Centro de Biología Molecular `Severo Ochoa'-Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Facultad de Ciencias, Madrid 28049, Spain
* Author for correspondence (e-mail: agbellido{at}cbm.uam.es)
Accepted 3 October 2002
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SUMMARY |
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Key words: Drosophila, vestigial, Wing development, Inductive assimilation, Cellular identity, `Mixed' tissues
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INTRODUCTION |
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vg encodes a nuclear protein of 453 amino acids with poor
homologies to other known proteins. It is expressed at low levels in the
primordial wing and haltere imaginal discs
(Williams et al., 1991). Later
in the development of both discs, the dorsoventral border of compartment is
defined by differential expression of apterous (ap)
(Diaz-Benjumea and Cohen,
1993
), and subsequent restricted activation of Notch
(N) (Irvine and Vogt,
1997
) and downstream genes such as wingless (wg)
(Kim et al., 1995
). The
activity of wg and N in the wing margin leads to the
expression of the vg through the activation of the vg
boundary enhancer (vg BE-lacZ)
(Kim et al., 1996
;
Williams et al., 1994
).
Subsequent to vg BE-lacZ activation, the expression of vg in
more proximal parts of the wing blade is regulated by the
decapentaplegic (dpp) pathway and by vg itself,
acting on the vg quadrant enhancer (vg QE-lacZ)
(Kim et al., 1996
). Reflecting
the activity of the vg enhancers, Vg is expressed in a gradient with
maximal concentrations in the wing margin and minimal in more proximal
territories of the wing (Williams et al.,
1991
). Vg interacts with the product of the gene
scalloped (sd), a protein with DNA recognition motifs
(Campbell et al., 1992
),
forming a transcriptional activation complex
(Halder and Carroll, 2001
;
Halder et al., 1998
). The
Vg-Sd complex is known to regulate the expression of downstream genes involved
in wing development (Halder and Carroll,
2001
; Halder et al.,
1998
; Kim et al.,
1996
; Klein and
Martínez-Arias, 1998
). The absence of Vg, Sd or both causes
lack of cell proliferation in the wing blade region where vg is
expressed (Williams et al.,
1991
; Williams et al.,
1993
). By contrast, the ectopic expression of vg may
cause the appearance of territories with cuticular and genetic expression
patterns that are characteristic of distal wing blade
(Halder et al., 1998
;
Kim et al., 1996
). It is
important to note that the ectopic expression of Sd alone does not induce
tissue transformations. Thus, Sd is necessary to bind the complex Vg-Sd to
DNA, but does not confer tissue specificity by itself
(Halder and Carroll,
2001
).
In order to analyse the capacity and the genetic requirements of the
ectopic expression of vg to initiate and drive the transformation of
tissue towards wing blade identity, we studied the autonomous and non
autonomous cuticular and gene expression patterns that appear after the
ectopic expression of vg during development. We drove vg
ectopic expression using different territorial Gal4 lines (G4/UAS system)
(Brand and Perrimon, 1993) or
by Flip-out (FLP/FRT system) recombination in clones
(de Celis and Bray, 1997
;
Ito et al., 1997
). The results
show that the morphogenetic effects of vg ectopic expression depend
on developmental timing and the genetic specification of a disc territory
where the overexpression takes place.
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MATERIALS AND METHODS |
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We used the following UAS lines: UAS-vgK
(Kim et al., 1996);
UAS-vgZ (Paumard-Rigal
et al., 1998
); UAS-wg
(Lawrence et al., 1995
);
UAS-wg dominant negative (UAS-wgDN)
(Klein and Martínez-Arias,
1999
); UAS-Dll
(Gorfinkiel et al., 1997
);
UAS-homothorax-GFP (UAS-hth-GFP) (provided by Fernando Casares);
UAS-nubbin (UAS-nub)
(Neumann and Cohen, 1998
);
UAS-sd (Campbell et al.,
1992
); UAS-Delta (UAS-Dl)
(Huppert et al., 1997
);
UAS-Serrate (UAS-Ser) and UAS-Notch constitutively
active (UAS-Nintra12.1)
(de Celis and Bray, 1997
);
UAS-thickvein constitutively active (UAS-tkvQ25)
(Lecuit et al., 1996
) or
dominant negative tkv (UAS-tkvDN)
(Haerry et al., 1998
);
UAS-Ras constitutively active (UAS-RasV12)
(Karim and Rubin, 1998
);
UAS-Ras dominant negative (UAS-Raf3.1DN)
(Kim et al., 1995
); UAS-P35
(provided by C. Lehner); UAS-ap
(Fernandez-Funez et al.,
1998
); UAS-fringe (UAS-fng)
(Kim et al., 1995
); and
UAS-GFP.
