Department of Genetics and Development, Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA
*Author for correspondence (e-mail: struhl{at}cuccfa.ccc.columbia.edu)
Accepted 7 December 2001
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SUMMARY |
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Key words: EGFR signaling, Pattern formation, Wing imaginal disc, Drosophila, Compartment boundary, Organizing activity
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INTRODUCTION |
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We show in the accompanying paper (Zecca and Struhl, 2002) that both the DV and wing-notum subdivisions are governed by Epidermal Growth Factor Receptor (EGFR) signaling, via the early heritable activation of ap and by the continuous maintenance of Iro-C gene expression. We now examine the identity and source of the signal(s) responsible for controlling both segregations, as well as the mechanisms by which these signals control the overall growth and patterning of the wing disc.
Our results implicate Vein (Vn), one of three known ligands for the EGFR, in the activation of both ap and Iro-C gene expression, and indicate that it is normally provided by cells located in the dorsoproximal region of the disc. However, ectopic expression of Vn in other regions of the disc does not induce ectopic ap or Iro-C expression, nor is it able to restore dorsal expression of these genes in the absence of endogenous Vn activity. These results indicate that Vn functions as a permissive rather than an instructive factor in establishing the DV and wing-notum subdivisions. For example, Vn may normally have to be processed by, or act in conjunction with, other dorsally expressed factors to generate the instructive EGFR ligand. Alternatively, Vn-EGFR signaling may be inhibited by factors expressed in more ventrally situated cells.
Our results also suggest an unexpected mechanism for controlling growth and patterning of the wing. Although both the ap and Iro-C genes are first expressed within overlapping dorsal domains of the early wing disc, the boundary of ap expression then shifts ventrally relative to that of Iro-C expression, indicating that the level of EGFR signaling declines in D compartment cells as they proliferate and move out of the range of the instructive EGFR signal. We find that this reduction is essential for D and V compartment cells to interact to form a new and stable source of organizing signals, such as the morphogen Wingless (Wg), which control subsequent wing development.
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MATERIALS AND METHODS |
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The genotype for vn mutant larvae are as follows: (i) y w hsp70-flp; ap-lacZ/+; Tub1>flu-GFP, y+>Gal4 UAS-rho vnL6/vndddRY. (ii) y w hsp70-flp; Tub
1>flu-GFP, y+>Gal4 UAS-rho vnL6/vndddRY mirr-lacZ. (iii) y w hsp70-flp; UAS-vn (or UAS-ap); Tub
1>flu-GFP, y+>Gal4 vnL6/vndddRY mirr-lacZ. (iv) y w hsp70-flp UAS-GFPnls; UAS-RasV12/+; Tub
1>Gal80, y+>Gal4 vnL6/vndddRY mirr-lacZ.
lacZ reporter lines and antibody staining
lacZ reporter lines and antisera used are described in the accompanying paper (Zecca and Struhl, 2002), except for wgen11 (Kassis et al., 1992
), a wg-lacZ reporter used only here (Fig. 3C). Imaginal discs from wandering third instar larvae were fixed and stained following standard protocols.
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RESULTS |
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To assess a possible role for Vn in organizing ap and Iro-C gene expression, we first asked whether ectopic Vn expression altered the normal domains of ap and the Iro-C gene mirror (mirr). Clones of cells expressing Vn under Gal4/UAS control were induced during the first larval instar (Materials and Methods). However, we could not detect any effect of such forced Vn expression on either ap and mirr-lacZ expression, even when multiple clones were scattered throughout a single disc (data not shown).
We next examined the consequences of ectopically expressing Vn in wing imaginal discs devoid of endogenous vn activity. vn mutant wing discs proliferate very poorly, if at all and do not express either ap or Iro-C genes (Simcox et al., 1996; Wang et al., 2000
). However, vn mutant discs that contained multiple Vn expressing clones appeared similar to wild-type discs in size as well as the patterns of ap and mirr-lacZ gene expression (Fig. 1A). Moreover, single Vn expressing clones could confer at least partial (Fig. 1B), if not extensive (Fig. 1D), rescue of growth as well as ap and mirr-lacZ expression in vn mutant discs.
