Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Facultad de Ciencias, 28049, Madrid, Spain
Authors for correspondence (e-mail:
abaena{at}cbm.uam.es
and
jcpastor{at}cbm.uam.es)
Accepted 26 September 2003
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SUMMARY |
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Key words: Peripodial epithelium, Drosophila, Squamous cells, Imaginal discs, Wing disc, Patterning, Wingless, Egfr
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Introduction |
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At the end of the third instar, the peripodial side of the wing imaginal
disc consists of several cell types with different morphology
(Fig. 1A) and gene expression
profiles (Fig. 1D,F). The
central territories in the peripodial side of the wing imaginal disc consist
of around 400 squamous cells that constitute its PE. Ultrabithorax
(Ubx) (Fig. 1D) and
puckered (puc), among other genes
(Gibson and Schubiger, 2001),
are expressed in the PE territory. Ubx, although expressed in all
posterior cells of the embryonic primordium of the wing imaginal disc, is
later restricted to its peripodial side
(Brower, 1987
). The cubic
distal cells show differential expression of zinc finger homeodomain
2 (zfh-2) (Fig.
1F) and dachsous (ds) (not shown), while
proximal cubic cells express the genes of the iroquois complex
(iro-C) (Fig. 1F).
These genes expressed in cubic cells are also expressed in the wing-notum side
of the disc, where their contribution to the patterning of the disc has been
studied. The expression of zfh-2 depends on Wg signalling
(Whitworth and Russell, 2003
)
and is required, together with ds, for wing hinge specification
(Clark et al., 1995
;
Whitworth and Russell, 2003
).
The iro-C genes, however, specify different territories in the notum
(Diez del Corral et al., 1999
)
and their expression there depends on Epidermal growth factor receptor (Egfr)
signalling (Wang et al., 2000
;
Zecca and Struhl, 2002
).
The complementary and mutually exclusive activities of the Wg and Egfr
signalling pathways are responsible for the subdivision of the wing-notum side
of the wing disc into proximal (notum) and distal (wing and hinge) territories
early during the second larval instar
(Baonza et al., 2000;
Klein, 2001
;
Wang et al., 2000
;
Zecca and Struhl, 2002
). These
signalling pathways are activated, respectively, by the diffusible ligands Wg
(Ng et al., 1996
;
Williams et al., 1993
) and
Vein (Vn) (Simcox et al.,
1996
). At this stage, the expression domain of wg is
restricted to a sector of anterior distal cells
(Williams et al., 1993
), while
the expression domain of vn is restricted to a central line of
proximal cells (Simcox et al.,
1996
). Loss of function of the Wg pathway prevents the development
of distal structures, thus allowing the expansion of Egfr activity to distal
territories. Conversely, ectopic Wg activity represses Egfr signalling in the
notum, causing wing duplications (Baonza et
al., 2000
), though loss of Egfr function does not allow expansion
of Wg signalling (Wang et al.,
2000
). Published reports only evaluate the activity of both
signalling pathways in the wing-notum side of the disc, despite the diffusive
ability of Wg and Vn. The wing field is later subdivided into wing blade and
wing hinge territories, by the combined action of Wg signalling and Vestigial
(wing blade) (Baena-Lopez and
Garcia-Bellido, 2003
; Klein
and Martínez-Arias, 1999
) or Wg signalling alone (wing
hinge) (Baena-Lopez and Garcia-Bellido,
2003
; Klein, 2001
;
Whitworth and Russell,
2003
).
In this article, we analyse the mechanisms of genetic specification of the wing disc PE. We show that the PE is a third developmental field set in the early development of the wing disc, different from the wing and notum fields, with distinct genetic requirements. We also show that Wg and Egfr signalling pathways are active up to the border between cubic and squamous cells in the peripodial side of the wing disc. Correct development of the PE does not require the activity of these signalling cascades. Furthermore, Wg or Egfr signalling transform the morphology and genetic specification of squamous cells into those characteristic of territories in the wing-notum side.