We used the following lacZ lines: vg QE-lacZ
(Kim et al., 1996); vg
BE-lacZ (Williams et al.,
1994
); wg-lacZ
(Kassis et al., 1992
);
aprk560 (ap-lacZ)
(Diaz-Benjumea and Cohen,
1993
); and sdETX4 (sd-lacZ)
(Anand et al., 1990
).
The lines used for the induction of overexpression mosaics by Flip-out were
y f36a FLP122; abx/Ubx FRT f+ FRT G4 UAS
lacZ (de Celis and Bray,
1997); and y FLP122; Act FRT y+ FRT G4
UAS-GFP: MKRS/ SM6A-TM6B (Ito et al.,
1997
).
Ectopic expression using territorial G4 lines and generation of
overexpression clones
The ectopic expression with different lines G4 was induced at 17, 25 and
29°C. Genetic of Flip-out to induce clones of overexpression of vg,
wg or both wg-vg: larvae were transferred from 25°C to
37°C for 7 minutes at different ages 36±12, 48±12 or
60±12 hours after laying egg AEL for vg clones, and
36±12 and 60±12 hours AEL for the wg-vg or wg
clones.
Inmunohistochemistry
Imaginal disc were dissected in 1xPBS and fixed in 4% PFA at 4°C
for 40 minutes, followed by 3x20 minute washes in 0.3% PBTriton and
3x20 minute washes in PBT-BSA. Incubation with primary antibody was
carried out overnight at 4°C. After repeating washes with PBT and PBT-BSA
the imaginal discs were incubated 2 hours at room temperature with the
secondary antibody. Imaginal discs were mounted in Vectashield.
We used the following primary antibodies: rabbit anti-Dll and anti-Vg (provided by Sean Carroll); mouse anti-Wg (Hybridoma Bank); guinea pig anti-Hth (gift of Fernando Casares); mouse anti-Cut (Hybridoma Bank); mouse anti-Bs (provided by M. Affolter); mouse anti-Nub (provided by S. Cohen); mouse anti-En (Hybridoma Bank); rat anti-Ser (Hybridoma Bank); rat anti-CD2 (Hybridoma Bank); and mouse or rabbit anti ß-gal (Amersham). We used rabbit and mouse Alexa 488, 546, Cy5 and guinea pig-Cy5 as secondary antibodies.
Microscopy and image treatment
For the processing of images in clear field and confocal microscopy we used
the programs Metaview (Meta Imaging Corporation Plus) and Photoshop 6.0 (Adobe
Corporation).
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RESULTS |
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It has been shown that the overexpression of vg driven by
different G4 lines, such as ptch-G4
(Paumard-Rigal et al., 1998)
(Simmonds et al., 1998
),
dpp-G4 (Kim et al., 1996
;
Klein and Martínez-Arias,
1998
; Klein and
Martínez-Arias, 1999
) and Dll-G4
(Fig. 1A,C)
(Halder et al., 1998
;
Weatherbee et al., 1998
),
ectopically induce histotypes and gene expression patterns characteristic of
the wing blade. We explored the maximal transformation phenotypes using the
driver Dll-G4. The ectopic expression of vg in the distal territories
of the appendages (proboscis, first and second pairs of legs and genitalia),
where Dll is expressed, leads to the transformation into tissues with
characteristic cuticular patterns and differentiation of the wing blade
(Fig. 1A,C). In the first and
second pairs of legs, these transformations include typical trichomes,
anteroposterior and dorsoventral wing margin chaetae
(Fig. 1A2-A4, C1-C3), veins and
campaniform sensillae (25/77) (Fig.
