Developmental Biology program, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
* Present address: Mount Sinai School of Medicine, 1 Gustav Levy Place, New York, NY, USA
Author for correspondence (email: cohen{at}embl-heidelberg.de)
Accepted 26 February 2002
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SUMMARY |
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Key words: Imaginal disc, Teashirt, Homothorax, Wing, Pattern formation, Drosophila melanogaster
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INTRODUCTION |
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Wg and Dpp are also required for wing development, but how they specify distal wing fate is less well understood. The wg1 mutant provides the most striking evidence for an early role of Wg in specification of the wing (Sharma and Chopra 1976; Morata and Lawrence 1977
). wg1 mutant flies often replace the wing with a duplication of the thorax. This is reflected in the imaginal discs by loss of expression of the wing pouch marker Nubbin and uniform expression of the body-wall marker Teashirt throughout the disc (Ng et al., 1996
). Using temperature-sensitive alleles of wg, it has been possible to show that this requirement is fulfilled during second instar (Couso et al., 1993
; Williams et al., 1993
; Ng et al., 1996
). Wg is initially expressed in a ventroanterior wedge in the wing disc. At this stage, Wg represses expression of the neuregulin-like ligand Vein in the ventral region of the disc (Schnepp et al., 1996
; Wang et al., 2000
). Vein activates the EGF receptor and controls the localized expression of Apterous, to specify dorsal cell identity. Vein does not regulate Wg expression but does suppress wing development in the notum, apparently by blocking responsiveness of notum cells to Wg. Ectopic expression of Wg in the notum causes transformation to wing fate (Ng et al., 1996
). Activation of Apterous in turn leads to activation of Notch signaling in cells along the dorsoventral (DV) boundary and to induction of wg and the vestigial boundary enhancer (Diaz-Benjumea and Cohen 1995
; Couso et al., 1995
; Rulifson and Blair, 1995
; Kim et al., 1995
; Kim et al., 1996
; de Celis et al., 1996
; Doherty et al., 1996
).
The antagonistic interaction between Vein and Wg in early stages is necessary for the separation of wing and notum, but is not sufficient to explain how the wing field is specified. Vein limits the ability of Wg to induce wing fate in the notum, but does not explain how the size and shape of the wing field are defined by ventrally expressed Wg. Control of Vestigial expression has been proposed to be an important step in this process (Klein and Martinez-Arias, 1998). To date the earliest positively expressed marker for wing fate is the POU-homeodomain protein Nubbin, which is induced in the presumptive wing field under Wg control in late second instar (Ng et al., 1995
; Ng et al., 1996
). At this stage, Vestigial is expressed in both presumptive wing and body wall territories. Wg activity also represses expression of Teashirt and Homothorax. Repression of Tsh is important for the establishment of distal wing fate, as ectopic expression of Tsh blocks the Wg expression at the DV boundary and interferes with wing pouch development (Casares and Mann, 2000
). Repression of Hth in the distal region is also important. Ectopic expression of Hth in the wing pouch causes defects in the wing (Ryoo et al., 1999
; Azpiazu and Morata, 2000
; Casares and Mann, 2000
). We examine the earliest stages of wing specification with reference to the expression of Nubbin, Tsh and Hth. We find that repression of Tsh in response to Wg and Dpp signaling occurs well in advance of both the onset of Nubbin expression and repression of Hth, which must therefore be considered secondary events in determination of the wing.
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MATERIALS AND METHODS |
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Fly stocks
nub1 is a regulatory mutant of nubbin that affects expression in the wing disc (Ng et al., 1995), vg83b27r is a null allele of vestigial (Williams et al., 1993
). act>CD2>Gal4 is described in (Pignoni and Zipursky, 1997
). apUG035 is a null allele of ap (Cohen et al., 1992
). UAS-armS10 was described in (Pai et al., 1997
). UAS-tkv* (Lecuit et al., 1996
) and UAS-Tsh (Wu and Cohen, 2000
) have been described previously. wg1 is a disc specific regulatory allele (Sharma and Chopra 1976
). wg17B40lacZ is a lethal allele caused by insertion of a P-element lacZ reporter (FlyBase).
Clonal analysis
nubbin mutant clones were induced at 72- to 96-hour-old larvae of genotype wHSflp; 30A nub1 FRT40A/arm-lacZ FRT40A. 30A homozygotes are viable and do not have defects in the wing. vestigial mutant clones were induced at 48-72 hours AEL in larvae of genotype wHSflp; FRT42 vg83b27r/FRT42 arm-lacZ.
