Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system with which to understand how such asymmetries are generated and how they contribute to formation of a complex structure. Early in development, the wing primordium is subdivided into a thin layer of peripodial epithelium (PE) and an apposing thickened layer of pseudostratified columnar epithelium (CE), known as the disc proper (DP). The DP gives rise to the wing blade, hinge and dorsal mesothorax, whereas the PE makes only a minor contribution to the ventral hinge and pleura. The mechanisms that generate this major asymmetry and its contribution to wing development are poorly understood. The Lines protein destabilizes the nuclear protein Bowl in ectodermal structures. Here, we show that Bowl accumulates in the PE from early stages of wing development and is absent from the DP. Broad inhibition of Bowl in the PE resulted in the replacement of the PE with a mirror image duplication of the DP. The failure to generate the PE severely compromised wing growth and the formation of the notum. Conversely, the activation of bowl in the DP (by removal or inhibition of lines function)resulted in the transformation of the DP into PE. Thus, we provide evidence that bowl and lines act as a binary switch to subdivide the wing primordium into PE and DP, and assign crucial roles for this asymmetry in wing growth and patterning.

INTRODUCTION

The Drosophila wing imaginal disc is an excellent model with which to investigate the fundamental mechanisms that generate a complex structure from an undifferentiated field of cells. One of the earliest events in wing development is its subdivision into two apposing surfaces, known as the peripodial epithelium (PE) and the disc proper (DP)(Cohen, 1993; Fristrom and Fristrom, 1993; Milner, 1984). Despite the central role of this symmetry-breaking event in setting up two fields of cells with distinct morphologies and developmental potentials, the mechanisms that generate this major asymmetry remain poorly understood.

The formation of the wing appendage depends on the establishment of the anteroposterior (AP), dorsoventral (DV) and proximodistal (PD) axes within the DP (reviewed by Blair, 1995; Dahmann and Basler, 1999; Klein, 2001; Lawrence et al., 1996; Mann and Morata, 2000). The AP and DV subdivisions are established by the transcription factors engrailed (en) and apterous (ap)(Blair et al., 1994; Diaz-Benjumea and Cohen, 1993; Lawrence and Morata, 1976; Morata and Lawrence, 1975),which program their respective compartment to induce expression of the Decapentaplegic (Dpp) and Wingless (Wg) morphogens adjacent to the AP and DV compartment boundaries (Basler and Struhl,1994; Diaz-Benjumea and Cohen,1995; Fleming et al.,1997; Panin et al.,1997; Tabata and Kornberg,1994; Williams et al.,1994; Zecca et al.,1995). Dpp and Wg, in turn, induce expression of target genes to pattern the AP and DV axes in a concentration-dependent manner(Lecuit et al., 1996; Nellen et al., 1996; Neumann and Cohen, 1996; Zecca et al., 1996). During the second instar, the wing undergoes a third major subdivision along the PD axis to form the wing distally and the notum proximally; this subdivision is mediated by the secreted Wg ligand and the Epidermal Growth Factor (EGF)receptor ligand, Vein. Wg is expressed in a wedge of ventroanterior cells(Couso et al., 1993; Klein and Arias, 1998; Ng et al., 1996; Williams et al., 1993), and induces the expression of the pdm1 homolog nubbin(nub) to initiate wing formation(Cifuentes and Garcia-Bellido,1997; Ng et al.,1995; Ng et al.,1996). Vein emanates from a patch of proximal cells to induce expression of pannier and members of the Iroquois-C gene complex to specify the medial and lateral parts of the notum(Simcox et al., 1996; Wang et al., 2000; Zecca and Struhl, 2002a; Zecca and Struhl, 2002b). As development proceeds, the wing field is further subdivided into the pouch distally, and the hinge proximally. The transcription factors vestigial (vg) and homothorax (hth), teashirt (tsh) and zfh2 are expressed in discrete subregions of the wing pouch and hinge, respectively, and control the identity of these subregions. (Mann and Morata,2000; Kim et al.,1996; Williams et al.,1991; Azpiazu and Morata,2000; Casares and Mann,2000; Whitworth and Russell,2003; Wu and Cohen,2002).

