Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain
* Present address: Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK
Author for correspondence (e-mail: jmodol{at}cbm.uam.es)
Accepted 22 May 2002
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SUMMARY |
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Key words: Iroquois complex, Dpp signalling, Imaginal wing disc, Notum development, Drosophila melanogaster
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INTRODUCTION |
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Still, the earliest subdivisions of the wing discs do not correspond to these territories but to compartments defined by cell lineage restrictions (García-Bellido et al., 1973) (reviewed by Mann and Morata, 2000
). The first subdivision, into anterior (A) and posterior compartments (P), is inherited from the embryo and it is established by the expression of the selector genes engrailed and invected in the P compartment. A dorsal-ventral (DV) compartmental subdivision, ortogonal to the AP one, occurs during the early-mid second instar. This subdivision is established by the expression of the gene apterous (ap) in the D compartment. The expression of a selector gene confers identity to the cells of a compartment. Cells from apposing compartments do not intermingle because of differential affinities. In addition, compartment borders are sources of signalling molecules that organize both cell proliferation and patterning of the entire disc (for reviews, see Brook et al., 1996
; Teleman et al., 2001
; Vincent and Briscoe, 2001
).
According to current thinking (reviewed by Klein, 2001), the subdivision of the wing disc along the proximal-distal axis into body wall (notum) and appendage (wing) is effected, during the early second instar, by the Wingless (Wg) and the EGFR signalling pathways. The Wg molecule accumulates in the most distal part of the disc (Couso et al., 1993
; Ng et al., 1996
) and instructs cells to repress the ubiquitously expressed zinc-finger transcription factor gene teashirt (tsh) (Wu and Cohen, 2002
) and activate wing-specific genes like nubbin (nub), vestigial (vg) and scalloped (sd) (Ng et al., 1996
; Williams et al., 1993
), thus specifying the wing blade territory. Specification of the medial region of the disc, which will give rise to the dorsal wing hinge, requires Wg, homothorax and tsh, although it is unclear how the actions of these genes are integrated (Azpiazu and Morata, 2000
; Casares and Mann, 2000
; Klein and Martínez-Arias, 1998
). More is known about the specification of the proximal region of the disc that gives rise to the notum. Here, the neuregulin Vein (Vn) molecule is expressed, activates the tyrosine kinase EGF receptor, and this autonomously turns on notum genes like the three members of the Iroquois Complex (Iro-C) (Simcox et al., 1996
; Wang et al., 2000
; Zecca and Struhl, 2002a
; Zecca and Struhl, 2002b
). The Iro-C genes, araucan (ara), caupolican (caup) and mirror (mirr), encode related homeodomain proteins conserved from worms to vertebrates (reviewed by Cavodeassi et al., 2001
). Their expression in the most proximal region of the second instar wing disc is essential for notum specification, since clones of Iro-C cells induced early within this territory acquire the identity of the adjacent distal region, namely, the proximal wing hinge and differentiate structures characteristic of this region (tegula, sclerites, etc) (Diez del Corral et al., 1999
). Thus, the early domain of expression of Iro-C defines the extent of the notum territory.
An antagonistic interaction between the Wg and the EGFR pathways might explain the confinement of Iro-C expression to the proximal region of the wing disc. Thus, it has been proposed that, in the second instar disc, Wg signalling represses vn. Since Wg signalling is strongest in the distal part of the disc, it should restrict expression of vn to the proximal part of the disc (Wang et al., 2000). Then, activation of the EGFR pathway would be maximal in this territory and this would activate Iro-C within it. Hence, the early domain of expression of Iro-C, and therefore the prospective notum territory, would ultimately be delimited by the negative input of Wg emanating from the distal region of the disc.