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Although Vn must be expressed dorsally to rescue mirr-lacZ and ap expression in vn mutant discs, Vn-expressing cells exert their rescuing activity non-autonomously. mirr-lacZ gene expression was restored not only in Vn-expressing cells but also in nearby, non-expressing cells over a range of at least 5-10 cell diameters (Fig. 1B-D). ap expression was also rescued in non-expressing cells, although in this case, the boundaries of ap expression could extend at least 20-30 cell diameters away from the clone border (Fig. 1B,D). However, as we show in the accompanying paper (Zecca and Struhl, 2002), ap expression depends on EGFR signaling only during early wing disc development, in contrast to Iro-C gene expression, which requires continuous EGFR input. Hence, the boundaries of ap expression in the mature disc do not reflect the direct response of cells to Vn activity at this late stage. Instead, we infer that Vn-expressing clones rescue ap expression at shorter range during early wing disc development, committing the descendents of these cells to continue to express ap, even if they subsequently move out of the range of Vn-dependent signaling.
In summary, Vn expression in the dorsal portion of the disc is necessary for the normal activation of the ap and mirr-lacZ genes. However, ectopic expression of Vn in the ventral portion of the disc does not induce ectopic ventral expression of either gene, nor does it restore their normal, dorsal expression in the absence of endogenous vn gene function. We conclude that Vn serves a permissive rather than instructive role in the activation of both ap and the Iro-C genes (see Discussion).
Ectopic activity of the EGFR ligand Spitz can organize ap and Iro-C gene expression
The spitz (spi) gene is expressed in most or all ectodermal cells, yielding a membrane bound, but inactive, EGFR ligand (Rutledge et al., 1992; Sturtevant et al., 1993
). This inert form of Spi is then processed to generate the active, secreted EGFR ligand, an event that is promoted by the transmembrane protein Rhomboid (Rho) (Schweitzer et al., 1995
; Golembo et al., 1996
; Bang and Kintner, 2000
) and normally occurs only late in wing disc development, when Rho is first expressed (Sturtevant et al., 1993
; Simcox, 1997
; Guichard et al., 1999
; Culi et al., 2001
). To assess whether localized production of an EGFR ligand is sufficient to organize the normal patterns of ap and Iro-C gene expression in the wing disc, we generated marked clones of Rho-expressing cells to create ectopic sources of active Spi during early wing disc development.
Initially, we examined the effects of Rho-expressing clones generated during the first larval instar in otherwise wild-type discs. Unlike clones of Vn-expressing cells, which have no effect on normal wing disc development, clones of Rho-expressing cells can cause dramatic reorganization, including the formation of ectopic D compartments (Fig. 2A) and ectopic notum primordia (Fig. 2B), or the abnormal expansion of the D compartment (Fig. 2B,C). Rho-expressing clones were typically associated with ectopic ap expression that can extend at least 10-20 cell diameters beyond the clone borders (Fig. 2B,C), whereas the non-autonomous effects on mirr-lacZ expression were more restricted, extending, at most, 5-10 cell diameters into neighboring, wild-type tissue (Fig. 2B). As already discussed for the rescuing activity of Vn-expressing clones, we attribute this difference in the range of the ap and Iro-C gene responses to the distinct early and continuous roles for EGFR signaling in directing their expression.
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Thus, the behavior of Rho-expressing clones provides a proof of principle that localized production of an active EGFR ligand is sufficient to organize the subdivision of the wing disc into DV compartments and wing-notum primordia. vn mutant discs apparently lack the capacity to generate such an instructive EGFR ligand, and hence fail to develop unless provided with exogenous Vn or Rho activity.
Reduced EGFR/Ras signaling is required for the DV compartmental interface to acquire organizer activity
During normal development, ap and the Iro-C genes are initially activated in overlapping dorsoproximal domains in response to EGFR/Ras signaling (Williams et al., 1993; Diez del Corral et al., 1999
; Wang et al., 2000
). However, the domain of ap expression then expands ventrally relative to that of Iro-C gene expression, into a region of low EGFR/Ras signaling, and the DV compartment boundary becomes a stable source of Wingless (Wg) and other signals (Williams et al., 1994
; Diaz-Benjumea and Cohen, 1995
; Fleming et al., 1997
; Panin et al., 1997
), which organize the subsequent growth and differentiation of the wing primordium (Zecca et al., 1996
; Neumann and Cohen, 1997
). In the course of extending our analysis of the role of EGFR signaling in organizing both the DV and wing-notum segregations, we also generated clones of cells that express an activated form of Ras (RasV12) in vn as well as wild-type discs. Such clones activate ap and the Iro-C genes in a strictly cell-autonomous manner (Zecca and Struhl, 2002
), and hence present a novel situation in which the boundary of ap expression cannot shift away from that of Iro-C gene expression. As we describe below, the behavior of such clones suggests that during normal development, the DV boundary must shift ventrally, into a domain of relatively low EGFR/Ras activity, in order to acquire wing organizer activity.