The contribution of the PE to wing growth and patterning has received far less attention. The PE has been implicated in disc eversion and fusion of adjacent discs to form a continuous adult cuticle during metamorphosis(Agnes et al., 1999; Fristrom and Fristrom, 1993; Milner et al., 1984; Pastor-Pareja et al., 2004; Usui and Simpson, 2000; Zeitlinger and Bohmann, 1999). Selective ablation of the PE resulted in smaller and malformed wings(Gibson and Schubiger, 2000),and inhibition of specific signaling pathways in the eye and wing PE resulted in patterning abnormalities and a reduction in disc size(Cho et al., 2000; Gibson and Schubiger, 2000; Pallavi and Shashidhara,2003), suggesting earlier roles for the PE in signaling to the DP to control its growth and patterning (reviewed by Gibson and Schubiger, 2001). Lineage analysis have suggested that peripodial cells stream laterally to populate the DP (McClure and Schubiger,2005; Pallavi and Shashidhara,2003). Despite these intriguing results, little is known about how the PE and the DP are specified or how they interact with each other.

Drumstick (Drm), Odd-skipped (Odd), Bowl, and Sister of Odd and Bowl (Sob)share a conserved Cys2His2 zinc-finger domain, and play diverse roles in patterning ectodermal structures. Bowl is a putative transcription factor (Wang and Coulter,1996). During embryogenesis, the protein Lines binds to and destabilizes Bowl (Hatini et al.,2005). Drm is a small peptide that binds to Lines and localizes it to the cytoplasm, permitting stabilization of Bowl in restricted domains (see Fig. 1L for the regulatory interactions connecting drm, lines and bowl)(Green et al., 2002; Hatini et al., 2005). During larval development, bowl contributes to leg and eye development(Bras-Pereira et al., 2006; de Celis Ibeas and Bray, 2003; Hao et al., 2003). drm mutants, however, do not display phenotypes in these tissues,suggesting that odd and/or sob, which are related to drm in structure and pattern of gene expression, may sometimes act redundantly with drm(Bras-Pereira et al., 2006; Hao et al., 2003). Although gain-of-function experiments support this hypothesis, loss-of-function and biochemical evidence are still lacking. Lines, however, destabilizes Bowl in all tissues examined (Green et al.,2002; Hatini et al.,2005; Iwaki et al.,2001; Johansen et al.,2003; Bras-Pereira et al.,2006; Hao et al.,2003).

We now report that lines and bowl specify alternative DP and PE fates during early stages of wing development. By blocking the specification of the PE, we were further able to provide definitive evidence that the PE is not required for the establishment of the AP, DV or PD patterning systems. The PE is instead required to support the growth and survival of the DP and the formation of the notum. Overall, our work elucidates a mechanism that generates a major asymmetry across the wing primordium and reveals crucial roles for this asymmetry in wing growth and patterning.