In this work, we further analyze the control of the early expression of Iro-C in the proximal region of the wing disc. Our findings argue against Wg signalling being the main negative regulator of Iro-C expression. Moreover, while we confirm that the Vn/EGFR signalling pathway is necessary for Iro-C activation, our data, in agreement with recent findings (Zecca and Struhl, 2002a), indicate that the availability of Vn does not restrict Iro-C expression to the prospective notum. This appears to be accomplished, instead, by signalling mediated by the BMP2/4 homolog Dpp. dpp is expressed in a stripe of A cells abutting the AP compartment boundary from very early larval stages (Burke and Basler, 1996
), an expression essential for growth and patterning of the wing disc in its AP axis (reviewed by Affolter et al., 2001
; Dahmann and Basler, 1999
; Podos and Ferguson, 1999
; Serrano and OFarrell, 1997
). However, Dpp had not been implicated, until now, in the initial territorial subdivision of the disc along the proximal-distal axis. We find that during the early-mid second larval instar, the Dpp pathway is active only in the wing and hinge territories. This activity defines, by repression, the distal border of the Iro-C domain and confines the expression of Iro-C to the notum territory. Later, dpp becomes expressed in the most proximal part of the notum and turns off Iro-C in this region. This downregulation is associated with the subdivision of the notum into medial and lateral regions (Calleja et al., 2000
).
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MATERIALS AND METHODS |
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Histochemistry
Imaginal discs were dissected and stained as described previously (Gómez-Skarmeta et al., 1995). Primary antibodies were: rabbit and mouse anti-ß-galactosidase (Cappel and Promega); rat anti-Iro-C (Diez del Corral et al., 1999
), which reacts with the Araucan and Caupolican proteins; rabbit anti-pMad (Tanimoto et al., 2000
); mouse anti-Omb (a gift from G. O. Pflugfelder); mouse anti-Wg, mouse anti-Nub and rabbit anti-Tsh (kindly provided by M. S. Cohen); guinea pig anti-Senseless (a gift from H. J. Bellen); mouse anti-Ptc (from I. Guerrero). Secondary antibodies were from the Jackson Laboratory and Amersham. Probe to detect vn mRNAs was prepared using the DIG RNA labelling kit (Roche), as described by the supplier. Whole-mount in situ hybridizations were performed essentially as described previously (Jiang et al., 1991
).
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RESULTS |
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With the aid of the Gal4 system, we confirmed that EGFR activity was necessary to activate notal Iro-C expression (Wang et al., 2000; Zecca and Struhl, 2002a
; Zecca and Struhl, 2002b
). Indeed, blocking EGFR signalling in the notum territory (UAS-rafDN/MS-248Gal4) inhibited Iro-C expression (Fig. 2G). This suggested that the availability of EGFR activity might define the distal limit of the Iro-C domain. However, overactivation of the EGFR pathway in proximal/intermediate regions of the disc by misexpressing UAS-vn or UAS-ras1V12 (a constitutive activation of the EGFR pathway) with several different drivers did not expand the Iro-C domain (Fig. 2D-F, compare with Fig. 4A,E). The inability of the misexpression of vn to modify the Iro-C domain of late third instar discs has recently been reported (Zecca and Struhl, 2002a
). We concluded that the EGFR signalling pathway, as activated by the Vn ligand, while important for Iro-C activation is not the main candidate to delimit the domain of Iro-C expression and, therefore, the notum territory.
The Dpp pathway is mostly active in the distal regions of the early wing disc
In third instar wing discs, the expression of dpp in both proximal and distal territories (see below) does not suggest a function in regulating the domain of Iro-C. However, in the second instar disc dpp is expressed in distal regions but it is absent from the Iro-C domain (Fig. 3A,B) (see also Burke and Basler, 1996; Masucci et al., 1990
). Dpp is a diffusible molecule and, therefore, we determined its range of activity by monitoring the phosphorylated form of the Mad protein (pMad), an intermediate of the Dpp transduction pathway (Tanimoto et al., 2000
). pMad accumulated in the cells near the source of Dpp, but it was reduced or absent within the Iro-C domain (Fig. 3C,D). Another useful indicator of Dpp activity is the type I TGFß receptor Thick veins (Tkv), since its expression is negatively regulated by Dpp signalling (Lecuit and Cohen, 1998
). In addition, high levels of Tkv can limit Dpp diffusion and help to confine the region in which the pathway will be activated (Lecuit and Cohen, 1998
). We find that the Iro-C domain is located within a region of high accumulation of Tkv (Fig. 3E,F), a result compatible with Dpp activity being strongly reduced or absent from that domain.