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Materials and methods |
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Clonal analysis
Gain-of-function clones were generated by the FLP/FRT technique
(Chou and Perrimon, 1992). All
the UAS fly strains were crossed with y w hsp70-flp; Act FRT
y+ FRT Gal4 UAS-GFP (Ito
et al., 1997
) and heat shocked for 7 minutes at 37°C at 24-48
or 48-72 hours after egg laying (AEL).
Wg gain-of-function clones were also generated by the MARCM technique
(Fig. 3B,E,G)
(Lee and Luo, 1999). y w
hsp70-flp Tubulin
1-Gal4 UAS-GFPnls; UAS-wgG;
Tubulin
1-Gal80 FRT2A/ FRT2A
(Struhl and Greenwald, 2001
)
larvae were heat shocked at 37°C for 15 minutes at 48-72 hours AEL.
|
Immunohistochemistry
Dissected larvae were fixed for 20 minutes in a 4% paraformaldehyde
solution in PBT (PBS/0.1% Tween 20) and immunostained with mouse anti-Ubx
(provided by Ernesto Sanchez-Herrero), rat anti-Iro-C (provided by Juan
Modolell), rat anti-Zfh-2 (provided by Martha Lundell), mouse anti-Armadillo
(Arm) (Hybridoma Bank), mouse anti-Discs large 1 (Dlg1) (Hybridoma Bank),
mouse anti-Wg (Hybridoma Bank), rabbit anti-Vg (provided by Sean Carroll),
rabbit anti-Distalless (Dll) (provided by Sean Carroll), rat anti-Ds (provided
by Michael Simon), mouse anti-Nubbin (Nub) (provided by S. Cohen), rat
anti-Tailup (Tup) (provided by Jim Skeath) or guinea pig anti-Eyegone (Eyg)
(provided by Natalia Azpiazu) antibodies in PBTBSA (PBT/0.3% BSA). Alexa
Fluor-488-, -546- (Molecular Probes) or Cy5 (Jackson
InmunoResearch)-conjugated secondary antibodies were used to detect primary
antibodies. F-actin staining was performed with Texas Red-coupled phalloidin
(Sigma). Cell nuclei were stained for 20 minutes in a 1 mM To-Pro-3 iodide
(Molecular Probes) solution in PBT. Imaginal discs were mounted in Vectashield
(Vector Laboratories, Inc).
Microscopy and image treatment
Images were acquired in a BioRad 2000 confocal microscope and treated with
the Metaview (Universal Imaging) and Photoshop 7.0 (Adobe Corp) image
programs.
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Results |
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Proximodistal (PD) territorial segregation in the wing-notum side of the
wing disc proceeds simultaneously to the morphological changes of peripodial
cells, and it is driven by the complementary and mutually exclusive activities
of the Wg and Egfr signalling pathways
(Baonza et al., 2000;
Klein, 2001
;
Wang et al., 2000
;
Zecca and Struhl, 2002
). In
the second instar, cubic cells express zfh-2 and iro-C in
distal and proximal domains, respectively
(Fig. 1E). The early expression
pattern of these genes, which is dependent on Wg and Egfr signalling
(Baonza et al., 2000
;
Klein, 2001
;
Wang et al., 2000
;
Whitworth and Russell, 2003
;
Zecca and Struhl, 2002
)
suggests that the mechanism of PD segregation is also active in the peripodial
side of the disc, although their expression pattern later evolves to overlap
in some regions (Fig. 1F,G). In
this context, proximal cubic cells would be part of the notum developmental
field, whereas distal cubic cells would be part of the wing developmental
field. Interestingly, Zfh-2 and Iro-C are not detected in the PE. Also, the
limit of expression of Zfh-2 and Iro-C is sharp and coincident with the limit
between cubic and squamous cells.
Wg and Egfr signalling activities are not required for PE
development
The absence of expression of Wg and Egfr signalling downstream genes
suggests that the activity of both signalling pathways might be repressed in
the prospective PE from embryonic development or early larval stages in order
to generate a difference between the wing and notum fields and the PE of the
disc. To evaluate the role of Wg and Egfr signalling in the development of the
peripodial side of the wing imaginal disc, we generated loss-of-function
clones for both signalling pathways at 24-48 and 48-72 hours after egg laying
(AEL).
