SEARCH

SEARCH BY CITATION

Keywords:

  • cell interactions;
  • signal transduction;
  • cell differentiation;
  • vein formation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The formation of longitudinal veins in the Drosophila wing involves cell interactions mediated by the conserved signaling pathways Decapentaplegic (Dpp), Notch, and epidermal growth factor receptor (EGFR). Interactions between Notch and EGFR taking place in the wing disc divide each vein into a central domain, where EGFR is active, and two boundary domains where Notch is active. The expression of decapentaplegic (dpp) is activated in the veins during pupal development, and we have generated Gal4 drivers using the regulatory region that drives dpp expression at this stage. By using these drivers, we studied the relationships between the Notch, EGFR, and Dpp signaling pathways that occur during pupal development. Our results indicate that the interactions between EGFR and Notch initiated in the imaginal disc are maintained throughout pupal development and contribute to determine the places where dpp is expressed. Once dpp expression is initiated, Dpp and EGFR activities in the provein maintain each other and, in cooperation, determine vein cell differentiation. Developmental Dynamics 232:738–752, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The Drosophila wing is decorated with four longitudinal veins (L2–L5) distributed in constant positions along its anterior–posterior axis. The cells forming the veins have the apical surface smaller than intervein cells, and the cuticle they secrete is more pigmented. Vein patterning is initiated in the larval stage and finalized during pupal development. The development of veins in the wing imaginal disc has been well characterized and involves the subdivision of the wing blade epithelium into provein and intervein regions (reviewed in de Celis, 1998, 2003). The Dpp and Hedgehog (Hh) signaling pathways regulate the expression of both vein- and intervein-specific genes along the anterior–posterior axis of the wing blade (de Celis, 2003). Thus, Hh function directly determines the position of the veins L3 and L4, as well as the spacing between them, by regulating the expression of the iroquois complex (iro-C) and knot (kn) in the L3 provein and the L3/L4 intervein, respectively (Gómez-Skarmeta and Modolell, 1996; Mullor et al., 1997; Vervoort et al., 1999; Mohler et al., 2000; Crozatier et al., 2002). The kn and iro-C genes encode transcription factors that mediate most aspects of Hh signaling in the patterning of the center of the wing (Gómez-Skarmeta et al., 1996; Vervoort et al., 1999; Crozatier et al., 2002).

In addition, Hh signaling activates the expression of dpp in a narrow stripe of anterior cells abutting the anterior–posterior compartment boundary (reviewed in Lawrence and Struhl, 1996). Dpp is secreted and can reach most of the wing epithelium, where it contributes to the patterning of the veins L2 and L5 by regulating the expression of the spalt, knirps (kni), and iro gene complexes (de Celis et al., 1996; Gómez-Skarmeta and Modolell, 1996; Biehs et al., 1998; Entchev et al., 2000; Teleman and Cohen, 2000). Interactions between the transcription factors encoded by these genes confine the expression of kni-C and iro-C to the veins L2 and L5, respectively (de Celis and Barrio, 2000). The expression of these genes in the veins is needed for vein formation and constitutes the first indication of vein fate commitment (Gómez-Skarmeta et al., 1996; Lunde et al., 1998). Simultaneous to the vein-specific expression of iro-C and kni-C, the expression of blistered (bs) becomes restricted to the developing interveins, where it prevents vein formation (Fristrom et al., 1994; Montagne et al., 1996; Roch et al., 1998). The restricted expression of provein-specific genes and the localization of bs expression to the interveins occur as the wing disc increases its size by proliferation, and both are completed before puparium formation.

The formation of veins with the correct width also requires the function of the Notch and epidermal growth factor receptor (EGFR) signaling pathways in the wing imaginal disc. EGFR activity promotes vein formation, whereas Notch signaling prevents it. Several genes belonging to the Notch and EGFR signaling pathways are expressed in provein territories in the wing disc and determine where these pathways are activated. For example, the Notch-ligand Delta (Dl) is preferentially expressed in the vein and activates Notch signaling in adjacent cells, leading to the expression of the Notch-downstream gene Enhancer of split mβ (E(spl)mβ) (Kooh et al., 1993; de Celis et al., 1997; Huppert et al., 1997). Vein cells also express several components of the EGFR pathway, such as rhomboid (rho) and Star, which participate in the activation of EGFR ligands (Sturtevant et al., 1993; Kolodkin et al., 1994; Sturtevant and Bier, 1995; Wasserman and Freeman, 1997; Guichard et al., 1999; Lee et al., 2001). EGFR signaling in the vein leads to the accumulation of phosphorylated mitogen-activated protein kinase (MAPK) and to the expression of the EGFR antagonist argos (arg; Golembo et al., 1996; Gabay et al., 1997; Martin-Blanco et al., 1999). Notch and EGFR signaling are mutually dependent at this stage. Thus, EGFR activity regulates Dl expression in the veins, and Notch signaling represses rho in the adjacent cells (Sturtevant and Bier, 1995; Huppert et al., 1997; de Celis et al., 1997). These interactions create two complementary domains of signaling in each imaginal provein: the vein, where EGFR is active, and the adjacent cells separating each vein from the intervein, where Notch signaling is active (de Celis et al., 1997).

Although during imaginal development the longitudinal veins and interveins are genetically specified, the cellular differentiation of the veins occurs during pupal development. Several changes in gene expression patterns related to the developing veins occur after pupariation. First, the expression of two transcription factors required for vein formation, Ventral veinless (Vvl) and Nubbin, becomes restricted to the veins (de Celis et al., 1995, and data not shown). Second, the expression of dpp is activated de novo in the pupal veins, where Dpp signaling contributes to their differentiation (Yu et al., 1996; de Celis, 1997). It is not clear how changes in the expression of signaling molecules and transcription factors are regulated after puparium formation, nor what interactions maintain signaling activities restricted to particular cellular domains at this stage. The use of temperature-sensitive alleles and heat shock constructs indicates that Notch and EGFR are also involved in vein formation during pupal development (Shellenbarger and Mohler, 1978; Guichard et al., 1999). At this stage, Notch is required to prevent vein differentiation, and EGFR signaling appears to play a novel late role in promoting intervein development (Martin-Blanco et al., 1999). The involvement of Notch, EGFR, and Dpp signaling in vein formation at the pupal stage makes vein development a suitable model system to analyze the relationships between signaling pathways in cell differentiation.

We have studied the contribution of EGFR, Notch, and Dpp signaling to vein formation during pupal development. To overcome the early requirements of these pathways in the establishment of vein territories in the imaginal disc, we generated Gal4 drivers expressed only during the pupal development of the veins. The expression in the developing pupal veins of activated or dominant-negative forms of elements belonging to each of these pathways interfere with vein differentiation in specific ways. The result of genetic combinations between members of different signaling pathways suggests that the roles and interactions between EGFR and Notch initiated in the imaginal disc are maintained throughout pupal development. The relationship between EGFR and Dpp signaling is complex, because activation of each pathway can partially rescue the loss of the other one. We show that once initiated, Dpp signaling in the vein becomes engaged in feedback regulatory loops with both EGFR and the transcription factors that regulate vein and intervein development.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Activation Domains of the Notch, EGFR, and Dpp Signaling Pathways Are Related to Vein Development During Pupal Development