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The Dpp pathway downregulates Iro-C in the medial notum
During the third instar, after Iro-C has specified the prospective notum, dpp is turned on in this territory and helps effect its patterning (Mullor et al., 1997; Sato and Saigo, 2000
; Tomoyasu et al., 2000
) (Fig. 5E,F, arrows). The activation of dpp in the proximal-most region of the prospective notum is accompanied by a gradual removal of Iro-C (Fig. 5E,F), a repression essential to specify the medial versus the lateral notum (Calleja et al., 2000
). Dpp was responsible for this downregulation, since it was prevented by decreasing (dppd12/dppd14 mutant; compare Fig. 4D with 4E,G) or abolishing (clones mutant for a null tkv allele; Fig. 5G) Dpp signalling. In contrast, constitutive activity of the Dpp pathway in cell clones autonomously inhibited Iro-C in the lateral notum, except in a region overlapping or very close to an endogenous source of Dpp (compare Fig. 5F with 5H; see also 5C, arrowhead). Thus, while in the medial notum there is a correspondence between Dpp expression and Iro-C repression, this correlation does not hold everywhere in the lateral notum, where the appearance of Dpp expression may not result in turning off Iro-C (Fig. 5F, arrowhead; Fig. 5H, circled). Interestingly, vn is also maximally expressed in the region of overlap of dpp and Iro-C expressions (Simcox et al., 1996
) (our unpublished data), and might antagonize, through the activation of EGFR signalling, the repression of the Iro-C genes by the Dpp pathway. We conclude that, in the third instar disc, the levels of Dpp signalling are critical to establish the medial-lateral subdivision of the notum by its negative regulation of Iro-C in the medial region. This negative regulation should be mediated by pannier (Calleja et al., 2000
), which is activated by dpp in the medial notum (Sato and Saigo, 2000
; Tomoyasu et al., 2000
).
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DISCUSSION |
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Indeed, we find that in the second instar disc dpp is expressed only in its distal regions and that the activity of the Dpp pathway, as measured by pMad accumulation, is reduced in or absent from the proximal part of the disc, where Iro-C is expressed. When Dpp signalling is reduced or abolished in dppd12/dppd14 discs or tkva12 clones, respectively, Iro-C expression is distally expanded. Conversely, increased dpp expression or constitutive activation of the pathway (UAS-tkvQD clones) repress Iro-C expression within the prospective notum. These data, together with previous findings (Wang et al., 2000), suggest that the Iro-C domain of expression, and hence the notum territory of the wing disc, is defined during the early larval stages by two antagonistic signals: Vn/EGFR, which activates Iro-C, and Dpp, which constrains its expression to the proximal region of the disc by repressing it in the neighbouring (hinge) territory (Fig. 6). While we have confirmed that Vn/EGFR is necessary for Iro-C activation, the localised expression of vn does not appear to be instrumental in confining Iro-C expression to this territory, since ectopic Vn does not significantly expand the Iro-C domain. After submission of this manuscript, Zecca and Struhl (Zecca and Struhl, 2002b
) similarly reported that misexpression of vn does not modify Iro-C expression. However, these authors additionally show, using overexpressing cell clones (Zecca and Struhl, 2002b
), that a constitutively activated form of EGFR can autonomously activate Iro-C in particular subregions of the prospective hinge, and that RasV12, a presumably stronger activation of the pathway, can do so anywhere within the prospective wing hinge and even in some clones within the wing blade. While we have not observed ectopic activation of Iro-C with UAS-ras1V12 expressed with several Gal4 lines, probably because we have used milder overexpressing conditions (i.e., larvae were cultured at 18°C, since in our hands they died at higher temperatures), these observations indicate that, at least in third instar wing discs, sufficiently strong activation of the EGFR/Ras pathway is able to activate Iro-C almost anywhere in the disc. Zecca and Struhl (Zecca and Struhl, 2002a