1A1,A3). In the third pair of legs, the transformations are to
haltere histotypes (Fig. 1A5)
(Halder et al., 1998
;
Weatherbee et al., 1998
).
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The adult transformations are correlated in imaginal discs with autonomous
expression of genes characteristic of wing blade. These gene expressions only
appear within GFP-expressing cells, as mobilised by Dll-G4. The ectopic wing
margin differentiated in the outgrowths correlates with the expression of
cut (ct) (not shown) and wg in the presumptive wing
margin in the disc (Fig. 1E-G).
Thus, as in the wild-type wing margin, wg represses the expression of
vg QE-lacZ (Fig. 1E).
The transformations have large ventral wing territories and small dorsal
territories encircled by the new wing margin
(Fig. 1A1-4,E,F). The
dorsoventral and anteroposterior transformations are correlated with the
differential expression of en and ap
(Fig. 1A,E,F,G), in the mature
imaginal discs. The expression of en is maintained in the wild-type
topology but the expression of ap is modified
(Fig. 1F,G). Thus, the ring of
ap in the leg is repressed in dorsal leg territories and expanded in
ventral ones (Fig. 1F). In the
first and second leg-transformed territories, the genes characteristic of wing
blade territories, such as spalt (sal), blistered
(bs) (Halder et al.,
1998; Weatherbee et al.,
1998
) and vg QE-lacZ
(Fig. 1G) are expressed. In the
third leg, where transformations are to haltere, some markers of wing
transformation (such as sal or vg QE-lacZ) are not expressed
(not shown), probably because of Ubx activity
(Halder et al., 1998
;
Shashidhara et al., 1999
;
Weatherbee et al., 1998
).
Ubx expression is never modified by overexpression of vg or
wg-vg driven by different G4 lines or in clones (not shown, see
below). All described transformations and expression of wing blade genes are
autonomously restricted to the Dll expression domain, visualised by
GFP.
In contrast to these G4 lines, which induce transformation phenotypes, the ectopic expression of vg driven by G4 lines is not associated with histotypic transformations, and only causes tissue-specific malformations. Thus, with pnr-G4, we observe defects in thorax closure, and with c253-G4 duplications of chaetae in the notum (not shown). We also failed to obtain transformations when the ectopic expression of vg alone in the eye was driven by vg-G4.
These results indicate that expression of vg is necessary but not sufficient for the initiation of the wing blade developmental pathway.
Cooperative effects of vg and wg in the ectopic
transformations
We analyzed why some G4 lines may lead transformations while others fail to
do it driving UAS-vg. We have found that the overexpression of
vg only induces transformations when the G4 line shares expression
domains with high levels of wg in early stages of larval development.
Thus, we have found that the overexpression of vg only induces
transformations when the G4 line shares expression domains with high levels of
wg in early stages of larval development. Furthermore, that
wg is necessary for the augment the transformation is confirmed by
experiments in the leg in which a dominant-negative form of wg
(wgDN) is ectopically co-expressed with vg, using
the driver Dll-G4. In these legs, the transformation is strongly reduced
(Fig. 1B,D). Without
transformation, these legs maintain distal tarsal structures (territories of
the legs with chaetae and without bracts), suggesting that the lack of
transformation is not simply a consequence of the low levels of Wg activity
(Fig. 1B). To test the
hypothesis of collaboration of wg and vg in wing blade
transformation, we co-expressed them in the expression domain of
vg-G4 in the eye (Fig.
