP), tsh and AgiR-(express in both PM and DP) GAL4 drivers.
426-GAL4 driver is expressed only in the presumptive hinge
(Fig. 7A) and pnr-GAL4
(Fig. 7D) is expressed only in
the notum. However, both are late-expressing GAL4 drivers, beginning from
early third instar stage. ap-GAL4 is an early expressing (second
instar) driver and is expressed in the entire dorsal compartment.
Overexpression of DN-Ras and Aos using 426- and pnr-GAL4 drivers did
not show any phenotype (Fig.
7B,C,E,F). Their overexpression using ap-GAL4 driver
resulted in severe reduction of the notum
(Fig. 7H,I). Thus, although the
effects on the viability of notum/hinge cells are consistent with the clonal
analysis of Zecca and Struhl (Zecca and
Struhl, 2002), we did not observe any cell fate transformations.
tsh-GAL4 and AgiR-GAL4 drivers are expressed in the notum
and hinge and also in the PM (Fig.
7J,M). Overexpression of DN-Ras and Aos using these drivers caused
loss of notum, severe with tsh-GAL4 [which is expressed in the entire
notum (Fig. 7K,L)] and partial
with AgiR-GAL4 [which is expressed only in a subset of lateral notum
cells (Fig. 7N,O)].
|
Dynamic expression of Aos in the peripodial membrane
Our results described above suggest a role for the Egfr pathway in the PM
to specify notum/hinge identity. Whenever the Egfr pathway is activated, it
induces the expression of Aos, which in turn acts as a negative regulator,
thus keeping the pathway under tight feedback regulation
(Golembo et al., 1996). Thus,
expression of Aos marks the activity of the Egfr pathway. If the Egfr pathway
active in the PM at any stage during development, it would be reflected in Aos
expression pattern. We examined the expression pattern of
aos-lacZ, which reflects endogenous Aos expression pattern
(Freeman et al., 1992
). In
third instar wing imaginal discs, Aos is expressed both in the pouch and the
notum (Fig. 8A). Double
staining with Ubx indicated that Aos is not expressed in PM cells
(Fig. 8B). Large cells that
express Aos in the notum are adepithelial cells (myoblasts), which are marked
by Cut (Ct) expression (Fig.
8C,D). We then examined if Aos-lacZ is expressed in
earlier stages, which may reflect activation of Egfr during wing disc
patterning. In early second instar wing discs, Aos is expressed in PM cells
overlaying the posterior notum (Fig.
8E). Double staining with Ct suggested that the large cells
expressing Aos are not myoblasts (Fig.
8E). Ct is expressed only in the anterior compartment and Aos is
expressed only in the posterior compartment. Expression of Aos in the PM
spreads in the midand late-second instar larval stages
(Fig. 8F,G). Double staining
with Ubx confirmed that Aos is expressed in PM cells, although its expression
is mostly restricted to the PM cells overlaying dorso-posterior mesonotum
(Fig. 8K). Only in the early
third instar wing discs is Aos expression first seen in the pouch
(Fig. 8L).
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Discussion |
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We have observed that PM and DP cells maintain a spatial relationship even
after the separation of their lineages. Thus, notum cells are clonally related
to PM cells overlaying the notum and pouch cells are clonally related to PM
cells overlaying the pouch. Morphologically, PM cells over the pouch and the
notum form two distinct groups. In the notum region, PM cells are densely
populated and they send out microtubule extensions to DP cells through the
disc lumen (Gibson and Schubiger,
2000; Cho et al.,
2000
). Over the wing pouch, PM cells appear to be less densely
arranged and are bigger and flatter. The expression patterns of various gene
markers further divide the PM into distinct spatial domains (compare the
expression patterns of lio-lacZ, odd-GAL4, 409-GAL4 and
AgiR-GAL4). These spatial domains may represent specific functional
domains. Taken together, these observations point to a more developmental
function for the wing disc PM, rather than just the provision of structural
support to the growing DP. As there is a large difference (1:80) in the number
of clonally related PM and DP cells, the PM may influence the growth and/or
patterning of a relatively larger region of the wing disc rather than
individual DP cells.
A significant finding of our study is the role of the PM in
wing/notum/hinge decision. The wing disc is initially divided into anterior
and posterior compartments by virtue of En expression only in a subset of disc
cells. Later, it is subdivided into three distinct groups of cells, wing,
notum and the hinge. This is marked by the expression of Wg in the presumptive
wing region (Ng et al., 1996),
Pnr in the presumptive notum (Calleja et
al., 2000
) and Tsh in the presumptive hinge
(Casares and Mann, 2000
;
Wu and Cohen, 2002
). PM cells
over the notum and the pouch may provide positional cues for notum/hinge-wing
decision. We have observed that the Egfr pathway functions in the PM to
specify notum-specific genes and/or to inhibit wing-specific genes.
Mis-expression of DNDER, DN-Ras, DN-Raf or Aos in the PM was enough to induce
notum/hinge-to-wing transformations. The dynamic expression pattern of Aos
marks the spatial and temporal pattern of Egfr activation. In the second
instar larval stage, when wing-notum decision is made, Aos is expressed
specifically in those PM cells that overlay the posterior notum. Once the
wing-notum decision is made, Aos expression recedes from PM cells and it
starts expressing in the notumassociated myoblasts and in the pouch. Although
at this stage we cannot rule out the possibility that the Egfr pathway is
required in both PM and DP cells to specify notum fate, our results suggest
that the Egfr pathway mediates interactions between PM and DP cells during the
notum/hinge specification.