MATERIALS AND METHODS

Fly strains and clonal analysis

Mitotic clones were induced using the FLP/FRT(Golic, 1991; Xu and Rubin, 1993) and the MARCM techniques (Lee and Luo,2001) at 24-36, 36-48, 48-72, 72-96 and 96-120 hours AEL, which correspond to early first, late first, second, early third and mid third instar. Flies of the genotype y w hs-FLP; FRT42D Ubi-GFP and y w hs-FLP; Ubi-GFP FRT40A (B. Edgar) were used to induce FLP/FRT clones and y w hs-FLP Tub-GAL4 UAS-GFP-6Xmyc-NLS; FRT42D Tub-Gal80 hs-CD2,y+ (gift of G. Struhl) to induce MARCM clones. The linesG2 (Bokor and DiNardo, 1996), lines2f(Nusslein-Volhard et al.,1984), bowl1(Wang and Coulter, 1996) and drm3 (Green et al.,2002) alleles were used to generate mutant clones. wg-lacZ (Kassis et al.,1992) and P{lacZ}oddrk111 were used to map domains of gene expression. UAS-Lines (9.2), UAS-Myc-Lines(8), UAS-Flag-Bowl (28)(Hatini et al., 2005), UAS-Drm (2.1) (Green et al.,2002), UAS-LinesRNAi (VDRC), were expressed in clones using y w hs-FLP; act5C>y+>GAL4 UAS-GFP(Pignoni and Zipursky, 1997),and in the PE using Ubx-GAL4(Pallavi and Shashidhara,2003).

Immunofluorescence and microscopy

Staining protocols have been described elsewhere(Hatini et al., 2005). Primary antibodies used were: rabbit anti-Vg (Kim et al., 1996), mouse anti-Nub(Ng et al., 1996), mouse anti-Dll (Vachon et al.,1992), mouse anti-Sal (gift of S. Cohen), rabbit anti-Tsh(Wu and Cohen, 2000), rat anti-Zfh2 (gift of M. Lundell), rabbit anti-β-galactosidase (Cappel),mouse anti-Wg (4D4, DSHB) (Brook and Cohen, 1996), rabbit anti-Bowl(de Celis Ibeas and Bray,2003), mouse anti-Ubx (White and Wilcox, 1984), rabbit anti-Hth(Pai et al., 1998), guinea pig anti-Hth (Casares and Mann,1998), rabbit anti-Al (gift of G. Campbell), rabbit anti-activated caspase 3 (Cell Signaling Technology), mouse anti-Arm (7A1, DSHB) and rabbit anti-phospho Histone H3 (Upstate Signaling). Confocal images were scanned using a Zeiss LSM510 in multi-tracking mode. β-Galactosidase activity stains are described elsewhere (Patel et al., 1989).

RESULTS

Bowl accumulates in the wing PE and is absent from the DP

To understand the role of bowl in wing development, we examined the dynamic pattern of Bowl distribution relative to Wg. During the second instar, Wg accumulates in a wedge of ventro-anterior cells in the DP(Fig. 1B, arrow)(Couso et al., 1993; Ng et al., 1996; Williams et al., 1993). At this stage, Bowl accumulated only in peripodial cells(Fig. 1A), and not in DP cells(Fig. 1B). At the early to mid third instar, Wg was upregulated along the DV compartment boundary(Fig. 1D,F, arrows). Bowl,however, was detected in the PE (Fig. 1C,E) and in the lateral margins of the notum (arrowheads in Fig. 1D,F). At the completion of larval development, Bowl was upregulated along the lateral margins of the notum (arrowheads in Fig. 1H)and the ventro-anterior margin of the PE (asterisks in Fig. 1G,H). However, Bowl was downregulated in the medial region of the PE and absent from its posterior margin. After pupariation, Bowl was detected at high levels in both the ventro-anterior hinge (asterisk in Fig. 1I) and the lateral margins of the notum (data not shown). An odd-lacZ reporter that is expressed similarly to Bowl(Fig. 1J) was detected in the axillary region and in a subset of proximal hinge derivatives in ventral nuclei in adult wings (Fig. 1K), suggesting that a subset of odd-lacZ- and Bowl-expressing cells are fated to contribute to the ventral hinge.