Flip-out clones
armS10-expressing clones were induced at 24-48, 48-72 and 72-96 hours (first, second or third instar stages) in larvae of the genotype y w act>CD2>Gal4/y w HSflpI; UAS-armS10/UAS-GFP. tkv*-expressing clones were induced in second and in third instar between 48-72 hours in larvae of the genotype: y w act>CD2>Gal4/y w HSflpI; UAS-tkv*/UAS-GFP.
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RESULTS |
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As Hth expression retracts from the wing pouch, it resolves into three distinct domains in the proximal region (Fig. 2D,E). Two of the Hth domains overlap with the proximal rings of Wg expression that are observed in the presumptive wing hinge region (Fig. 2F) (Neumann and Cohen, 1996a). It has been reported previously that Hth is regulated by Wg at these late stages (Azpiazu and Morata, 2000
; Casares and Mann, 2000
). Both rings of Wg expression are distal to the Tsh expression domain (Fig. 2G). The proximalmost ring of Hth, which is regulated by secreted Wg, overlaps the edge of the Tsh domain (arrow Fig. 2E). At this stage, Vestigial and Nubbin expression are centered on the stripe of Wg expression at the DV boundary. Vestigial expression is limited to the distal wing pouch and does not extend as far as the first ring of Hth expression (Fig. 2F). Nubbin extends more proximally, overlapping the first and second rings of Hth and the first ring of Wg expression (Fig. 2G). Tsh expression is proximal to the outer ring of Wg expression, which runs through the base of the wing hinge (Neumann and Cohen, 1996a
). Thus, the border of Tsh expression coincides with border between wing and the body wall, whereas Hth is expressed in rings in the wing hinge as well as more proximally in the notum.
Vestigial and Nubbin do not repress Tsh in the wing pouch
The nascent wing pouch is first marked by the repression of Tsh during the second instar. This appears to occur before Wg expression is activated at the DV boundary (Fig. 1). This would suggest that Wg expression at the DV boundary is unlikely to be responsible for repression of Tsh. To test this, we examined Tsh expression in apterous null mutant discs. apterous activity is required to initiate signaling between dorsal and ventral compartments and to induce Wg expression along the DV boundary. Earlier expression of Wg in the ventral anterior wedge is not affected in apterous mutant discs (Ng et al., 1996). Tsh expression is repressed normally in the rudimentary wing pouch of apterous mutant discs (Fig. 3A), indicating that Wg expression at the DV border is not required in order for Tsh to be repressed. Vestigial expression is also not induced at the DV border in apterous mutant discs (Williams et al., 1993
), suggesting that Vestigial may not be required for Tsh repression. Consistent with this, we find that Tsh expression is repressed normally in the wing pouch of vestigial null mutant wing discs (Fig. 3B). Likewise, clones of cells mutant for vestigial did not show ectopic expression of Tsh, though they did show ectopic expression of Hth (Fig. 4A). The vestigial mutant clones were induced in early second instar, prior to specification of the wing pouch. Comparable results were obtained with clones induced later in development, during second or early third instar and examined in mature third instar discs (not shown) (Azpiazu and Morata, 2000
). We also tested clones expressing Vestigial for repression of Tsh. As expected, clones expressing Vestigial did not affect the level of Tsh expression, but reduced Hth levels to some extent (Fig. 4B). Taken together, these observations indicate that Vestigial contributes to repression of Hth, but not to repression of Tsh.
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Wg and Dpp repress Tsh at early stages
Repression of Tsh appears to be the first identifiable step in specification of wing fate. Previous reports have indicated that ectopic expression of Tsh in the wing pouch interferes with wing development. For example, Wg expression at the DV boundary was strongly reduced by Tsh expression, whereas ectopic expression of Hth had a much weaker effect on Wg expression (Casares and Mann, 2000). Ectopic expression of Tsh in the wing pouch also induced Hth expression in some cells (Casares and Mann, 2000
; Azpiazu and Morata, 2000
). Wg expression at the DV boundary is necessary for growth and patterning of the wing pouch (Zecca et al., 1996
; Neumann and Cohen, 1996a
; Neumann and Cohen, 1997
), so repression of Tsh is necessary to allow subsequent wing development.