Clones expressing the intracellular domain of E-cadherin
(E-Cadhintra5) autonomously lack Wg signalling because of
sequestering of Armadillo (Arm) (Sanson et
al., 1996), the transcriptional effector of Wg signalling,
consistent with the cytoplasmic accumulation of Arm we observe
(Fig. 2A).
ECadhintra5 overexpression early in the prospective wing field
abolishes its development, causing notum duplications and a complete lack of
Wg function (Sanson et al.,
1996
; Sharma and Chopra,
1976
). These clones do not survive or are smaller in size than
controls in the wing-notum side of the disc, but are normal in size in the PE
(Fig. 2B). In addition, these
clones eliminate the expression of zfh-2 in distal cubic cells of the
peripodial side (Fig. 2C).
|
The above results show that the activities of the Wg and Egfr signalling pathways are required for the normal development of cubic cells, but not for the proliferation, survival and genetic specification of the PE. Finally, we do not observe modifications in cell morphology when E-Cadhintra5 and DN-Raf3.1 are co-expressed in cubic cells (not shown). This indicates that the development of the PE has specific genetic requirements that preclude an expansion of the peripodial field to adjacent wing and notum territories in the absence of Wg and Egfr activities.
Ectopic Wg signalling transforms the cell morphology and fate of the
PE into those of the wing hinge
To further evaluate the role of Wg signalling in the development of the
peripodial side of the wing disc, we generated wg expressing clones
at 24-48 and 48-72 hours AEL. In these clones, the shape of PE cells changes
from squamous into the columnar morphology characteristic of cells in the
wing-notum side of the disc (Fig.
3A,B,E). The transformation is not only cell autonomous but
affects cells non-autonomously within a range of 7-15 cellular diameters away
from the clone (Fig. 3B). The
severity of the morphological change decreases with distance to the clone,
which suggests a dependence on Wg concentration
(Fig. 3B). By contrast,
autonomous activation of Wg signalling in Arm overexpressing
clones only results in autonomous transformations of the cellular morphology
and genetic identity of PE cells (Fig.
3D). The increase in cell density induced by both Wg and
Arm
in the PE may be due not only to the change in cell shape,
but also to a higher rate of proliferation, evident when the size of these
clones is compared with controls.
In addition to cell morphology changes, ectopic Wg in the PE induces autonomous and non-autonomous expression of characteristic wing hinge genetic markers such as ds (Fig. 3E) and zfh-2 (Fig. 3F), and represses Ubx in the PE (Fig. 3C). Wg-expressing clones never induce the expression of wing blade or notum markers such as vestigial (vg), distalless (dll), iro-C and eyegone (eyg) (not shown). These data show that ectopic Wg signalling transforms the PE cells into cells of the wing hinge. Interestingly, ectopic expression of Wg induces autonomous expression of nubbin (nub), a transcription factor expressed in the proximal hinge and wing blade. However, in contrast to the strictly nuclear wild-type localization of Nub, these clones show high levels of Nub in the cytoplasm of the transformed cells (Fig. 3G). This result points to an incomplete transformation of the PE into wing hinge, which suggests that the PE is refractory to this transformation. The ability of ectopic Wg to transform the PE into wing hinge is independent of the developmental time when the clones are generated, suggesting that repression of Wg activity may be essential for the correct development of the PE. Clones expressing Wg also induce zfh-2 expression in proximal cubic cells (Fig. 3F) and repress the expression of iro-C (not shown), which again suggests that the mechanism that drives PD segregation in the wing-notum side is also active in cubic cells.
Considering a possible transmission of Wg signalling through the lumen of
imaginal discs (Cho et al.,
2000; Gibson et al.,
2002
; Gibson and Schubiger,
2000
; Gibson and Schubiger,
2001
), we evaluated whether ectopic expression of Wg in one side
of the wing disc affects the opposite side. Ectopic Wg in cells of the
wing-notum side does not change the specification (Ubx expression) or
morphology of squamous cells (not shown). Changes in the reverse direction do
not take place either (not shown). This shows that Wg signalling is not
transmitted through the lumen of the wing imaginal disc. Finally, the ectopic
expression of Wg transforms peripodial cell morphology in the wing, haltere
and leg imaginal discs, but does not change the squamousity in the PE of the
eye-antenna imaginal disc (not shown).