The adult wing has four longitudinal veins (L2–L5), each formed by a characteristic number of cells in width (Fig. 1A). The expression of the transcription factors Vvl and Bs (DSRF) is localized during pupal development to veins and intervein territories, respectively (de Celis et al., 1995; Roch et al., 1998), and only one row of cells along the vein/intervein boundary coexpresses Vvl and Bs (Fig. 1B,C). The domains of activity of the Notch, EGFR, and Dpp signaling pathways, monitored by the expression of E(spl)mβ, argos, and P-Mad, respectively, are related to Vvl and Bs expression during pupal development. Notch activation (E(spl)mβ expression) is restricted to the lateral regions of the veins and extends some cells into the interveins (Fig. 1B,D). In this manner, higher levels of E(spl)mβ expression occur in vein cells expressing only Vvl, in cells expressing both Vvl and Bs, and in the intervein cells adjacent to the vein that express only Bs (Fig. 1B,D). Lower levels of E(spl)mβ are detected in other intervein cells expressing Bs (Fig. 1B,D). Dpp activation, monitored by the expression of the phosphorylated nuclear protein Mad (P-Mad), is limited to the veins and occurs in the same cells as Vvl (Fig. 1E–G). EGFR activation (arg expression) is also restricted to the vein, in a domain complementary to Bs (Fig. 1H,I). The activation of Notch and Dpp shows some overlap in the vein cells adjacent to the interveins (Fig. 1J,K). Although the extent of expression domains changes in time, their relative positions are maintained throughout pupal development (data not shown).

thumbnail image

Figure 1. Expression of P-Mad, argos-lacZ, and E(spl)mβ-CD2 in relation with Bs and Ventral veinless (Vvl) in wild-type pupal wings. A: Adult wild-type wing showing the longitudinal veins (L2–L5). B–D: Expression of Vvl (green), Bs (DSRF, red), and E(spl)mβ-CD2 (E(spl), blue) in a pupal wing 24 hr after puparium formation (APF). The overlap between Vvl and DSRF is indicated by arrowheads. C: Red and green channels showing the domains of Vvl (green) and Bs (DSRF; red). D: Red and blue channels showing the overlap (arrowhead) between the expression of E(spl)mβ-CD2 (E(spl), blue) and Bs (DSRF, red). E–G: Expression of Vvl (green in E,G) and P-Mad (PMad, red in E,F) occurs in the same cells. H: Complementary expression of argos-lacZ (arg, red) and Bs (DSRF, green) in the L4 region of a pupal wing 30 hr APF. I: Tangential section of a pupal wing 40 hr APF, showing expression of argos-lacZ (arg, red) and Bs (DSRF, green). Individual green and red channels are shown below. J,K: Expression of E(spl)mβ-CD2 (E(spl), red in J) and P-Mad (PMad, green in J,K), showing higher accumulation of E(spl)mβ-CD2 in cells with lower levels of P-Mad (arrowheads).

Download figure to PowerPoint

Generation and Expression of a Pupal Gal4 Driver

The function of Notch, EGFR, and Dpp are required during both imaginal and pupal development. This makes difficult to discriminate their requirements at these two different stages. In addition, the use of temperature-sensitive alleles or heat-shock constructs has yielded contradictory results in the case of EGFR, and it has been suggested that its activity is required in late pupae to prevent vein differentiation (Martin-Blanco et al., 1999). Furthermore, the phenotypes obtained using temperature-sensitive alleles or heat-shock constructs are generally very weak (Shellenbarger and Mohler, 1978; Sturtevant and Bier, 1995; Yu et al., 1996; Guichard et al., 1999) and could not reveal accurately the requirements of the affected genes. To overcome the early requirements of Notch, EGFR, and Dpp during imaginal disc growth and patterning, we generated a Gal4 driver by fusing regulatory regions taken from the dpp gene to a bicistronic lacZ-Gal4 gene (see Experimental Procedures section). This Gal4 driver (shv3kpn-Gal4) is expressed preferentially in the longitudinal veins L2, L3, L4, and distal L5 from at least 14 hr APF (Fig. 2A) until at least 40 hr APF (Fig. 2B). The expression of βGal is restricted to the veins, but the expression of reporter UAS constructs is also detected at lower levels in some intervein cells (compare Fig. 2C and C′). Thus, maximal expression is present in cells expressing Vvl and not expressing Bs (Fig. 2D–F,G–I). Of interest, the initiation of shv3kpn-Gal4 expression during pupal development is independent of the differentiation of veins, although the levels of expression are modified in response to modifications in EGFR and Dpp signaling (see below). In experiments using the shv3kpn-Gal4 driver, the imaginal development of the veins is normal and the phenotypes of genetic combinations with any UAS line can be unequivocally ascribed to alterations occurring during pupal development.

thumbnail image

Figure 2. Expression of the shv3Kpn-Gal4/LacZ line. A–C′: Expression of green fluorescent protein (GFP, green in A–C) and β-Gal (red in C,C′) in shv3kpn-Gal4-lacZ/UAS-GFP pupal wings 14 hr after puparium formation (APF, A), 40 hr APF (B), and 24 hr APF (C–C′). The expression of β-Gal is strong in veins L2, L3, L4, and distal L5 and weak in the proximal L5 vein. In addition, the expression of GFP driven by the Gal4 protein is also detected in intervein cells localized parallel to the posterior wing margin and in groups of cells in other interveins (arrowhead). D–F: Higher magnification of the L3 and L4 veins, showing the expression of Tau-lacZ (βGal, red in D,E) and Ventral veinless (Vvl, green in D,F) in shv3kpn-Gal4/UAS-taulacZ pupal wings 24 hr APF. E: Arrowheads point to intervein cells expressing the UAS-taulacZ reporter construct. G–I: Expression of tau-GFP (GFP, green) and Bs (red) in shv3kpn-Gal4lacZ/UAS-tau-GFP pupal wings 36 hr APF. H: Higher magnification of the vein L3. I: Tangential section of H showing the complementary domains of Bs and tau-GFP expression in the dorsal (D) and ventral (V) wing surfaces.

Download figure to PowerPoint

Modifications of Notch and EGFR Signaling Activity in the Developing Pupal Veins

The results of ectopic expression in the pupal veins of any member of the Notch or EGFR pathways indicate that their functions in the imaginal disc are maintained during pupal development. Thus, when dominant-negative forms of Notch are expressed in the developing veins, the result is a strong thickening of the veins (Fig. 3A). This finding indicates that the activity of Notch is required to maintain the correct thickness of the veins after this is determined in the imaginal disc, in agreement with data obtained using temperature-sensitive alleles (Shellenbarger and Mohler, 1978). Ectopic expression in the veins of the intracellular fragment of Notch (Nintra) causes the elimination of veins (Fig. 3B) and the ectopic expression of E(spl)mβ in the veins (Fig. 3M). Accordingly, the expression of any E(spl) gene in the veins also prevents vein differentiation (Fig. 3C, and data not shown). These results indicate that Notch activity and E(spl) expression must be confined to the boundary provein cells during pupal development.

thumbnail image

Figure 3. Phenotypic consequences of altered Notch and epidermal growth factor receptor (EGFR) signaling during pupal development. A–C: Adult wings of flies shv3kpn-Gal4/UAS-NDN (N[DN], A), shv3kpn-Gal4/UAS-Nintra (Ni, B), and shv3kpn-Gal4/UAS-E(spl)mβ (E(spl), C). The inset in A shows a higher magnification of the L3 vein, showing several cells differentiating more than one trichome (arrows). D–F: Adult wings of flies shv3kpn-Gal4/UAS-EGFRDN (EGFR[DN], D), shv3kpn-Gal4/UASRasV12 (Ras*, E), and shv3kpn-Gal4/UAS-rafact (Raf*, F). Insets in E and F show higher magnification of the medial region of the L3 and L4 veins, showing abnormal trichome differentiation; arrows indicate the position of the distal L3 sensillum. G: Adult wing of flies shv3kpn-Gal4/UAS-MKP3 (MKP3). H,I: Adult wings of flies shv3kpn-Gal4/UAS-EGFR (EGFR, H) and 1348-Gal4/UAS-EGFR (1348/EGRF, I). Note that expression of EGFR in the interveins (Gal4-1348; de Celis, 1997) causes ectopic veins similar to those of shv3kpn-Gal4/UAS-EGFR. J–L: Expression of P-Mad (P-Mad, green in J,K) and E(spl)mβ (E(spl), red in J,L) in shv3kpn-Gal4/UAS-EGFR pupal wings (24 hr after puparium formation [APF]). Ectopic domains of expression appear in the interveins (arrows in J) in positions related to the expression of the shv3kpn-Gal4 driver. M: Expression of E(spl)mβ-CD2 in shv3kpn-Gal4/UAS-Nintra pupal wings 24 hr APF showing ectopic expression within the veins L3 and L4 (arrows).