All the observations on notum/hinge-to-wing transformations in this report
and elsewhere (Wang et al.,
2000; Baonza et al.,
2000
) are restricted to the posterior compartment. However,
ectopic expression of Wg can cause notum-to-wing transformation in both the
anterior and posterior compartments (Ng et
al., 1996
). En is expressed in large number of PM cells that
overlay part of the anterior compartment. Ubx, which is expressed only in the
posterior compartment of T2 (parasegment 5), is expressed in all PM cells. In
addition, overexpression of Hh in PM cells does not induce ectopic
dpp-lacZ expression in those cells (P.K., unpublished
observations). These observations suggest posterior identity of all PM cells.
Is this the reason for observed notum/hinge-to-wing transformations only in
the posterior compartment? If the answer is yes, how is the notum specified in
the anterior compartment? Further investigation is needed to determine the
compartmentalization within the PM and compartment-specific interactions
between PM and DP.
Interestingly, the only other function so far attributed to the wing disc
PM is dorsal closure of the notum (Agnes et
al., 1999). Does the PM have a role in patterning other regions of
the wing disc? At later stages during wing patterning, the Egfr signaling is
required to specify apterous (ap) expression in the dorsal
compartment, and thereby to specify dorsoventral axis formation
(Wang et al., 2000
). In
addition, it is required to specify the vein and intervein development
(Diaz-Benjumea and Hafen,
1994
). The Egfr pathway is also implicated in signaling from the
dorsoventral organizer (Nagaraj et al.,
1999
). However, we did not see any phenotype in the wing pouch
following the expression of DN-DER, DN-Ras, DN-Raf or Aos in PM cells. It is
possible that the Egfr pathway is functional from DP itself to specify Ap
expression. Alternatively, as the Egfr pathway plays a more permissive role
than instructive role in specifying Ap expression
(Zecca and Struhl, 2002
),
lowering of its activity in the PM alone may not be sufficient to affect DV
patterning events. After the specification of dorsal and ventral compartments,
fng and Ser interact to activate Notch (N) in the DV
boundary. Ser is known to express and function in the PM of eye imaginal discs
(Gibson and Schubiger, 2000
;
Cho et al., 2000
). Ectopic
expression of a dominant-negative form of Ser (DN-Ser) in the eye disc PM
affects ommatidial patterning (Gibson and
Schubiger, 2000
). Ectopic expression of DN-Ser in the wing disc PM
using Ubx-GAL4 did not affect wing patterning (data not shown). These
results suggest differences in the function of the PM in eye and wing
discs.
Precise genetic ablation of PM cells during wing patterning may provide
insights, if the PM has a role in patterning other regions of the wing disc.
We did try to ablate wing disc PM cells genetically using UAS-FLP,
Ubx-GAL4 and UASFLPout-Ricin
(Hidalgo et al., 1995).
However, in all our experiments animals were invariably early larval lethal,
presumably owing to Ricin expression in other tissues. To circumvent this
problem, one would need more precise GAL4 driver that, while being specific to
the PM in wing discs, is not expressed in other tissues. Nevertheless, our
results on signaling from DP to regulate growth properties of PM cells and the
Egfr signaling from PM to DP cells to specify notum establish bi-directional
signaling between the two epithelial layers.
Although our results suggest a role for the PM in specifying notum
development, further investigation is required to determine how the Egfr
pathway is activated in PM cells. Does DP play a role in activating the Egfr
pathway in PM cells, which in turn specifies notum development in the DP? This
would be analogous to oocyte-follicle cell interactions, wherein Gurken
expressed in the oocyte is responsible for the activation of Egfr pathway in
the follicle cells. Subsequently, follicle cells signal to the oocyte, which
results in the re-organization of the cytoskeleton of the latter.
Identification of the ligand for Egfr in PM cells and the signal that goes
from PM to DP may lead us to the mechanism by which it is activated in PM
cells. Because only PM cells over the notum send out microtubule extensions,
any molecule that signals to DP to specify notum development may depend on
these processes. Such microtubule extensions have also been observed in the
eye disc and are shown to be required for proper signaling from PM to DP.
Overexpression of a dominant-negative form of glued (DN-Glu), a component of
microtubule-binding motor complex proteins in the eye disc PM causes delay in
the progression of the morphogenetic furrow
(Gibson and Schubiger, 2000).
However, we did not see any effect of expressing DN-Glu in the wing disc PM
using Ubx-GAL4 driver (P.K., unpublished observations). The role, if
any, of microtubule extensions in the wing disc would probably be independent
of the motor complex. Further investigation is needed to identify signal
molecules involved in PM to DP interactions.
Identification of signal transduction pathways that mediate interactions
between different types of epithelial sheets during normal development may
provide us with clues to understand development of tissues and pathological
situations leading to metastasis. In vitro studies using human cell lines
suggest a role for Egfr/Ras signaling in cell motility and tumour invasion
(Krueger et al., 2001;
Mahoney et al., 2002
;
Lotz et al., 2003
). We have
reported that the Egfr pathway mediates signaling between squamous and
columnar epithelia. Further studies on the interaction between PM and DP may
help identifying several key factors mediating cancer progression.
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ACKNOWLEDGMENTS |
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