How is Tsh repressed in the wing during early wing development? We have previously reported that ectopic expression of Wg can repress Tsh in the notum and lead to ectopic wing formation and that wg1 mutants cause uniform expression of Tsh throughout the mature wing disc (Ng et al., 1996). This suggested that Wg activity is required in second instar to repress Tsh. To verify this, we examined Tsh expression in early wg1/wg17en40lacZ discs and found that Tsh was not repressed (Fig. 5A,B). We note that wg17en40lacZ-expressing cells also expressed Tsh in these discs. This indicates Tsh is indeed ectopically expressed in wg-expressing cells. Tsh was not repressed at all in two out of six second instar wg1/wg17en40lacZ wing discs. Tsh was incompletely repressed in two out of six wing discs and repressed normally in two out of six discs. This is consistent with the frequency with which adult wings were missing and replaced by duplicated notum structures in this genotype (26/80 possible wings were affected in 40 wg1/wg17en40lacZ adult flies). Replacement of the wing by a duplication of notum structures is obvious in mature third instar discs (Fig. 5C).
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vein is another candidate for mediating the effects of Wg signaling on Tsh expression. vein is a ligand for the EGF receptor that is normally expressed in the presumptive notum region and is required for the specification of dorsal and notum fate (Wang et al., 2000). Specification of the wing pouch by Wg correlates with repression of vein expression in the ventral region of the wing disc. We note that although vein is not expressed in the ventral pleural region of the wing disc, Tsh expression is also repressed in ArmS10-expressing clones in this region (Fig. 6D, double arrowhead). Thus, we consider it unlikely that the repression of Tsh by Wg signaling is mediated by repression of Vein. This view is supported by the observations of Wang et al. (2000
), who found that Tsh is expressed in the notum of Egfrts and vnts mutant wing discs.
In view of the finding that Wg and Dpp work together to induce Dll and repress Hth in the leg disc, we wondered whether Dpp signaling is also required for Tsh repression in the wing. To test this, we expressed a constitutively active form of the Dpp receptor Thickveins in clones of cells (Tkv*) (Lecuit et al., 1996). Tsh expression was repressed in some Tkv*-expressing clones in the lateral region of the disc when clones were induced in early second instar (Fig. 7) (larvae dissected 3 days after clone induction). Hth expression was also repressed by Tkv* at this stage, but to a lesser extent. Clones induced in late second-early third instar larvae did not repress Tsh (not shown). These observations suggest that Dpp signaling cooperates with Wg signaling in repression of Tsh in the nascent wing pouch.
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DISCUSSION |
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We have presented evidence that repression of Tsh in the earliest phase of wing specification appears to be required for subsequent Notch-dependent induction of Wg at the DV boundary. Casares and Mann (Casares and Mann, 2000) have reported that clones of cells lacking Hth activity cause outgrowth of extra wing tissue along the DV boundary. Our results suggest that this is unlikely to be due to an early role of Hth in specification of the size of the wing field because Hth is expressed in the early presumptive wing well after Tsh is repressed. Instead, Hth appears to act in the second stage in conjunction with Tsh to limit the region in which Notch can activate Wg at the DV boundary. Wg expression at the DV boundary extends proximally into the domain of Hth expression in the anterior wing hinge but does not extend into the Tsh domain. In ectopic expression experiments, Hth has a limited ability to repress Notch-dependent activation of Wg on its own, but is able to do so when co-expressed with Tsh (Casares and Mann, 2000
). These observations support the view that Hth cooperates with Tsh during later stages to repress Wg activation. We note that this does not exclude a role for Hth as a co-factor in conjunction with Tsh at earlier stages, but if so, the positional information would seem to derive from regulation of Tsh expression.
Interestingly, Hth and Tsh can also repress the vestigial quadrant enhancer, which depends on Wg and Dpp signaling in phase 3 (Casares and Mann, 2000). Homothorax, Tsh and Vestigial appear to form a loop of mutual repression at this stage, as Vestigial also represses expression of Hth (Azpiazu and Morata, 2000
). Together, these observations suggest that Wg and Dpp have a complex regulatory interaction with Hth. Their activities repress it in the pouch, perhaps through activation of Vestigial and Scalloped. At the same time, the outer rings of Wg expression are required for Hth expression in the wing hinge (Casares and Mann, 2000
; Azpiazu and Morata, 2000
). We suggest that regulation of Hth may be secondary to regulation of Tsh in specification of the wing field.
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ACKNOWLEDGMENTS |
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