Download figure to PowerPoint

Changes in EGFR activity during pupal development also have strong effects on vein formation. Dominant-negative versions of EGFR, or its downstream effectors Ras and Raf, eliminate veins (Fig. 3D, and data not shown), whereas overexpression of activated-Ras and Raf cause the formation of thicker veins (Fig. 3E,F). Some stretches of the vein L4 are lost after overexpression of activated-Raf (Fig. 3F). Why the L4 vein responds differentially to the missexpression of activated-Raf is not known but has also been observed in other experimental situations (Martin-Blanco et al., 1999). As expected, ectopic expression of the MAPK antagonist MAPK phosphatase-3 (MKP3) also causes the elimination of the veins (Fig. 3G). Ectopic expression of normal EGFR causes the formation of veins of normal thickness in the interveins (Fig. 3H). Most likely, these veins differentiate due to the low level of expression of the shv3Kpn-Gal4 driver in the interveins, because the same phenotype results from expression of EGFR in the interveins (Fig. 3I). The ectopic veins formed by misexpression of EGFR have similar expression patterns of E(spl)mβ and P-Mad than the normal veins, suggesting that EGFR activity is able to initiate vein differentiation during pupal development (Fig. 3J–L). In addition to effects on vein formation, ectopic expression of dominant-negative Notch and activated-Ras affect trichome differentiation (insets in Fig. 3A,E). Thus, NotchDN causes many vein cells to differentiate more than one trichome, and activated-Ras affects trichome size and density (cell size), as well as cuticle pigmentation. These data suggest that normal levels of Ras activity are necessary for the correct cellular differentiation of the vein.

To drive expression only in the vein and discard any nonautonomous effect due to the basal intervein expression of the shv3Kpn-Gal4 driver, we used a different shv-Gal4 driver (shv3.7KX-Gal4) expressed only in the L5 vein (Fig. 4A–D). Misexpression of dominant-negative forms of Ras, Raf, and EGFR in the L5 vein eliminates its differentiation, whereas ectopic expression of activated forms of Ras and Raf cause the formation of a thicker L5 vein (Fig. 4E,F, and data not shown). Conversely, dominant-negative forms of Notch produce a thicker L5 vein and activated forms of Notch eliminate this vein (Fig. 4G,H). These results confirm the autonomous requirement of Notch and EGFR signaling in the veins for their formation and differentiation.

thumbnail image

Figure 4. Phenotypic consequences of altered Notch and epidermal growth factor receptor (EGFR) signaling in the L5 vein. A–D: L5 vein-specific expression of the shv3.7KX-Gal4/lacZ driver. A:shv3.7KX-Gal4/lacZ/UAS-tauGFP pupal wings 24 hr after puparium formation, showing the expression of GFP (green in upper panel in A) and βGal (red in lower panel in A). B: Higher magnification of the driver expression in the L5 vein (green fluorescent protein [GFP], green) and Bs (red). C,D: Z-section showing the complementarily expression of Bs (red in C) and GFP (green in C,D). E,F: Adult wings of flies shv3.7KX-Gal4/UAS-EGFRDN (EGFR[DN], E) and shv3.7KX-Gal4/UAS-RasV12 (Ras*, F). G,H: Adult wings of flies shv3.7KX-Gal4/UAS-NDN (N[DN], G) and shv3.7KX-Gal4/UAS-Nintra (Ni, H).

Download figure to PowerPoint

The phenotypic consequences of altered Notch and EGFR activities are correlated with changes in the expression of vein and intervein genes. Thus, Bs expression is present in vein territories that fail to differentiate as a result of Notch activation or EGFR loss-of-function (Fig. 5A,C,D) and is eliminated from the thicker veins caused by dominant-negative Notch (Fig. 5E). Conversely, the vein-specific expression of Vvl is lost from vein territories that fail to differentiate after Notch activation or EGFR inactivation (Fig. 5B,F). The changes in Bs and Vvl expression caused by alterations in Notch and EGFR functions during pupal development indicate that the maintenance of both vein and intervein domains of gene expression is permanently under regulation by these two signaling pathways. The expression of E(spl)mβ and rho is directly regulated by Notch and EGFR, respectively (de Celis et al., 1997; Roch et al., 1998). We observe that changes in Notch or EGFR function modify both E(spl)mβ and rho expression (Fig. 5F–H), indicating that the activities of these signaling pathways are mutually dependent during pupal development.

thumbnail image

Figure 5. Expression patterns in pupal wings with altered Notch and epidermal growth factor receptor (EGFR) signaling. A–C: Expression of Bs (green in A,C) and Ventral veinless (Vvl; red in A,B) in shv3kpn-Gal4/UAS-Nintra pupal wings. Distal L2, L3, and L4 are shown. D: Expression of Bs in shv3.7KX-Gal4/UAS-EGFRDN pupal wings, showing the presence of Bs protein in the vein L5 (arrow). E: Expression of Bs (green) in shv3kpn-Gal4/UAS-NDN pupal wings. F: Expression of E(spl)mβ ((E(spl), green) and Vvl (red) in shv3Kpn-Gal4/UAS-EGFRDN (arrows). G,H: Expression of rho mRNA in shv3Kpn-Gal4/UAS-EGFRDN (G) and shv3kpn-Gal4/UAS-Nintra pupal wings (H). All wings are 24–28 hr after puparium formation.

Download figure to PowerPoint

It has been shown that the activity of Notch is needed to restrict the expression of key components of the EGFR pathway, such as rho, to the vein during imaginal development (Sturtevant and Bier, 1995; de Celis et al., 1997). In addition, mosaic analysis of combinations between Notch and EGFR lethal alleles suggest that the thickened veins caused by Notch insufficiency require EGFR activity (de Celis and Garcia-Bellido, 1994). However, clonal analysis does not inform about the time when this interaction takes place (imaginal vs. pupal development). To study the relationships between Notch and EGFR during pupal development, we analyzed the phenotype of combinations of gain of Notch and EGFR functions and also of loss of Notch and EGFR functions in misexpression experiments. To validate this approach, we first made combinations between several elements of the EGFR pathway whose molecular relationships are well known. As expected, ectopic expression of MKP3 (see Fig. 3G) suppresses the effect of activated-Ras (compare Fig. 6A and B). Ectopic expression of activated-Raf (Fig. 6C) suppresses the loss of veins caused by dominant-negative EGFR (Fig. 6D, compare with Fig. 6C and 3D). In contrast, ectopic expression of the same dominant-negative EGFR suppresses the thicker vein phenotype characteristic of rhomboid expression (Fig. 6E). These results suggest that it might be feasible to infer genetic relationships making use of our shv-Gal4 driver in genetic combinations with elements of independent signaling pathways. In all combinations tested involving activation of Notch (such as Nintra and E(spl) expression, both causing loss of veins) and EGFR (expression of rho and EGFR, causing thicker and ectopic veins), the resulting phenotypes correspond to the activation of EGFR (Fig. 6F,G, and data not shown). The phenotype of combinations where both Notch and EGFR activities are reduced (NDN and EGFRDN) corresponds to that of reductions of EGFR alone (data not shown). Taken together, these results indicate that, during pupal development, Notch contribution to vein formation is to limit the extent of EGFR activation.