; Zecca and Struhl, 2002b
), based on these observations and in the absolute necessity of EGFR activity for Iro-C expression and notum development (Simcox et al., 1996
; Wang et al., 2000
; Zecca and Struhl, 2002a
; Zecca and Struhl, 2002b
), propose that this pathway would be maximally activated in the proximal region of the disc and would thus turn on and maintain Iro-C expression in the prospective notum. However, direct comparative measurements of the activity of the pathway (for instance, by examining the levels of phosphorylated MAP kinase) (Gabay et al., 1997
) in the prospective notum and in other region of the second and third instar wing disc have not been performed. Thus, it is not clear whether there is indeed a gradation of EGFR pathway activity along the proximal-distal axis of the disc (Wang et al., 2000
; Zecca and Struhl, 2002a
; Zecca and Struhl, 2002b
) and, if present, whether it does help effect the notum/hinge subdivision. Regardless of its presence and functional significance, our data indicates that the Dpp pathway, by negatively regulating the expression of Iro-C, is an important player in establishing the early distal border of the Iro-C domain and, therefore, the notum-hinge subdivision. In the wild type, this repression would be sufficient to counteract the activation by the EGFR pathway and prevent Iro-C expression in the hinge. However, strong experimental overactivity of the EGFR pathway would overrule the repression by Dpp and allow ectopic expression of Iro-C in the hinge and wing (Zecca and Struhl, 2002a
; Zecca and Struhl, 2002b
). Whether these two pathways act antagonistically and in parallel on the Iro-C or negatively regulate each other is not known. Note, finally, that in the early dppd12/dppd14 discs, Iro-C expression, while occurring in an expanded domain, is still excluded from the prospective wing territory (Fig. 4B). This suggests the presence of additional repressors and/or the absence of essential activators within this domain.
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Signalling pathways in notum development
From very early in the development of the wing disc, the Hedgehog (Hh) signalling pathway is active in a stripe of anterior cells adjacent to the AP compartment border. This stripe extends from the proximal-most part of the disc to the tip, as revealed by Hh targets like ci and ptc (Dahmann and Basler, 1999; Podos and Ferguson, 1999
; Serrano and OFarrell, 1997
) (our unpublished data). Surprisingly, we have observed that in these early discs dpp, another target of Hh, is not activated in their proximal region. This suggests the presence of uncharacterized negative regulators that block transcription of dpp in this territory. Although the complementary domains of Iro-C and pMad (Fig. 3C,D) might suggest that Iro-C could be one of these negative regulators, we find that misexpression of the Iro-C gene araucan does not downregulate dpp (F. C., unpublished).
Hh, Dpp and Wg signalling pathways intervene in the patterning of both the body trunk and the appendages of Drosophila. However, it is clear that the cellular responses to these signals are different in the mesothoracic body wall versus the wing and the legs (reviewed by Morata and Sánchez-Herrero, 1999). Indeed, in the wing disc, the pathways are essential for the specification and growth of the wing, but not so for the notum, which can develop to a large extent in the absence of these signals. Hh, Dpp and Wg are required for late events such as the notal medial/lateral subdivision and bristle patterning (Morata and Sánchez-Herrero, 1999
). Our findings reinforce this view since the Dpp pathway appears to be inactive within the notum territory during its specification (it only helps delimit its extent). Thus, to our knowledge, only the EGFR pathway is indispensable for the specification and growth of the notum.
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ACKNOWLEDGMENTS |
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