2B). Whereas the ectopic expression of vg or wg
alone does not show cuticular transformations (not shown), the co-expression
of both causes wing outgrowths with histotypical characteristics of wing blade
(Fig. 2A). The transformation
is autonomously associated with the expression of wing blade genetic markers
as nub, vg (Fig. 2C,D)
and Dll (not shown). Surprisingly, in the transformed territories the
expression of wg is lower than we would expect of an overexpression
using the G4/UAS system (Fig.
2D) (see Discussion). These results demonstrate that that
wg and vg collaborate to initiate wing development in
imaginal discs other than the wing.
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Other experimental or genetic conditions that affect ectopic
vg transformations
We have explored other experimental conditions that might allow vg
to induce wing transformations.
We searched for other genes, in addition to wg, that might
cooperate with vg, using vg-G4 expression in the eye. First, we
studied: (1) the effects of one or several UAS-vg doses and G4
induction temperatures (not shown); (2) variations in the stoichiometry of Vg
with Sd (Paumard-Rigal et al.,
1998; Simmonds et al.,
1998
) co-expressing several doses of the corresponding UAS; (3)
co-expression of vg with genes involved in the specification of the
proximodistal axes of the wing, such as nub, Dll and hth
(Abu-Shaar and Mann, 1998
;
Azpiazu and Morata, 2000
;
Cifuentes and Garcia-Bellido,
1997
); (4) co-expression with genes involved in dorsoventral wing
margin specification, such as ap
(Diaz-Benjumea and Cohen,
1993
) and fng (Irvine
and Wieschaus, 1994
); (5) co-expression of vg with UAS
p35 to rescue possible cell death (Hay et
al., 1994
); and (6) co-expression of vg with several
constructs of genes that are involved in signalling pathways during
development, such as UAS-Dl, UAS-Ser,
UAS-Nintra12.1, UAS-tkvQ25,
UAS-RasV12, UAS-tkvDN,
UAS-Raf3.1DN.
Secondly, we tested whether the size of the territory of ectopic expression was critical in the process of transformation, overexpressing vg with other G4 lines such as pnr-G4 and c253-G4.
We failed, in all instances, to enhance the histotypic transformation caused by overexpressing vg alone, and therefore conclude that neither the extension of the territory ectopically expressing vg nor the amount of overexpression is significantly relevant to the extent of transformation to wing. Thus, only wg in collaboration with vg seems to be specifically relevant in the promotion of wing development.
Phenotypes induced by the ectopic expression of vg in
clones: temporal and genetic limitations to the initiation of a wing
developmental program
Gene expression driven by a given G4 line occurs simultaneously in all the
cells of the territory at a given developmental stage, allowing collaborative
effects between cells. By contrast, the ectopic expression in clones provides
temporal and positional limits to the transformation. In clonal mosaics,
individual mutant cells are confronted with wild-type cells, allowing the
study of autonomous and non-autonomous effects in cell proliferation,
cuticular patterning and gene expression. We have monitored the cuticular and
genetic effects of vg ectopic expression in Flip-out clones [labelled
with forked (f) or GFP], in the wing, haltere, leg and
eye-antenna imaginal discs.
In the wing blade and wing hinge, vg clones induce tubular,
perpendicular outgrowths to the wing surface
(Fig. 3A). The outgrowths
include vg-expressing cells and surrounding wild-type cells
(Fig. 3A). vg clones
are frequently located at the tip of the outgrowth but they can also grow
along the lateral zones (Fig.
3A). In those clones that appear in central parts of the wing, all
the cells of the outgrowth and the clone always show a differentiation
corresponding to wing blade trichomes (Fig.
3A). Clones near the wing margin may differentiate marginal
chaetae (not shown). The size of the clones and the non-autonomous part of the
outgrowth depend on their distance to the wing margin
(Liu et al., 2000).
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vg clones in the presumptive wing blade do not modify the
wild-type expression of genes expressed, such as Dll, bs and
nub (not shown). However in the wing hinge, clones of vg
overexpression autonomously repress proximal genes such as hth
(Fig. 3C)
(Azpiazu and Morata, 2000;
Casares and Mann, 2000
;
Liu et al., 2000
) and activate
antagonist distal genes such as Dll
(Azpiazu and Morata, 2000
;
Liu et al., 2000
), vg
QE-lacZ (Fig. 3D),
nub (Liu et al.,
2000
) and bs (Fig.