Lines accumulates in nuclei in the DP and in the cytoplasm in the PE

drm is expressed in restricted domains, where it inhibits Lines,in part, by localizing it to the cytoplasm(Hatini et al., 2005). To probe the pattern of Lines activity in the wing imaginal disc, we induced expression of a `weak' Myc-Lines transgene that minimally affects wing development (UAS-Myc-Lines 8) in `FLP-out' cell clones using a combination of the FLP/FRT and the GAL4/UAS techniques(Pignoni and Zipursky, 1997). Myc-Lines accumulated in the cytoplasm in clones in the lateral margins and the medial region of the PE (Fig. 2A,B, respectively). However, Myc-Lines accumulated in nuclei in the DP (Fig. 2C). Thus, the distribution of Lines and Bowl in the PE and the DP was reciprocal. In the PE where Bowl accumulates, Lines was cytoplasmic and inactive. Reciprocally, in the DP where Bowl was absent, Lines was nuclear and active.

Lines inhibits Bowl accumulation in the DP

We also expressed a Flag-Bowl transgene in Flp-out clones to determine whether Bowl was destabilized in the DP. Indeed, Flag-Bowl accumulated only in cell clones that were generated in the medial and ventro-anterior margin of the PE where Bowl is normally detected(Fig. 2D-D′; arrow and arrowhead in D′, respectively). However, Flag-Bowl was not detected in DP clones, suggesting that endogenous Lines destabilized Bowl in this region. To test this idea, we examined Bowl accumulation in lines mutant clones and in drm-expressing clones and detected a stabilization of Bowl in these clones (Fig. 2E,F, respectively). Reciprocally, Bowl was destabilized in lines-expressing cell clones generated in the PE(Fig. 2G). However, Bowl accumulation was unaffected in drm mutant clones generated in the PE(data not shown), consistent with the proposal that odd and/or sob may sometimes act redundantly with drm to stabilize Bowl(Bras-Pereira et al., 2006; Hao et al., 2003). We were unable to test sob function as sob mutant alleles are not available. However, Bowl accumulation was unaffected in odd mutant clones. We also attempted to induce homozygous clones for the drm(P2)deficiency to generate drm odd sob triple mutant clones but failed to recover these clones. Therefore, additional studies will be necessary to define the multiple inputs necessary to stabilize Bowl in the PE.

Fig. 1.

Bowl accumulates in the PE and is absent from the DP. Spatiotemporal expression pattern of Bowl (red), wg-lacZ (green) or odd-lacZ (green). Optical sections at the plane of the PE (A,C,E) and DP (B,D,F). (A,B) 48 hours AEL; (C,D) 72 hours AEL; (E,F) 96 hours AEL. wg-lacZ was detected in the DP in a wedge of ventral cells (arrow in B). wg was upregulated along the DV compartment boundary (arrows in D and F). Bowl accumulated broadly in the PE (A,C,E) and was absent from the DP (B,D,F), except for the lateral margins of the notum (arrowheads in D and F). (G,H) 120-144 hours AEL; Bowl accumulated in the anterior margin of the PE (asterisks in G and H) and the lateral margins of the notum (arrowheads in H). However, Bowl was downregulated in the medial and posterior regions of the PE. (I)Everting discs; Bowl accumulated in the ventro-anterior hinge (asterisk) and in the lateral margins of the notum (not shown in this image). (J) odd-lacZ was detected in a similar pattern at this stage (arrowheads indicate the lateral margins of the notum, asterisks the ventro-anterior hinge). (K) Adult wings; odd-lacZ was detected in ventro-proximal hinge nuclei. (L) A schematic diagram illustrating the regulatory interactions linking drm, lines and bowl.

Fig. 1.