We first examined whether the severe proliferation defect associated with vn mutant wing discs could be rescued by inducing RasV12-expressing clones during the first larval instar. In contrast to our results with Vn- and Rho-expressing clones, both of which can dramatically stimulate the growth of vn mutant discs (Fig. 1 and Fig. 2D,E), we obtained only a few vn mutant discs that showed a modest increase in size (Fig. 3A). Moreover, this increase appeared to be due to a cell-autonomous rescue of proliferation within the RasV12-expressing clone, rather than to a long-range, non-autonomous induction of growth, as observed for Vn-expressing and Rho-expressing clones (compare Fig. 3A,B; see also Fig. 1 and Fig. 2D,E). Nevertheless, such RasV12-expressing clones autonomously expressed ap, indicating that the clone border constitutes an ectopic DV compartmental interface (Fig. 3A). We therefore wondered if RasV12-expressing clones fail to rescue vn mutant discs because D cells within the clone cannot interact properly with surrounding V cells to induce organizing signals. Given the technical difficulties of assaying RasV12 clones in vn mutant discs, we chose to test this possibility by analyzing the behavior of RasV12-expressing clones in otherwise wild-type discs.
As we observed in vn mutant discs, RasV12-expressing clones generated in wild-type, first instar discs autonomously expressed ap, but did not appear to stimulate growth or patterning of surrounding cells (Fig. 3C). To assay whether the ectopic DV compartment boundaries associated with such clones have organizing activity, we examined the expression of wg (as visualized by the expression of either Wg protein or a wg-lacZ gene), which is normally induced in a thin stripe of cells flanking the DV compartment boundary by reciprocal signaling interactions between D and V compartment cells. In contrast to the normal DV boundary, we found that wg expression was not induced in ap-expressing and non-ap-expressing cells that abut along the borders of RasV12-expressing clones (Fig. 3C), although low levels of wg expression could sometimes be detected in cells scattered throughout the clone. These clones also blocked normal wg expression when they extended to the endogenous DV boundary (not shown). The results obtained with RasV12-expressing clones contrast with those obtained with Vn-expressing clones in vn mutant discs, as well as with Rho-expressing clones in either vn mutant or wild-type discs. Such Vn- or Rho-expressing clones maintained Iro-C gene expression at short range but induced the formation of larger domains of ap-expressing cells that typically extended several cell diameters further away from the clone, and hence into domains of lower EGFR signaling (Fig. 1, Fig. 2C,D). Like the normal DV compartment boundary, cells that flank the borders of these ectopic ap expression domains expressed Wg (Fig. 3B; data not shown; see also Fig. 2C). Thus, we infer that cells flanking the ectopic DV boundaries associated with RasV12-expressing clones fail to express Wg, because high levels of Ras activation in the ectopic D cells disrupt the reciprocal signaling events normally responsible for Wg induction along the DV boundary.
wg is only one of several genes that are normally induced in cells flanking the DV boundary and are required for the organizing activity of the boundary. Another is vestigial (vg), a selector-like gene that specifies wing blade identity (Kim et al., 1996) and is upregulated both by reciprocal signaling interactions between D and V compartment cells and by Wg signaling in cells located further from the DV boundary (Williams et al., 1994
; Kim et al., 1995
; Kim et al., 1996
; Zecca et al., 1996
). We find that RasV12-expressing clones autonomously fail to express vg (as visualized by the expression of Vg protein, Fig. 3D), providing another indication that high levels of EGFR/Ras activity in D cells interfere with the reciprocal signaling interactions which normally induce the expression of boundary genes, such as vg and wg along the DV compartment interface (see Discussion).
We conclude that the mere presence of an apON-apOFF interface is not sufficient to induce wg and vg expression and organize wing development. Instead, it appears that the apON-apOFF interface must be located in a region of low EGFR signaling to acquire organizer activity, a condition that is satisfied during normal development by the ventral expansion of the D compartment away from the source of high EGFR signaling. In agreement with this, we find that clones of ectopic ap-expressing cells can rescue wing growth and differentiation in vn mutant discs [Fig. 3E; similar observations have also been reported by Wang et al. (Wang et al., 2000)]. Thus, the only requirement for Vn-dependent EGFR signaling in controlling the development of the wing primordium appears to be activation of ap expression and the consequent establishment of the D compartment. Once this occurs, high levels of EGFR signaling are no longer required, but are instead detrimental, as they prevent the DV boundary from acquiring organizer activity.