3C; Table 1)
(Liu et al., 2000
). In some
cases, the ectopic expression of distal genes may appear non-autonomously
outside the clone, up to a distance of several cell diameters
(Fig. 3B,C;
Table 1)
(Liu et al., 2000
). Vg, or the
vg enhancer lacZ reporters, are never detected
non-autonomously in vg clones located outside of the wing blade.
Whereas early vg clones can show co-expression with wg (not
shown), later in development, wg expression is displaced outside of
the clone several cell diameters (Fig.
3A) (Liu et al.,
2000
). The absence of wing margin cuticular elements in adult
vg clones and ct or vg BE-lacZ
(Fig. 3E;
Table 1) expression in the
discs suggests that the clones are specified as wing blade, not including wing
margin territories. vg clones in the wing imaginal disc show a
correlated and autonomous expression of sd-lacZ within the clone
(Fig. 3F). In tissues other
than the wing, the ectopic expression of vg may drive the expression
of its transcriptional partner (not shown)
(Halder and Carroll, 2001
;
Halder et al., 1998
).
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In territories other than the wing, vg clones show transformations toward wing histotype only when they are initiated in specific positions and stages of development. Thus, ectopic expression of vg in clones is only associated with wing outgrowth phenotypes when it is initiated in territories that normally express high levels of wg (Fig. 4C, Fig. 5C,H). For example, vg clones in the notum, only show wing histotype when they are initiated very early in development (36±12 hours AEL), whereas in the eye and leg imaginal discs, transformations may appear later (48±12 hours AEL). Clones initiated later in development or in territories with low levels of wg expression cause cuticular abnormalities (Fig. 5B) but not transformations towards wing histotype.
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In tissues other than the wing, clones of vg ectopic expression associated with transformation differentiate only wing blade trichomes (Fig. 4A,B, Fig. 5A,G), in contrast to the overexpression of vg with G4 lines in the same territories. The wing blade trichomes in some tissues such as notum or legs may appear `mixed', with tissue-specific chaetae in a `salt and pepper' distribution (Fig. 4B, Fig. 5A). The adult `mixed' cuticular patterns are correlated for each examined tissue with the specific expression of some wing blade genes (Table 1). Thus, we never detected the expression of nub in the notum, whereas it is induced in the leg or eye imaginal discs (Fig. 5I); and bs is never detected in the eye, whereas it is induced in the leg (Fig. 5D). Paradoxically, in contrast to `salt and pepper' distribution of the adult cuticular structures detected in the transformations, the ectopic expression of specific wing blade genes only occurs in subsets of cells within the clones (Fig. 4D, Fig. 5D,E). Moreover, the cells expressing wing blade genes are compacted and located anywhere within the clones of vg (Fig. 4D, Fig. 5D,E). The discrepancy between cuticular pattern and gene expression suggests that vg can not displace all endogenous identity signals or, alternatively, that there are non-autonomous influences of surrounding cells on of the clone expressing wing blade genes.
In tissues other than the wing, the histotype transformations and expression of wing blade gene are cell autonomous. But in the notum, vg clones that straddle the DV boundary and are initiated in territories with high levels of wg (Fig. 4C) and occasionally cause non-autonomous expression of wing gene markers such as bs (Fig. 4D). This phenomenon of non-autonomous induction of tissue to express wing blade genes, we called `inductive assimilation' (see Discussion). In these clones, the wild-type expression of wg is displaced, but where ap expressing and non-expressing cells are confronted in the clone, wg expression is autonomously enhanced, as in the wing margin (Fig. 4C). This reflects the possibility that vg clones may recruit the expression of ap before the wild-type specification of DV wing margin, generating ectopic DV wing margins.