Ectopic Wg and Vg transform the cell morphology and fate of the PE
into those of the wing blade
Cells expressing Wg and Vg in the wing-notum side of the disc acquire the
identity of wing blade cells (Baena-Lopez
and Garcia-Bellido, 2003;
Klein and Martínez-Arias,
1999
). We asked whether PE cells, besides the ability to be
transformed into hinge, can acquire the more distal fate of the wing blade.
Ectopic expression clones of Vg in the PE do not modify the identity or the
morphology of squamous cells (not shown), which reinforces the idea that PE
cells lack Wg activity. Peripodial cells co-expressing Wg and Vg, by contrast,
acquire the identity of the wing blade, as suggested by the cell-autonomous
induction of the wing genetic marker dll
(Fig. 4C). Wg-Vg co-expression
changes the shape of squamous cells into columnar
(Fig. 4A,B). The effect of
Wg-Vg clones on cell morphology is not only autonomous, expanding more cell
diameters away than in clones expressing Wg only. This region of
non-autonomous transformation around the clones expresses exclusively
wing-hinge genetic markers, such as zfh-2
(Fig. 4B) or nub
(Fig. 4A,C). In contrast to
clones overexpressing Wg, the autonomous and non-autonomous expression of
nub induced by Wg-Vg clones is always nuclear
(Fig. 4A,C), an indication that
Vg might contribute to the translocation or maintenance of Nub into the
nucleus.
|
Ectopic Egfr signalling transforms the cell morphology and fate of
the PE into those of the notum
The role of Egfr signalling in the development of the peripodial side of
the wing imaginal disc was studied by generating clones expressing Vn, a
diffusible Egfr ligand (Simcox et al.,
1996), at 24-48 hours AEL. Eight out of 25 peripodial clones
expressing Vn showed peripodial cells transformed from squamous into columnar
(Fig. 5A), although not all the
cells of the clones were affected to the same extent. The transformation also
affected non-autonomously some cells outside these eight clones, though, as
described for the autonomous transformation, its penetrance is not complete
(Fig. 5A). Accordingly,
Ubx expression is repressed in some peripodial cells
(Fig. 5A). It should be noticed
that Vn overexpression is also unable to induce completely penetrant
wing-to-notum transformations when expressed in the prospective wing field
(Wang et al., 2000
). A fully
penetrant transformation, however, is achieved when a constitutively active
form of the Ras protein (RasV12)
(Karim and Rubin, 1998
) is
expressed in peripodial cells (Fig.
5B-D). Clones expressing RasV12 change the
squamous morphology of PE cells both autonomously and non-autonomously
(Fig. 5B,C), which suggests
that Egfr activation induces Vn expression, a positive loop also observed in
the expression of Vn in the notum (Wang et
al., 2000
). The increase in cell density observed in
RasV12 clones may be associated to an excess in cell
proliferation.
|
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Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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The first differences at the morphological level between cells of the
peripodial and wing-notum side of the wing disc arise during the second larval
instar, though a previous genetic heterogeneity may already exist (discussed
below). The phenotype of clones lacking Wg and Egfr signalling, as well as the
expression patterns of downstream genes (zfh-2 and iro-C) in
wild-type discs, indicate that the activity of both signalling cascades is
required in the peripodial side of the disc only for normal development of
cubic cells surrounding the PE, but not in the PE itself. Ectopic activation
in the PE of the Wg or Egfr pathways causes squamous cells to adopt the
columnar morphology characteristic of the cells of the wing-notum side
(Fig. 6A-C). This
transformation does not involve changes in the apicobasal polarity of the
cell, as seen by normal localization of Arm
(Fig. 3A,
Fig. 5B), F-actin and Discs
large 1 (Dlg1) (not shown). The transformation induced by ectopic Wg
signalling affects exclusively the cells of the clone when this pathway is
activated by the expression of the autonomous activator Arm.