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DISCUSSION |
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Our results challenge this view. We show that localized expression of Vn in the dorsoproximal region of vn mutant discs is both necessary and sufficient to rescue normal wing development. However, we also demonstrate that ventral expression of Vn fails to rescue the development of vn mutant discs and that indiscriminate expression of Vn throughout the disc does not reorganize ap or Iro-C expression. Taken together, these results indicate that Vn performs a permissive, rather than an instructive, role in controlling ap and Iro-C gene expression. Finally, we demonstrate that ectopic dorsal expression of an active form of Spi can substitute for the absence of vn function during wing disc development. This last result confirms that Vn is normally required for the generation of an instructive EGFR ligand in the dorsal region of the disc, and poses the question of what this ligand is.
One possibility is that Vn itself is the ligand, but that it must be converted from an inert (or weakly active) precursor to an active (or more potent) form. Accordingly, the instructive cue would be a dorsally localized processing or activating factor that executes this conversion, serving a role analogous to that of Rho in activating Spi. This processing activity is unlikely to be Rho itself, because Rho does not appear to be required for either the DV or wing-notum segregations (Simcox, 1997). In principle, a Vn-converting activity could be provided by one of several additional Rho-like proteins that have recently been identified (Wasserman et al., 2000
). However, Vn differs from Spi in that nascent Vn is likely to be a secreted protein, whereas nascent Spi is a transmembrane protein from which the EGF-containing extracellular domain is cleaved and released to generate the active EGFR ligand. Hence, Vn and Spi might be activated by distinct molecular mechanisms, with only Spi and structurally related Spi-like ligands requiring the action of Rho family proteins. Curiously, substitution of the single EGF repeat in Vn by that in Spi creates a potent, Rho-independent ligand for the EGFR (Schnepp et al., 1998
). Hence, Vn might normally be converted to an active form by a mechanism that modifies the structure or conformation of its EGF repeat.
An alternative possibility is that the instructive EGFR signal is a presently unidentified EGFR ligand or co-factor that acts in conjunction with Vn (e.g. by forming a heterodimer with Vn or by potentiating EGFR activity by some other mechanism). Because indiscriminate expression of Vn does not ectopically activate either ap or the Iro-C genes, we would expect that such a ligand or co-factor would be locally expressed in the dorsal region of the disc. At least one additional spi-like gene has recently been identified in the Drosophila genome (Wasserman et al., 2000), providing a possible candidate for a Vn co-ligand. We note that we obtained a single vn mutant disc in which a clone of laterally situated Vn-expressing cells appeared to act non-autonomously to activate ap expression in a nearby, more dorsal patch of cells (Fig. 1C). Although only one such exceptional disc was observed, it raises the possibility that Vn secreted by one cell can act jointly with a factor expressed by other cells to generate the instructive EGFR ligand. Such non-autonomy is consistent with a mechanism in which Vn functions in conjunction with another ligand, or in which Vn is converted from a weak to potent EGFR ligand after it is secreted.
A third possibility is that nascent Vn does not require processing to become an effective EGFR ligand, but instead that its ability to activate the EGFR is restricted to dorsoproximal regions of the wing disc by repressors generated in more ventrally situated cells. Because activated Spi induces both ap and Iro-C gene expression in the ventral region of the disc, such repressors might be selective for Vn itself, rather than being general inhibitors of EGFR activity. Alternatively, activated Spi might be a more potent activator of EGFR activity than Vn, allowing it to drive EGFR signaling in the presence of repressors that render Vn ineffective. Wg could, in principle, be such a repressor, as it is active ventrally and represses Vn-dependent EGFR signaling in early wing discs (Wang et al., 2000). However, during later development, Wg is expressed in a broad stripe in the presumptive notum that overlaps the domain of Iro-C gene expression in the prospective lateral notum. Hence, at least at this stage, Wg does not appear to block the Vn-dependent activation of the EGFR that maintains Iro-C gene expression.