Bowl accumulates in the PE and is absent from the DP. Spatiotemporal expression pattern of Bowl (red), wg-lacZ (green) or odd-lacZ (green). Optical sections at the plane of the PE (A,C,E) and DP (B,D,F). (A,B) 48 hours AEL; (C,D) 72 hours AEL; (E,F) 96 hours AEL. wg-lacZ was detected in the DP in a wedge of ventral cells (arrow in B). wg was upregulated along the DV compartment boundary (arrows in D and F). Bowl accumulated broadly in the PE (A,C,E) and was absent from the DP (B,D,F), except for the lateral margins of the notum (arrowheads in D and F). (G,H) 120-144 hours AEL; Bowl accumulated in the anterior margin of the PE (asterisks in G and H) and the lateral margins of the notum (arrowheads in H). However, Bowl was downregulated in the medial and posterior regions of the PE. (I)Everting discs; Bowl accumulated in the ventro-anterior hinge (asterisk) and in the lateral margins of the notum (not shown in this image). (J) odd-lacZ was detected in a similar pattern at this stage (arrowheads indicate the lateral margins of the notum, asterisks the ventro-anterior hinge). (K) Adult wings; odd-lacZ was detected in ventro-proximal hinge nuclei. (L) A schematic diagram illustrating the regulatory interactions linking drm, lines and bowl.

Broad expression of lines in the PE replaces the PE with the DP and inhibits wing growth

To investigate the role of lines in wing development, we next overexpressed a lines transgene that strongly destabilizes Bowl(UAS-Lines 9.2) in the PE where Lines is repressed, or removed lines function in the DP where Lines is active. First, we broadly expressed lines with Ubx-GAL4(Pallavi and Shashidhara,2003) to broadly destabilize Bowl in the PE using the GAL4/UAS technique (Brand and Perrimon,1993). We found that the flattened morphology of wild-type wing discs (Fig. 3A) was replaced with a spherical morphology (Fig. 3B), in which the thin PE (Fig. 3A′,C,D′-D‴) was replaced with the thickened pseudostratified CE that forms the DP (Fig. 3B′,E′-E‴). Coincidentally, the expression of the peripodial markers Bowl, Ubx and Eya was lost in these discs(Fig. 4B,C; data not shown). Conversely, Lines was broadly nuclear (Fig. 4D), indicating that the PE (where Lines is enriched in the cytoplasm) was replaced with the DP (where Lines is enriched in nuclei). The Ubx>Lines wing discs were also roughly 10-30% the size of wild-type wing discs (compare Fig. 3A with 3B; experimental discs, 39 mm2, n=7, s.d.=18 mm2; wild type, 147 mm2, n=7, s.d.=21 mm2). These discs survived beyond the larval stages and elongated during metamorphosis (see Fig. S1 in the supplementary material) but failed to contribute to adult structures. We obtained similar phenotypes by driving expression of UAS-Lines with C311-GAL4 and Tsh-GAL4 (see Fig. S2 in the supplementary material).

Organ growth depends largely on a balance between cell proliferation and cell death. Therefore, to determine the reason for the reduction in wing size,we examined the expression of Phospho-Histone H3 (PH3) and activated caspase 3(Ice) in Ubx>Lines discs to detect dividing and apoptotic cells, respectively (Ryoo et al.,2002). We detected similar levels of proliferating cells(Fig. 3G), but elevated levels of apoptotic cells in these discs (Fig. 3F) compared with age-matched controls (not shown). We therefore propose that the PE supports wing growth by promoting cell survival in the DP.

The AP, DV and PD patterning systems are established and maintained in the absence of the PE

We considered the possibility that the reduction in wing size resulted from the loss of positional identities along the wing PD axis. We therefore analyzed Vg (Kim et al., 1996; Williams et al., 1991), Nub(Cifuentes and Garcia-Bellido,1997; Ng et al.,1995; Ng et al.,1996) and Zfh2 expression(Whitworth and Russell, 2003)to determine whether the pouch, the hinge or the notum were affected. In wild type, Vg is restricted to the distal pouch, whereas Zfh2 is restricted to the hinge (Fig. 4E). Nub is detected in the pouch and in part of the dorsal hinge(Fig. 3D,D″,D‴). In experimental discs, Nub (Fig. 3E,E″-E‴), Vg and Zfh2(Fig. 4F,J) were detected in both layers of the disc epithelium, indicating that the PE was replaced with a mirror image duplication of the DP. Only a small region near the disc stalk did not express Zfh2 (arrowhead in Fig. 4F), indicating that the notum, which is Zfh2 negative, was nearly lost. The reduction in notal growth could partially account for the reduction in overall wing size.