However, the transformations are both cell autonomous and non-autonomous when
ectopic activity is induced by the overexpression of the diffusible ligands Wg
and Vn, though this latter in a non-fully penetrant way. The transformations
induced by RasV12 were also both autonomous and
non-autonomous, which may be explained by the fact that these clones could
also induce the expression of Vn. The above observations suggest an
involvement of both signalling pathways in the control of cell morphology in
the imaginal disc.
|
The ability of Wg and Egfr ectopic signalling to change the fate of peripodial cells is independent of the developmental time when the clones are generated. However the PE must be subject to a progressive genetic specification as larval development proceeds. This is evidenced by the fact that Wg-Vg expression clones generated early are able to reproduce the developmental program of the wing field to a greater extent than the clones induced later, which implies that the PE is less competent to adopt this fate later in development.
Taking together the above results, we conclude that the PE constitutes a
developmental field in early wing imaginal discs, different from the wing and
notum fields. This peripodial field does not require the activity of the Wg
and Egfr signalling pathways. Moreover, ectopic Wg and Egfr signalling are
able to expand the wing and notum fields at the expense of the PE. So, it
seems that reduced levels of Wg and Egfr signalling are a prerequisite for the
development of the peripodial field. The reduced levels of Wg and Egfr
signalling in the PE cannot be explained by absence of the receptors or any
other elements required for the transduction of the Wg and Egfr signals, given
the ability of the Wg and Vn ligands to induce transformations. Two possible
mechanisms could account for the absence of Wg and Egfr activity in the PE.
(1) Normal diffusion of the Wg and Vn ligands is unable to activate the
pathway in the PE (Fig. 6D).
(2) The normal decay of concentration of the ligands from their sources in the
wing-notum side could lower this concentration down to zero
(Fig. 6D) or below a
hypothetical threshold (Fig.
6E). These possibilities, however, are challenged by the fact that
Wg and Egfr signalling do not define the limit of the peripodial field, as the
PE does not expand when we simultaneously eliminate the activities of the Wg
and Egfr pathways in cubic cells. This suggests that a pre-existing genetic
heterogeneity sets the limits of the prospective peripodial field. Although
our data do not reveal the mechanisms that suppress Wg and Egfr signalling in
the PE, they seem to favour a second model in which the PE is refractory to
these signals (Fig. 6F-H). This
could be due to a barrier to the diffusion of the ligands in the PE
(Fig. 6F) or to repression of
the signal downstream of the receptor level
(Fig. 6G). Both a barrier to
diffusion of the ligands and downstream repression could be overcome if the
amount of ligand or downstream signalling is experimentally elevated.
Suppression of the Wg and Egfr signalling pathways in late embryonic
development, in addition, is a necessary step for the specification of the
wing disc primordium and its segregation from the leg disc
(Kubota et al., 2000;
Kubota et al., 2003
). It seems
possible, therefore, that the repression of these signalling pathways,
inherited from the embryo, is later restricted to the peripodial side of the
disc; alternatively, the repression of Wg and Egfr signalling in the PE might
arise later as a non-related event. Furthermore, the early repression of these
signals in the wing and notum fields could be, from an evolutionary
perspective, a suitable mechanism for the generation of a peripodial field in
ancestral uninvaginated discs
(Svácha, 1992
;
Truman and Riddiford,
1999
).
Some recent reports have focused on a role of the PE of the eye and wing
discs in the patterning of the other side of the disc
(Cho et al., 2000;
Gibson et al., 2002
;
Gibson and Schubiger, 2000
),
showing the ability of Dpp and Hh proteins expressed in the PE to affect the
development of the other side of the disc. Our results, however, rule out
translumenal communication of the Wg and Egfr signals. Translumenal
communication of intercellular signals, therefore, is not the simple outcome
of the apposition of the two sides of the disc or general secretion of ligands
into the disc lumen. On the contrary, transmission of a signal through the
disc lumen would require specific mechanisms for every different signalling
pathway.
Finally, the comparative analysis among the peripodial epithelia of different imaginal discs shows that the development of each PE has characteristic genetic requirements, at least after their specification, as evidenced by the inability of Wg and RasV12 clones to transform the morphology of peripodial cells in the eye-antenna disc. The possibility of a common mechanism for the generation of the peripodial field in all imaginal discs, however, still remains.
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ACKNOWLEDGMENTS |
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Footnotes |
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