thumbnail image

Figure 6. Epistatic relationships between epidermal growth factor receptor (EGFR) and Notch signaling. A–D: High magnification of the distal L3/L4 region of adult wings with modified EGFR signaling. A,B: The phenotype of thicker veins caused by ectopic expression of RasV12 (shv3kpn-Gal4/UAS-rasV12, Ras*, A) is suppressed by coexpression of mitogen-activated protein kinase–specific phosphatase-3 (MKP3, shv3kpn-Gal4/UAS-rasV12+UAS-MKP3; Ras*+MKP3, B). D: The phenotype of vein loss caused by expression of the dominant-negative EGFR (see Fig. 3D) is suppressed by coexpression of activated-Raf (shv3kpn-Gal4/UAS-rafact+UAS-EGFRDN, Raf*+EGFR[DN], D). C: Control wing with expression of activated-Raf (shv3kpn-Gal4/UAS-rafact, Raf*) is shown. E: Suppression of the thicker veins caused by ectopic expression of rhomboid by coexpression of dominant-negative EGFR (Rho+EGFR[DN]). F: Suppression of the vein loss caused by ectopic expression of E(spl)mβ by coexpression of rhomboid (shv3kpn-Gal4/UAS-E(spl)mβ+UAS-rho (E(spl)+Rho). This phenotype is indistinguishable from that caused by ectopic expression of rho alone (not shown). G: Adult wing of shv3kpn-Gal4/UAS-Nintra+UAS-EGFR (Ni+EGFR). This phenotype is very similar to that caused by ectopic expression of UAS-EGFR (see Fig. 3H). H,H′: Expression of Bs (red) and tau-GFP (green fluorescent protein [GFP], green) in shv3kpn-Gal4/UAS-EGFRDNUAS-tau-GFP pupal wings. H′: Longitudinal section along the length of vein L4, showing the coexpression of Bs (red in middle panel) and tau-GFP (green in lower panel), indicating that the expression of the driver is maintained even through vein differentiation is prevented (see Fig. 3D). I: Phenotype of vein loss caused by expression of MKP3 (shv3Kpn-Gal4/UAS-MKP3). J,K: Expression of lacZ RNA in shv3Kpn-Gal4/UAS-MKP3+UAS-tau-lacZ 24–28 hr after puparium formation pupal wings (MKP3+lacZ; J) and shv3Kpn-Gal4/UAS-tau-lacZ pupal wings of the same age (lacZ; K). Note that, although the pattern of the shv3Kpn-Gal driver is retained when vein differentiation is prevented (see I), the levels of expression are clearly reduced.

Download figure to PowerPoint

Of interest, expression of the Gal4 driver used in these experiments remains in the veins when a dominant-negative EGFR receptor is expressed in these territories, even though Bs is expressed there and vein differentiation is prevented (Fig. 6H,H′). However, the levels of expression of the shv3Kpn-Gal4 driver (measured by in situ hybridization to minimize problems of perdurance of the βGal and GFP reporter proteins) are clearly reduced when EGFR activity is compromised (Fig. 6J,K). The existence of trans-effects on the levels of driver expression suggests that the results of genetic combinations should be taken with caution, as a reduction in the levels of Gal4 protein might affect differentially the expression of independent UAS constructs.

Expression of dpp Downstream Genes in Pupal Wings

Dpp signaling plays a central role during vein differentiation in the pupal stage (Yu et al., 1996; de Celis, 1997). The expression of dpp and the domain of Dpp signaling activity in the pupal wing are restricted to the developing veins (de Celis, 1997; see Fig. 1F). Several genes belonging to the Dpp pathway, such as the antagonist dad, the transcription factor Brinker (brk), and the transmembrane receptor Thick veins (tkv) are regulated in part by Dpp signaling in the imaginal disc. Dpp activates the expression of dad in a broad central domain of the wing, whereas the expression of brk and tkv is eliminated or reduced, respectively, in the same central region (Tsuneizumi et al., 1997; de Celis, 1997; Minami et al., 1999). These expression patterns are maintained until 10 hr after puparium formation (Fig. 7A–C). Later, when dpp is expressed in the veins, the expression of tkv is maximal in the borders of the veins (Fig. 7D,D′), and dad expression is activated de novo in broad longitudinal stripes of cells corresponding to the veins (Fig. 7E,E′). At the same time, the expression of brk is eliminated from the veins and becomes restricted to the interveins in a pattern indistinguishable from that of Bs and complementary to E(spl)mβ (Fig. 7F,F′). Therefore, the distribution of dad and brk is related with the presence and absence of P-Mad, respectively, suggesting that Dpp activity contributes to the expression of key components of its signaling pathway during pupal development.

thumbnail image

Figure 7. Expression of tkv, dad, and brk in relation to E(spl)mβ. A–C: Expression of tkv-lacZ (tkv, red in A), dad-lacZ (dad, red in B), and brk-lacZ (brk, red in C) in pupal wings 6 hr after puparium formation (APF). In all panels, the expression of E(spl)mβ-CD2 (E(spl)) is shown in green. D,D′: Expression of tkv-lacZ (tkv, red in D,D′) and E(spl)mβ-CD2 (E(spl), green in D) in 24–28 hr APF pupal wings. E: Expression of dad-lacZ (dad, red in E,E′) and E(spl)mβ-CD2 (E(spl); green in E) in 24–28 hr APF pupal wings. F: Expression of brk-lacZ in 24–28 hr APF pupal wings. D′,E′,F′: Shown are the single red channels.

Download figure to PowerPoint

Consequences of Altered Dpp Signaling in the Veins and Interveins

Because of the requirements of dpp for growth and patterning of the wing disc during imaginal development, it has not yet been possible to assign a direct role for several components of the pathway in vein differentiation. We analyzed whether dad and brk are required for vein formation by taking advantage of the expression of Gal4 in the pupal veins under the regulation of the shv enhancer. Misexpression of dad and brk causes loss of veins, as expected if Brk and Dad are antagonistic to Dpp signaling during pupal development (Fig. 8B,C). Expression of activated-tkv (tkvQD) and dpp in the pupal veins causes the formation of thicker veins and ectopic veins (Tkv* in Fig. 8D), and the differentiation of most intervein tissue as vein (Dpp in Fig. 8E). This last phenotype is accompanied by a reduction in the size of the wing, most likely due to the smaller apical surface of vein cells. Cell size and pigmentation of the ectopic vein tissue is similar to that characteristic of wild-type vein cells, in contrast to the vein differentiation caused by activated-Ras (see Fig. 3E), suggesting that Dpp and EGFR signaling contribute differentially to vein–cell differentiation. The extravein formation due to ectopic expression of dpp could be associated in part to the intervein expression of the Gal4 driver used and not to diffusion of Dpp from the veins (see below). The expression of activated-Tkv in the interveins causes many groups of intervein cells to differentiate into vein histotype (Fig. 8G). This effect is reversed when dad is expressed simultaneously in the interveins (data not shown).