EGFR signaling and the control of growth and patterning of the wing primordium
All cells within the wing imaginal disc require a minimum level of EGFR/Ras activity to sustain a normal rate of proliferation (Diaz-Benjumea and Garcia-Bellido, 1990; Diaz-Benjumea and Hafen, 1994
; Prober and Edgar, 2000
). It is not known whether this activity reflects the basal activity of the EGFR/Ras transduction pathway, or the response of the receptor to a specific ligand. However, it is clear that this low level of EGFR/Ras activity does not require Vn dependent EGFR signaling, as we and Wang et al. (Wang et al., 2000
) have shown that ectopic expression of Ap in vn mutant discs can rescue growth and differentiation of the wing primordium. This result demonstrates that the absence of wing development in vn mutant discs is an indirect consequence of the failure to establish an apON-apOFF interface.
During normal development, the ap and Iro-C genes are initially activated in overlapping dorsoproximal domains in response to EGFR signaling, and hence, at this early stage, it appears that most or all D compartment cells are exposed to relatively high levels of EGFR/Ras signaling (Fig. 4). Thereafter, as the wing disc grows, ventrally situated D compartment cells inherit the on state of ap expression, even as they populate areas of the disc progressively farther from the domain of high EGFR/Ras signaling and sustained Iro-C expression. We suggest that the progressive reduction of EGFR/Ras activity in these ventrally situated D cells enables them to interact with neighboring V compartment cells to induce Wg and Vg expression and stimulate growth of the wing primordium. By contrast, early induced clones of RasV12-expressing cells autonomously express ap and experience persistent high levels of Ras activation, as indicated by sustained expression of the Iro-C genes. As a consequence, the ectopic DV boundary cannot shift outside of the domain of high EGFR/Ras signaling. We show here that cells flanking this ectopic DV boundary fail to engage in the reciprocal induction of Wg and Vg expression or to stimulate growth. Hence, the apON-apOFF interface may normally have to shift to a region of relatively low EGFR activity for the DV boundary to acquire wing organizer activity (Fig. 4).
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How might EGFR signaling regulate the capacity of the DV compartment boundary to function as an organizer? One possibility is that high levels of EGFR/Ras activity block the ability of cells to transduce Notch signals. During normal development, D and V cells engage in a positive auto-feedback loop of Delta/Notch and Serrate/Notch signaling that drives the reciprocal induction of Wg and Vg expression on both sides of the DV compartment boundary (Diaz-Benjumea and Cohen, 1995; Kim et al., 1995
; Fleming et al., 1997
; Panin et al., 1997
). Hence, if high levels of EGFR/Ras activity block Notch signal transduction, then persistent high levels of Ras activity on even one side of the DV boundary would suffice to disrupt the feedback loop and block the reciprocal induction of Wg and other boundary genes. Accordingly, the DV boundary might have to be located in a region of low EGFR activity in order to allow reciprocal Notch signaling to induce the expression of these, and perhaps other, organizer genes.
Another possibility is that the apON-apOFF interface may only be able to function as an organizer when cells on both sides are of prospective wing type. Prior to the initial activation of ap and the Iro-C genes, the nascent wing disc appears to be subdivided into mutually antagonistic domains of EGFR and Wg signaling that at least transiently define the incipient notum and wing primordia (Ng et al., 1996; Wang et al., 2000
). Because ap and the Iro-C genes are initially activated in response to a common source of EGFR signaling, most or all D cells at this stage may be of notum type (Fig. 4). It is only later, when ventrally situated D cells move out of range of Vn-dependent EGFR signaling and switch to being of wing type, that inductive interactions occur across the DV boundary to create a new and stable source of Wg signaling (Fig. 4). We suggest that cells on both sides of the DV boundary may have to be of wing type for the boundary to have organizer activity. One possible reason for why this might be the case is that vg, the selector-like gene that defines the wing state, is itself an integral component of the reciprocal signaling mechanism that allows D and V cells to induce the expression of DV boundary genes (Williams et al., 1994
; Kim et al., 1996
; Guss et al., 2001
). As we show here and in the accompanying paper (Zecca and Struhl, 2002
), high levels of EGFR/Ras signaling actively maintain Iro-C gene expression (and hence the notum state) and block vg expression (Wang et al., 2000
). Hence, the DV boundary may normally have to shift ventrally, into a domain of low EGFR/Ras signaling and high Wg signaling that defines the incipient wing state, to allow the positive feedback loop of inductive signaling to initiate across the DV compartment boundary. Once this loop is established, it would provide a stable source of Wg and other signals generated along the DV boundary that govern the subsequent growth and differentiation of the wing blade (Fig. 4).
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ACKNOWLEDGMENTS |
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