As in the G4 experiments, the expression of en is not modified in vg clones, and the disc therefore retains the embryonic AP compartment specification. However, ap expression is altered in clones expressing ectopically vg in a tissue-specific way. Thus, ap expression is not modified in the wing imaginal disc or haltere, whereas in leg discs, ap expression is induced in ventral territories and repressed in dorsal ones (Fig. 5E,F).
Similar to en expression, Ubx is not modified in clones expressing ectopically vg or vg and wg simultaneously (wg-vg), and therefore, the segmental identity dependent on Ubx expression is maintained. In the haltere, vg or wg-vg clones have otherwise the same autonomous and non-autonomous effects than in the wing (not shown).
The activity of wg pathway together with vg is
insufficient to promote a wing developmental program in clones
In order to test the interaction of the wg pathway with
vg in clones, we have studied the cuticular and gene expression
patterns shown by cells expressing either vg or wg alone,
and co-expressing wg and vg (wg-vg) simultaneously.
The phenotypes of wg or wg-vg clones have been monitored in
the wing, haltere, leg and eye-antenna imaginal discs.
In the wing, the overexpression of wg in clones does not cause
outgrowths and all cells of the clone differentiate into wing margin sensory
elements (not shown). These results confirm the proposition of Klein and
co-workers (Klein et al.,
1997; Klein and
Martínez-Arias, 1998
) that the overexpression of
wg induces the cells to acquire characteristics of wing margin.
However, these cells fail to express vg BE-lacZ or other wing margin
genes such as ct (not shown). In these clones, some wing blade
markers such as Dll (not shown)
(Zecca et al., 1996
),
vg (Fig. 6B)
(Zecca et al., 1996
) or
nub (Fig. 6C) are
autonomous and non-autonomously expressed, whereas other wing blade genes such
as bs are autonomous and non-autonomously repressed (not shown).
|
In the notum, overexpression of wg in clones is not associated with histotype transformations or `mixed' tissues (Fig. 6A), but may autonomously and non-autonomously express wing blade genes such as Dll (not shown) and nub (Fig. 6C) (Table 1). In wg clones, the non-autonomous gene expression is restricted to the nearest surrounding cells of the clone (see below and Discussion). The absence of transformation detected in these clones overexpressing wg is correlated with the absence of vg expression (Fig. 6B).
In the eye or leg imaginal discs, overexpression of wg in clones
usually does not activate the ectopic expression of vg or show
cuticular transformations (Table
1), but causes specific cuticular perturbations and gene
expression alterations as shown elsewhere
(Lee and Treisman, 2001;
Royet and Finkelstein, 1997
;
Struhl and Basler, 1993
;
Theisen et al., 1996
).
In the wing blade, the co-expression wg-vg in clones leads to the formation of tubular and perpendicular outgrowths. The size of the outgrowths is larger than in vg clones and is dependent on the distance from the wing margin (Fig. 7A,B). All the cells of the wg-vg clones are differentiated into wign margin sensory elements, as occurs in wg clones, whereas non autonomous territories of the outgrowth are differentiated into wing blade trichomes (Fig. 7A,B). In contrast to clones of wg, in which we detect homogenously high levels of Wg (Fig. 6D), clones of wg-vg show low levels of Wg in some cases (Fig. 7E; Table 1), which are still sufficient to promote the autonomous differentiation of wing margin sensory elements. wg-vg clones do not express vg BE-lacZ or ct (not shown) (Table 1). In the wing blade, the activation or repression of wing blade genes is equal to that observed in the wg overexpression clones.
|
In contrast to clones of either vg or wg alone, wg-vg clones in the wing hinge and notum cause transformation phenotypes everywhere. All the cells autonomously differentiate into wing margin and non-autonomously differentiate into wing blade trichomes (Fig. 7C). These transformations are correlated with the autonomous and non-autonomous expression of the wing blade genes studied (Fig. 7D,F,G; Table 1). In wg-vg clones, the expression of vg and other wing blade genes is autonomous and non-autonomous, but, in wg-vg clones the non-autonomous expression is extended to larger cell distances surrounding the clone than in wg clones (compare Fig. 6C with Fig. 7F and Table 1). In contrast to vg clones, the expression of bs in wg-vg clones is reduced, possibly because of their genetic specification as similar to cells of the wing margin region (Fig. 7G; Table 1).