thumbnail image

Figure 8. Phenotypic consequences of ectopic expression of dpp, brk, dad, and tkv in the veins and interveins. A: Wild-type (WT) control wing. B: Ectopic expression of dad in the veins (shv3Kpn-Gal4/UAS-dad, dad). C: Ectopic expression of brk in the veins (shv3Kpn-Gal4/UAS-brk, brk). D: Ectopic expression of activated-Tkv in the veins (shv3Kpn-Gal4/UAS-tkvQD, tkv*). E: Ectopic expression of dpp in the veins (shv3Kpn-Gal4/UAS-dpp, dpp). The overall reduction in size is mainly due to higher than normal cell density. F: Ectopic expression of dpp-GFP in the veins (shv3Kpn-Gal4/UAS-dpp-GFP, dpp-GFP). GFP, green fluorescent protein. G: Ectopic expression of activated-Tkv in the interveins (1348-Gal4/UAS-tkvQD, 1348/tkv*). D,E,G: Insets show higher magnification of the L3–L4 medial region. The position of the distal L3 sensilla is indicated by arrowheads. H,H′: Expression of Bs (red in H,H′) and P-Mad (P-Mad, green in H) in shv3Kpn-Gal4/UAS-dad pupal wings. I: Expression of P-Mad (P-Mad, green) and E(spl)mβ-CD2 (E(spl), red) in shv3Kpn-Gal4/UAS-dad pupal wings. J: Expression of Bs (red) and tau-GFP (GFP, green) in shv3Kpn-Gal4/UAS-dad UAS-tauGFP pupal wings, showing the coexpression of Bs and the driver in wings where vein differentiation is prevented (see B). Transverse sections of this wing are showed in N. K–M: Expression of P-Mad in pupal wings expressing activated-Notch (Kshv3Kpn-Gal4/UAS-Nintra, K), dominant-negative Notch (shv3Kpn-Gal4/UAS-NDN, L), and dominant-negative epidermal growth factor receptor (shv3Kpn-Gal4/UASEGFRDN EGFR; M). All wings are 24–28 hr after puparium formation.

Download figure to PowerPoint

The expression of the vein- and intervein-specific genes vvl and bs is affected by changes in Dpp signaling activity according to the associated changes in vein formation (Fig. 8H,I, and data not shown). Of interest, the reduction of Dpp activity by increased expression of dad has also consequences on the activity of other signaling pathways required for vein development. Thus, the expression of downstream components of both the Notch and EGFR signaling pathways is reduced or eliminated from the veins, indicating that one consequence of Dpp activity is the maintenance of Notch and EGFR signaling in the developing veins (Fig. 8I, and data not shown). The domain of Dpp signaling is also modified when EGFR and Notch activities are affected. Thus, the expression of P-Mad is eliminated from the veins when Notch is activated or EGFR reduced (Fig. 8K,M) and occurs in a broader domain when Notch activity is compromised (Fig. 8L). These effects are most likely related with the modifications of dpp expression in the developing pupal veins associated to changes in the activity of the EGFR pathway (de Celis, 1997). The expression pattern of the driver used in these experiments does not change when Dad prevents vein differentiation (Fig. 8J,N). However, the levels of expression, measured by in situ hybridization, are reduced (see Fig. 10Q), the same result observed in experiments expressing dominant-negative EGFR. These observations indicate that the fragment of the dpp regulatory region used in the Gal4-shv3Kpn construct is inefficiently turned off once it has been activated.

thumbnail image

Figure 10. Genetic relationships among elements the Dpp, epidermal growth factor receptor (EGFR), and Notch signaling pathways. A–L: High magnification of the wings distal end of flies with the following genotypes: wild-type wing (WT, A), shv3Kpn-Gal4 UAS-dpp (Dpp, B), shv3Kpn-Gal4/UAS-dad (Dad, C), shv3Kpn-Gal4 UAS-dad/UAS-dpp (Dpp+Dad, D), shv3kpn-Gal4/UAS-MKP3 (MKP3, E), shv3Kpn-Gal4 UAS-tkv* (Tkv*, F), shv3Kpn-Gal4/UAS-MKP3+UAS-tkv* (MKP3+Tkv*, G), shv3Kpn-Gal4 UAS-EGFRDN/UAS-tkv* (EGFR[DN]+Tkv*, H), shv3Kpn-Gal4 UAS-RasV12 (Ras*, I), shv3Kpn-Gal4 UAS-RasV12+UAS-dad (Ras*+Dad, J), shv3Kpn-Gal4 UAS-Nintra/UAS-tkvQD (Ni+tkv*, K), and shv3Kpn-Gal4 UAS-EGFRDN/UAS-dpp (EGFR[DN]+Dpp, L). The position of the L3 sensillum is indicated in each panel by an arrowhead. The arrow indicates ectopic vein tissue in the intervein. M–O: Expression of Bs (green in M and N) and P-Mad (red in M,O) in shv3Kpn-Gal4 UAS-EGFRDN/UAS-tkvQD pupal wings 24 hr after puparium formation (APF). P: Expression of Bs (green) and P-Mad (red) in shv3Kpn-Gal4 UAS-Nintra/UAS-tkvQD pupal wings 24 hr APF. Note in M and P the complementarity between the expression of Bs and P-Mad. Q: Expression of lacZ RNA in 24–28 hr APF pupal wings of the following genotypes: shv3Kpn-Gal4/UAS-tau-lacZ (above, WT) and shv3Kpn-Gal4/UAS-dad+UAS-tau-lacZ (below, Dad). Note that the pattern of the shv3Kpn-Gal driver is retained when vein differentiation is prevented (see C), although the levels of expression are clearly reduced compared with the wild-type control.

Download figure to PowerPoint

The expression of dpp only in the L5 vein causes the differentiation of a thicker L5 vein (Fig. 9A,B). This phenotype is accompanied by the formation of broader than normal domains of both P-Mad and E(spl)mβ expression (Fig. 9C–E). The cells where E(spl)mβ is not activated, however, remain restricted to the center of the vein, suggesting that Notch signaling activates E(spl)mβ expression over a broader domain due to the presence of P-Mad, even though it is not able to prevent vein differentiation. Of interest, the diffusion of a GFP-tagged Dpp protein from the site of expression is very limited (Fig. 9F–H), suggesting that Dpp is acting as a short-range signal during vein differentiation. The expression of either brk or dad suppress the extravein phenotype caused by ectopic dpp or activated-Tkv (Fig. 10C and data not shown), confirming that both brk and dad act downstream of the activated receptor. Furthermore, the suppression of the dpp ectopic-expression phenotype by cell-autonomous components of its signaling pathway suggests that Dpp diffusion plays a minor role during the pupal development of longitudinal veins.

thumbnail image

Figure 9. Ectopic expression of dpp in the vein L5. A: Phenotype of L5 thickening due to ectopic expression of dpp in the L5 vein (shv3.7KX-Gal4/UAS-dpp). B: Expression of the shv3.7KX-Gal4 line in shv3.7KX-Gal4/UAS-GFP pupal wings. C–E: Expression of E(spl)mβ-CD2 (E(spl), red in C,D) and P-Mad (PMad, green in C,E) in shv3.7KX-Gal4/UAS-dpp pupal wings. F–H: Expression of Dpp tagged with GFP (Dpp-GFP, green in F,H) and LacZ (βGal, red in F,G) in the L5 vein of shv3.7KX-Gal4/UAS-dpp/GFP. LacZ reveals the expression of the driver and is localized in the same cells accumulating Dpp-GFP. GFP, green fluorescent protein.

Download figure to PowerPoint

Functional Relationships Between the Notch, EGFR, and Dpp Signaling Pathways in the Pupal Veins

The functional relationships between the EGFR, Notch, and Dpp signaling pathways during pupal development were further analyzed in misexpression experiments using pair-wise combination of UAS lines with the shv3kpn-Gal4 driver. The expression in the veins of dominant-negative EGFR, MKP3, and activated-Notch causes loss of veins phenotypes (Fig. 10E and see Fig. 3B,D). Ectopic expression of activated-Tkv in these pupal wings rescue to a large extent the formation of veins (Fig. 10G, H, and K, respectively). Similarly, the ectopic veins that differentiate when activated-MAPK is expressed using the shv3kpn-Gal4 driver (shv3kpn-Gal4/UAS-sem; data not shown) do not differentiate when Dpp signaling is reduced (shv3kpn-Gal4/UAS-sem+UAS-dad; data not shown). These results suggest that Dpp is able to trigger vein differentiation when EGFR activity is compromised. However, we also observed a correction of the loss of veins phenotype caused by dad when activated-Ras is simultaneously coexpressed (Fig. 10J, compare with 10C and 10I). Finally, when Dpp and EGFRDN are coexpressed in the veins, we observed partial suppression of vein formation in the position of the normal vein (Fig. 10L, arrowhead) accompanied by ectopic vein tissue in the intervein (Fig. 10L, arrow).