Fig. 2.

The Lines and Bowl proteins accumulate in a reciprocal pattern across the PE and the DP. Clones expressing (A-C) Myc-Lines (weak insertion), (D)Flag-Bowl, (F) Drm or (G) Lines (strong insertion) were marked with nuclear GFP reporter, which accumulates to high levels in nuclei and to lower levels in the cytoplasm. (E) lines mutant clones were GFP-negative. Arrows in A-C,E-G indicate magnified areas shown in insets. (A-B″)Optical sections at the plane of the PE; Myc-Lines was enriched in the cytoplasm in the lateral margins of the PE (A-A″), and in the simple squamous PE (B-B″). DP clones below the PE are visible in these images.(C-C″) However, Myc-Lines was enriched in nuclei in the DP.(D,D′) Flag-Bowl accumulated only in clones generated in the PE (arrow in D′) and the ventro-anterior hinge (arrowhead in D′). Flag-Bowl failed to accumulate in clones generated in the DP. Bowl was stabilized in lines mutant cell clones (E-E″) and in drm expressing clones (F-F″) in the DP, and lost from the lines-expressing cell clones generated in the lateral margins of the PE (G-G″, arrow in G) and in the squamous PE (not shown). Clones were induced at 24-48 h AEL in A-D and at 72-96 h AEL in E-G.

Fig. 2.

The Lines and Bowl proteins accumulate in a reciprocal pattern across the PE and the DP. Clones expressing (A-C) Myc-Lines (weak insertion), (D)Flag-Bowl, (F) Drm or (G) Lines (strong insertion) were marked with nuclear GFP reporter, which accumulates to high levels in nuclei and to lower levels in the cytoplasm. (E) lines mutant clones were GFP-negative. Arrows in A-C,E-G indicate magnified areas shown in insets. (A-B″)Optical sections at the plane of the PE; Myc-Lines was enriched in the cytoplasm in the lateral margins of the PE (A-A″), and in the simple squamous PE (B-B″). DP clones below the PE are visible in these images.(C-C″) However, Myc-Lines was enriched in nuclei in the DP.(D,D′) Flag-Bowl accumulated only in clones generated in the PE (arrow in D′) and the ventro-anterior hinge (arrowhead in D′). Flag-Bowl failed to accumulate in clones generated in the DP. Bowl was stabilized in lines mutant cell clones (E-E″) and in drm expressing clones (F-F″) in the DP, and lost from the lines-expressing cell clones generated in the lateral margins of the PE (G-G″, arrow in G) and in the squamous PE (not shown). Clones were induced at 24-48 h AEL in A-D and at 72-96 h AEL in E-G.

Fig. 3.

Overexpression of lines in the PE replaces the PE with DP.(A,C,D) Wild type; (B,E-G) Ubx-GAL4 UAS-Lines(Ubx>Lines); arrowheads in A,B,D,E indicate plane of z-section in A′, B′, D′-D‴ and E′-E‴, respectively. (A-B′) Armadillo (Arm);cellular outlines are detected in apical sections; bright dots reveal adherence junctions in z-sections. Images in A and B are at the same magnification. (A-A′) Wild type; (B-B′) Ubx>Lines; the PE was replaced with a mirror image duplication of the pseudostratified CE and the disc was severely reduced in size. (C-E‴) Dapi (blue); Nub (red) is restricted to the DP. (C,D) Wild type; optical sections at the level of the PE (C), and the DP(D). (D′-D‴) The DP (left layer) expresses Nub and apposes the PE(right layer). (E-E‴) Ubx>Lines; the PE (right layer) was replaced with a mirror-image duplication of the DP. Nub was detected symmetrically in both layers of these discs. (F) Ice (red)accumulated in clusters of apoptotic cells (arrows in F). Apoptosis is minimal in wild-type discs (data not shown). (G) Phospho-Histone H3 (PH3; red)stain reveals homogenous distribution of actively dividing cells in experimental discs.