In imaginal discs other than the wing, the co-expression of wg-vg in clones shows `mixed' phenotypes (Fig. 8A,B) and gene expression specificities similar to clones expressing vg ectopically (Fig. 8C,D; Table 1), again revealing regional restrictions to the induction of transformations and specific limitations of tissue to activation of wing blade gene expression. In contrast to clones of wg alone, wg-vg clones show transformation phenotypes, probably because of the presence of vg expression. As in vg overexpression clones, wg-vg clones modify neither ap nor en expression (Table 1). These results suggest that the co-expression of wg-vg remains insufficient to promote a wing developmental program outside the wing imaginal disc.
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DISCUSSION |
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The ectopic expression of vg or wg-vg in clones may cause outgrowths with wing histotypic characteristics or patterning perturbations in the notum, leg or eyes. The transformed tissues show `mixed' phenotypes or `mosaic' territories where, in a `salt and pepper' distribution, wing blade trichomes co-exist with notum or leg chaetae. Adult cuticular `mixed' phenotypes are correlated with the ectopic expression of wing blade genes in particular combinations (Table 1). However, expression of wing blade genes is detected only in some compact groups of cells within the clones. These results indicate that either vg or wg-vg are insufficient by themselves to displace all endogenous signals of identity, or that reciprocal non-autonomous influences between clonal cells and surrounding cells exist, reducing the expression of wing blade genes to groups of cells within clones. The change of wing blade genes expression in compact groups of cells in the disc and `mixed' (salt and pepper) cuticular phenotypes in the adult could result from cell interactions during patterning and cell rearrangements in pupal stages.
Transformations induced by overexpression of vg or wg-vg in clones and G4 territories are, as a rule, cell autonomous, except in the wing hinge, notum and corresponding tissues in the haltere. In the wing hinge the cells of the outgrowths outside the vg clones differentiate into wing blade territories and show gene expression patterns characteristic of the wing blade cells located between the proximal vg expression and the internal ring of wg in the wild-type disc. This suggests that the non-autonomous effects in vg clones could reproduce the wild-type intercalary growth induced by the confrontation of cells expressing proximal genes with distal genes. In the notum, vg clones located simultaneously in territories expressing and not expressing ap, and initiated in the wg expression domain, may non-autonomously recruit surrounding cells to express characteristic wing blade genes at long cell distances, as wg-vg clones do. Thus, vg together with wg expression is necessary to induce and extend the transformation over long distances outside the clones. In contrast to vg or wg-vg clones, wg clones do not show non-autonomous transformation phenotypes and expression of wing blade genes at long distances. The issue of whether the recruitment process is caused by Wg diffusion, or whether it results from intercalary growth induced by the confrontation between cells expressing proximal genes (genes of the notum) and cells expressing distal genes (wing blade genes), remains unresolved.
The expression of selector genes like Ubx and en is not modified by overexpression of vg or wg-vg, but is inherited and maintained. However, the expression of the selector gene ap can be modified or inherited in some tissues, such as the legs, to give DV identity.
The comparative analysis of vg with other morphogenetic genes suggests that vg acts as Dll, pnr or iro, rather than as a `master' or `selector of tissue' gene: vg is simply a component of the genetic combination that is necessary to initiate and drive wing blade development where vg is normally expressed. Interestingly, the function of vg, in addition to conferring territorial identity, may also non-autonomously recruit surrounding cells (`inductive assimilation'), changing their specific cuticular and gene expression patterns. This is related to its function as a local organiser of growth when it is expressed among cells with different positional or regional fates. Later in development, vg, in combination with other genes, activates an inventory of downstream wing genes that specify more discrete territories within the wing blade such as veins, interveins and sensory elements.
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ACKNOWLEDGMENTS |
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