The expression of P-Mad, which is reduced when dominant-negative EGFR or activated-Notch are expressed in the veins, is rescued when activated-Tkv is coexpressed with EGFRDN or Nintra (Fig. 10M, N, and P, respectively). These observations suggest that EGFR and Notch signaling do not affect directly the phosphorylation of Mad. Interestingly, the domains of bs and vvl expression are also modified in all these combinations and became in register with P-Mad, indicating that the main role of EGFR and Notch in this aspect of vein formation is to contribute to the regulation of dpp expression during pupal development (Fig. 10M,O,P, and data not shown for Vvl).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

To interfere with Notch, EGFR, and Dpp function specifically during pupal development, we made use of two Gal4 lines driving expression of different components of these signaling pathways in the pupal veins. In these experiments, the formation of vein and intervein territories in the wing disc occurs normally, which allows us to evaluate unambiguously the functional requirements and interactions taking place during pupal development. One drawback of our analysis is that the expression of these drivers is affected by the genetic background, occurring at lower than normal levels when the function of EGFR or Dpp is reduced. Furthermore, all our phenotypic and expression pattern analysis were carried out in a wild-type genetic background; therefore, the endogenous components of the relevant signaling pathways affecting vein formation are still in place in wings expressing dominant-negative or activated constructs. Modification of endogenous components might affect the phenotypic consequences of misexpression of modified constructs, particularly in a process such as vein formation where feedback loops play a key role. Despite these considerations, we find very strong phenotypes of both loss of and thicker veins, depending on the genetic combination considered. These phenotypes are similar but much stronger than those obtained using temperature-sensitive alleles and heat-shock constructs. Furthermore, there is consistency between the expected and actual results in genetic combinations involving elements of any signaling pathway. Thus, downstream components of the EGFR, Notch, and Dpp pathways always suppress the phenotype of the more upstream components in pair-wise combinations such as Ras*/MKP3, EGFRDN/Raf*, NDN/E(spl), Tkv*/dad. We believe that this consistency validates the use of the drivers, even though the interpretation of the phenotypes can be difficult due both to the behavior of the Gal4 line used and the modification to the endogenous components of each signaling pathway.

Functions of EGFR Signaling During Pupal Vein Development

The interactions between the EGFR and Notch signaling pathways occurring in the imaginal disc are necessary for the differentiation of veins with normal thickness. We find that, in most cases tested, increasing the activity of the EGFR pathway in the pupal veins results in the formation of thicker veins. Conversely, expression of dominant-negative forms of any element of the EGFR pathway throughout pupal development always causes the elimination of the veins. These results are in agreement with previous data using temperature-sensitive alleles and heat-shock constructs (Guichard et al., 1999) and indicate that the main role of EGFR during pupal development is to promote vein differentiation. The expression of activated-Raf causes the loss of some stretches of the L4 vein. This phenotype is surprising because it occurs only in one vein, and it is not observed when activated forms of other components of the EGFR pathway are overexpressed. We do not know why the vein L4 responds differentially to overexpression of activated-Raf. Loss of veins have been observed when the EGFR pathway is activated late during pupal development, and this observation has lead to suggest that EGFR function is required to prevent vein formation at this stage (Martin-Blanco et al., 1999). Because the Gal4 driver we use is expressed throughout pupal development, we cannot support the above interpretation. Rather, we suggest that, in some experimental situations, the use of activated forms of members of the EGFR pathway leads to, contrary to expectation, a reduction in the activity of the pathway. This finding could occur as a consequence of EGFR activating negative regulators of the pathway such as argos, kekkon-1 (reviewed in Perrimon and McMahon, 1999), and MKP3 (Ruiz-Gómez et al., 2004). Under normal physiological conditions, the expression of these negative regulators controls the intensity and duration of signaling (Perrimon and McMahon, 1999). In some experimental situations, however, strong activation of negative regulators could lead to a stronger than normal reduction of signaling. The loss of veins as a consequence of reduced EGFR activity is not related to cell death, because inhibitors of apoptosis such as puckered and p35 do not modify this phenotype (data not shown). Rather, when EGFR is reduced during pupal development, cells specified as veins acquire expression patterns typical of the intervein and differentiate as interveins. In addition to maintaining wing cells committed toward a vein differentiation program through pupal development, EGFR signaling appears to play a role in the actual differentiation of the vein. This finding is based in the morphological alterations to the cuticle observed when the activity of the pathway is highly increased, such as when activated-Ras is expressed in the veins. In this case, vein cells differentiate smaller trichomes that appear densely packed, and their pigmentation is more pallid than that of normal vein cells.

Interactions Between Notch and EGFR in Vein Formation and in Other Developmental Processes

The loss of veins caused by ectopic activation of Notch in the vein or by ectopic expression of the E(spl) genes during pupal development is completely reversed by the coexpression of rho in the same places. Similarly, the thicker veins characteristic of loss of Notch function do not differentiate when EGFR activity is compromised. These observations indicate that Notch and EGFR pathways act antagonistically during pupal development, with EGFR placed downstream of Notch with regard to vein formation. As it happens during imaginal development, the function of Notch would be needed only to draw up the boundaries of EGFR activation through the regulation of key members of the EGFR pathway (de Celis et al., 1997). The antagonism between the Notch and EGFR signaling pathways has been observed in several developmental processes, although the molecular mechanism underlying this interaction is not necessarily the same. For example, EGFR is needed for the singling out of muscle progenitors, whereas Notch restricts the number of these cells (Buff et al., 1998; Halfon et al., 2000; Carmena et al., 2002). It appears that, during the selection of muscle progenitors, contrary to what happens in the veins, sufficiently strong activation of either pathway is capable of overcoming the effect of the other (Carmena et al., 2002). This ability suggests that Notch and EGFR signals act in parallel and must be balanced precisely to permit the proper segregation of muscle progenitors (Carmena et al., 2002). Thus, the logic of Notch/EGFR interactions in the segregation of muscle progenitors appears to be through competition of their transcriptional outputs for a common target gene, rather than the interdependent deployment of complementary spatial domains of signaling observed in the pupal veins.

Dpp Signaling Maintains Vein Commitment During Pupal Development

dpp is expressed and required in the developing veins only during pupal development. The change in dpp expression from the anterior–posterior compartment boundary to the veins brings about a redeployment of several components of the Dpp pathway, such as P-Mad, tkv, brk, and dad to the developing veins. The novel expression domains of these genes are related to P-Mad accumulation and, therefore, to Dpp signaling. Thus, during both embryonic and imaginal development, Dpp regulates the expression of key components of its pathway that in turn influence the levels and domain of Dpp signaling (Jazwinska et al., 1999; Minami et al., 1999; Funakoshi et al., 2001). The expression of dpp in the veins depends on the establishment of vein territories and requires EGFR and Notch activities (de Celis, 1997). In this way, dpp expression prefigures the vein pattern in the pupal wing, acting as a late read-out of the positional information system that localizes the veins. A comparable role is exerted by rho in the imaginal disc (Sturtevant et al., 1993; Sturtevant and Bier, 1995). In contrast to the long-range activity of secreted Dpp during imaginal development, it appears that Dpp in the veins functions as a short-range signal. This finding is based in the similarity between the expression domains of dpp and P-Mad, the rescue of dpp ectopic expression by its downstream antagonist dad and the failure to detect a tagged GFP-Dpp molecule away from the place of synthesis. The accumulation of Tkv in the cells separating the vein from the adjacent interveins is a likely mechanism to limit the diffusion of Dpp in the pupal wing.

Misexpression in the veins of several components of the Dpp pathway allowed us to evaluate their functional requirements during vein differentiation in the pupal stage. Increasing the levels of dad and expressing brk in the veins leads to loss of veins. In contrast, expression of activated-Tkv and dpp causes the formation of ectopic veins that can substitute most of the intervein territories. These results confirm that Dpp is necessary and sufficient to initiate vein differentiation in the pupal wing. Dpp signaling itself, measured by P-Mad expression, is independent of the activity of the EGFR and Notch pathways. In this way, the changes in P-Mad expression observed in pupal wings when EGFR signaling is reduced are reversed when activated-Tkv is expressed in the veins. Therefore, the modifications in P-Mad expression observed in Notch and EGFR mutant backgrounds are caused by changes in dpp expression and not by direct interferences with Mad phosphorylation. The places of P-Mad expression are related with the presence of the vein and intervein transcription factors Vvl and Bs, respectively. This correspondence is observed in wild-type wings and also in all experimental situations where Dpp signaling and P-Mad expressions are modified. The coincidence between P-Mad and Vvl expression domains, as well as the complementarity between P-Mad and Bs expression domains, appears independent of the activities of the EGFR and Notch pathways. Although we cannot exclude a model in which EGFR and Dpp signaling inputs cooperate to regulate vein-specific gene expression during pupal development, we propose that Dpp directly regulates gene expression patterns associated to vein territories during pupal development.

The differentiation of ectopic veins typical of dpp overexpression is not always perfect, as their corrugation is not as well defined as that of wild-type veins. Similarly overexpression of activated-Ras and Raf not only affect vein size, but also vein differentiation, causing weaker pigmentation and defects in trichome morphology. These observations suggest that the cellular differentiation of the veins requires correct levels of both Dpp and EGFR signaling. Cooperation in cell differentiation between these two pathways might underlie the difficulties in determining their hierarchical relationships during pupal development. Thus, genetic combinations between several elements of the EGFR and Dpp pathways gave mainly intermediate phenotypes where clear-cut epistasis cannot be strictly determined. For example, activated-Tkv expression can rescue the loss of veins characteristic of severe reductions of EGFR signaling (MKP3 and EGFRDN), but the resulting phenotypes of these combinations are weaker than the corresponding to expression of activated-Tkv alone. Activated-Ras, which causes a strong phenotype of vein thickening and trichome differentiation, is capable of rescuing to some extent the loss of veins caused by increased dad expression in the veins. Similarly, dominant-negative EGFR can eliminate the normal veins in combination with dpp. We suggest that Dpp and EGFR maintain each other's domains of activity during pupal development, and cooperate in the regulation of the differentiation program characteristics of vein cells. The main contribution of EGFR signaling during earlier stages of pupal development would be to define and to maintain the domains of dpp expression.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Genetic Strains

We have used the reporter lines argos-lacZ, dad-lacZ (Tsuneizumi et al., 1997), brk-lacZ (Minami et al., 1999), tkv-lacZ and E(spl)mβ-CD2 (de Celis et al., 1998), and the Gal4 line Gal4-1348 (de Celis, 1997). We also used the following UAS lines: UAS-GFP (Ito et al., 1997); UAStau-GFP and UAStau-lacZ (Hidalgo et al., 1995); UAS-Necd (Lawrence et al., 2000); UAS-rho and UAS-Nintra (de Celis et al., 1997); UAS-dad (Tsuneizumi et al., 1997); UAS-brk (Minami et al., 1999); UAS-dpp (Staehling-Hampton and Hoffmann, 1994); UASdpp-GFP (Teleman and Cohen, 2000); UAS-tkvQD (Nellen et al., 1996); UAS-EGFR, UAS-EGFRDN, UAS-rafDN, UAS-rasV12, and UAS-rasDN (Buff et al., 1998); and UAS-rafact (Brand and Perrimon, 1993). The MAPK activated form Sevenmaker was also used (UAS-sem; Martin-Blanco et al., 1999). To express MAPK phosphatase 3 (MKP3), we used a P-UAS insert in the 5′ end of the MKP3 gene (Ruiz-Gomez et al., 2004). Unless otherwise stated, crosses were done at 25°C. Wings were mounted in lactic acid-ethanol (1:1) and photographed with a Spot digital camera and a Zeiss Axioplan microscope.

Generation of Shortvein Gal4 Constructs

Two adjacent fragments of 3.7 kb (KpnI/XhoI) and 3 kb (KpnI) from the shortvein (shv) region of dpp were subcloned first in pBS, and the KpnI/XbaI fragments were subcloned in the pZGL vector (Hart and Bienz, 1996). This vector contains a dicistronic Gal4/lacZ gene separated by an internal ribosomal entry site. P-element–mediated transformation (Rubin and Spradling, 1982) was performed with 0.3 μg/μl of DNA of the transforming plasmid and 0.15 μg/μl of pUChsπΔ2-3 as transposase source. The recipient stock was y w1118.

Immunocytochemistry

We used rabbit anti-phosphorylated Mad (P-Mad; Tanimoto et al., 2000) and anti–β-galactosidase (Cappel), mouse monoclonal anti-Bs and anti-CD2 (Serotec), and rat anti-Vvl (Llimargas and Casanova., 1997). Secondary antibodies were from Jackson Immunological Laboratories (used at 1/200 dilution). Pupal wings were dissected, fixed, and stained as described in de Celis (1997). Confocal images were captured using a Bio-Rad confocal microscopy. In situ hybridization was carried out as described in de Celis (1997). In situ using lacZ as a probe were carried out at the same time and developed simultaneously in wild-type and mutant pupal wings.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank R. Barrio, S. Campuzano, A. Glavic, E. Sanchez-Herrero, and J. Sutherland for critical reading of the manuscript. We also thank two anonymous referees for their constructive criticism. J.F.d.C. was funded by a grant from the Ministry of Science and Technology. S.S. was a postdoctoral fellow of the Comunidad Autónoma de Madrid and enjoyed an EMBO short-term fellowship during the initial stages of this work. An institutional grant from the Ramón Areces Foundation to the CBMSO is also acknowledged.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Biehs B, Sturtevant MA, Bier E. 1998. Boundaries in the Drosophila wing imaginal disc organise vein-specific genetic programs. Development 125: 42454257.
  • Brand AH, Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401415.
  • Buff E, Carmena A, Gisselbrecht S, Jimenez F, Michelson A. 1998. Signalling by the Drosophila epidermal growth factor receptor is required for the specification and diversification of embryonic muscle progenitors. Development 125: 20752086.
  • Carmena A, Buff E, Halfon MS, Gisselbrecht S, Jimenez F, Baylies MK, Michelson AM. 2002. Reciprocal regulatory interactions between the Notch and Ras signaling pathways in the Drosophila embryonic mesoderm. Dev Biol 244: 226242.
  • Crozatier M, Glise B, Vincent A. 2002. Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser. Development 129: 42614269.
  • de Celis JF, 1997. Expression and function of decapentaplegic and thick veins during the differentiation of the veins in the Drosophila wing. Development 124: 10071018.
  • de Celis JF. 1998. Positioning and differentiation of veins in the Drosophila wing. Int J Dev Biol 42: 335343.
  • de Celis JF. 2003. Pattern formation in the Drosophila wing: the development of the veins. Bioessays 25: 443451.
  • de Celis JF, Barrio R. 2000. Function of the spalt/spalt-related gene complex in positioning the veins in the Drosophila wing. Mech Dev 91: 3141.
  • de Celis JF, Garcia-Bellido A. 1994. Roles of the Notch gene in Drosophila wing morphogenesis. Mech Dev 46: 109122.
  • de Celis JF, Llimargas M, Casanova J. 1995. ventral veinless, the gene encoding the Cf1a transcription factor, links positional information and cell differentiation during embryonic and imaginal development in D. melanogaster. Development 121: 34053416.
  • de Celis JF, Barrio R, Kafatos F. 1996. A gene complex acting downstream of dpp in Drosophila wing morphogenesis. Nature 381: 421424.
  • de Celis JF, Bray S, Garcia-Bellido A. 1997. Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing. Development 124: 19191928.
  • de Celis JF, Tyler DM, de Celis J, Bray SJ. 1998. Notch signalling mediates segmentation of the Drosophila leg. Development 125: 46174626.
  • Entchev EV, Schwabedissen A, Gonzalez-Gaitan M. 2000. Gradient formation of the TGF-beta homolog Dpp. Cell 103: 981991.
  • Fristrom D, Gotwals P, Eaton S, Kornberg T, Sturtevant MA, Bier E, Fristrom JW. 1994. blistered; a gene required for vein/intervein formation in wings of Drosophila. Development 120: 26612686.
  • Funakoshi Y, Minami M, Tabata T. 2001. mtv shapes the activity gradient of the Dpp morphogen through regulation of thick veins. Development 128: 6774.
  • Gabay L, Seger R, Shilo B-Z. 1997. In situ activation of Drosophila EGF receptor pathway during development. Science 277: 11031106.
  • Golembo M, Schweitzer R, Freeman M, Shilo BZ. 1996. argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop. Development 122: 223230.
  • Gómez-Skarmeta JL, Modolell J. 1996. araucan and caupolican provide a link between compartment subdivisions and patterning of sensory organs and veins in the Drosophila wing. Genes Dev 10: 29352945.
  • Gómez-Skarmeta JL, Diez del Corral R, de la Calle E, Ferrer-Marcó D, Modolell J. 1996. araucan and caupolican, two members of the novel Iroquois complex, encode homeoproteins that control proneural and vein-forming genes. Cell 85: 95105.
  • Guichard A, Biehs B, Sturtevant MA, Wickline L, Chacko J, Howard K, Bier E. 1999. rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development. Development 126: 26632676.
  • Halfon MS, Carmena A, Gisselbrecht S, Sackerson C, Jiménez F, Baylies M, Michelson AM. 2000. Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103: 6374.
  • Hart K, Bienz M. 1996. A test for cell autonomy, based on di-cistronic messenger translation. Development 122: 747751.
  • Hidalgo A, Urban J, Brand AH. 1995. Targeted ablation of glia disrupts axon tract formation in the Drosophila CNS. Development 121: 37033712.
  • Huppert S, Jacobsen T, Muskavitch MAT. 1997. Feedback regulation is central to Delta-Notch signalling required for Drosophila wing vein morphogenesis. Development 124: 32833291.
  • Ito K, Awano W, Suzuki K, Hiromi Y, Yamamoto D. 1997. The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124: 761771.
  • Jazwinska A, Kirov N, Wieschaus E, Roth S, Rushlow C. 1999. The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96: 563573.
  • Kolodkin AL, Pickup AT, Lin DM, Goodman CS, Banerjee U. 1994. Characterisation of Star and its interactions with sevenless and EGF receptor during photoreceptor cell development in Drosophila. Development 120: 17311745.
  • Kooh PJ, Fehon RG, Muskavitch AT. 1993. Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development. Development 117: 493507.
  • Lawrence PA, Struhl G. 1996. Morphogens, compartments, and pattern: lessons from Drosophila? Cell 85: 951961.
  • Lawrence N, Klein T, Brennan K, Martinez-Arias A. 2000. Structural requirements for Notch signalling with Delta and Serrate during the development and patterning of the wing disc of Drosophila. Development 127: 31853195.
  • Lee JR, Urban S, Garvey CF, Freeman M. 2001. Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila. Cell 107: 161171.
  • Llimargas M, Casanova J. 1977. ventral veinless, a POU domain transcription factor, regulates multiple transduction pathways required for tracheal branching in Drosophila. Development 124: 32733281.
  • Lunde K, Biehs B, Neuber U, Bier E. 1998. The knirps and knirps-related genes organize development of the second wing vein in Drosophila. Development 125: 41454154.
  • Martin-Blanco E, Roch F, Noll E, Baonza A, Duffy J, Perrimon N. 1999. A temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing. Development 126: 57395747.
  • Minami M, Kinoshita N, Kamoshida Y, Tanimoto H, Tabata T. 1999. brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes. Nature 398: 242246.
  • Mohler J, Seecoomar M, Agarwal S, Bier E, Hsai J. 2000. Activation of knot specifies the 3-4 intervein region in the Drosophila wing. Development 127: 5563.
  • Montagne J, Groppe J, Guillemin K, Krasnow MA, Gehring WJ, Affolter M. 1996. The Drosophila serum response factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered. Development 122: 25892597.
  • Mullor JL, Calleja M, Capdevila J, Guerrero I. 1997. Hedgehog activity, independent of Decapentaplegic, participates in wing disc patterning. Development 124: 12271237.
  • Nellen D, Burke R, Struhl G, Basler K. 1996. Direct and long range action of a DPP morphogen gradient. Cell 85: 357368.
  • Perrimon N, McMahon AP. 1999. Negative feedback mechanisms and their roles during pattern formation. Cell 97: 1316.
  • Roch F, Baonza A, Martin-Blanco E, Garcia-Bellido A. 1998. Genetic interactions and cell behaviour in blistered mutants during proliferation and differentiation of the Drosophila wing. Development 125: 18231832.
  • Rubin GM, Spradling AC. 1982. Genetic transformation of Drosophila with transposable element vectors. Science 218: 348353.
  • Ruiz-Gomez A, Lòpez-Varea A, Molnar C, de la Calle-Mustienes E, Ruiz-Gómez M, Gómez-Skarmeta JL, de Celis JF. 2004. Conserved cross-interactions between Ras/MAPK signalling and the dual-specificity phosphatase MKP3. Dev Dyn (in press).
  • Shellenbarger DL, Mohler JD. 1978. Temperature-sensitive periods and autonomy of pleiotropic effects of l(1)Nts1, a conditional Notch lethal in Drosophila. Dev Biol 62: 432446.
  • Staehling-Hampton K, Hoffmann FM. 1994. Ectopic decapentaplegic in the Drosophila midgut alters the expression of five homeotic genes, dpp, and wingless, causing specific morphological defects. Dev Biol 164: 502512.
  • Sturtevant MA, Bier E. 1995. Analysis of the genetic hierarchy guiding wing vein development in Drosophila. Development 121: 785801.
  • Sturtevant MA, Roark M, Bier E. 1993. The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signalling pathway. Genes Dev 7: 961973.
  • Tanimoto H, Itoh S, ten Dijke P, Tabata T. 2000. Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Mol Cell 5: 5971.
  • Teleman AA, Cohen SM. 2000. Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103: 971980.
  • Tsuneizumi K, Nakayama T, Kamoshida Y, Koernberg T, Christian J, Tabata T. 1997. Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389: 627631.
  • Vervoort M, Crozatier M, Valle D, Vincent A. 1999. The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr Biol 9: 632639.
  • Wasserman JD, Freeman M. 1997. Control of EGF receptor activation in Drosophila. Trends Cell Biol 7: 431436.
  • Yu K, Sturtevant MA, Biehs B, Francois V, Padgett R, Blackman RK, Bier E. 1996. The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development. Development 122: 40334044.