Fig. 3.

Overexpression of lines in the PE replaces the PE with DP.(A,C,D) Wild type; (B,E-G) Ubx-GAL4 UAS-Lines(Ubx>Lines); arrowheads in A,B,D,E indicate plane of z-section in A′, B′, D′-D‴ and E′-E‴, respectively. (A-B′) Armadillo (Arm);cellular outlines are detected in apical sections; bright dots reveal adherence junctions in z-sections. Images in A and B are at the same magnification. (A-A′) Wild type; (B-B′) Ubx>Lines; the PE was replaced with a mirror image duplication of the pseudostratified CE and the disc was severely reduced in size. (C-E‴) Dapi (blue); Nub (red) is restricted to the DP. (C,D) Wild type; optical sections at the level of the PE (C), and the DP(D). (D′-D‴) The DP (left layer) expresses Nub and apposes the PE(right layer). (E-E‴) Ubx>Lines; the PE (right layer) was replaced with a mirror-image duplication of the DP. Nub was detected symmetrically in both layers of these discs. (F) Ice (red)accumulated in clusters of apoptotic cells (arrows in F). Apoptosis is minimal in wild-type discs (data not shown). (G) Phospho-Histone H3 (PH3; red)stain reveals homogenous distribution of actively dividing cells in experimental discs.

We also considered the possibility that the reduction in wing growth resulted from the disruption of the AP or the DV patterning systems. We therefore examined the expression of the Wg(Fig. 4G-H) and Dpp (see Fig. S3 in the supplementary material) morphogens and their respective transcriptional targets Dll (Fig. 4I,J) and Sal (Fig. 4K,L). In wild type, Wg is detected in the pouch along the DV compartment boundary, in the hinge in two concentric rings, and in the notum in a band that transverses the AP axis(Fig. 4G). Wg and Dpp, in turn,respectively induce expression of Dll (Fig. 4I) and Sal (Fig. 4K) in broad domains within the pouch(Lecuit et al., 1996; Nellen et al., 1996; Neumann and Cohen, 1996; Zecca et al., 1996). In experimental discs, Wg was detected in both layers of the disc epithelium, in ring-like structures that encircled the pouch (short arrow) and the hinge(long arrows) (Fig. 4H). dpp was expressed orthogonally to Wg (see Fig. S3 in the supplementary material). Dll and Sal were also detected in both layers of these discs (Fig. 4J,L,respectively), indicating that the Wg and Dpp morphogens were able to signal broadly to induce their targets. We thus conclude that at least part of the AP, DV and PD axes are established independently of the PE. These findings imply that the PE supports wing growth in parallel to these systems, and plays a crucial role in promoting the growth of the notum.

Fig. 4.

The DV, AP and PD patterning systems are elaborated in the absence of the PE. Analysis of Ubx>Lines discs with molecular markers for the PE and for the AP, DV and PD patterning systems. (A,E,G,I,K)Wild type; (B-D,F,H,J,L) Ubx>Lines; maximal intensity projections. Arrowheads in D,H,J,L indicate the plane of z-sections.(A) Wild type; Ubx (red) and Bowl (green) are expressed in the PE in nested domains. Ubx occupies a broad central domain. Bowl occupies a broader domain. (B-D) Ubx>Lines; expression of the peripodial markers Bowl (B) and Ubx (C) is absent; arrow in B indicates a leg where Bowl accumulation is not affected. (D) Lines was broadly nuclear.(E) Wild type; Vg (red) marks the pouch; Zfh2 (green) marks the hinge.(F) Ubx>Lines; Vg and Zfh2 were expressed in adjacent domains in both layers of the wing disc. The Zfh2-negative notum formed a tiny rudiment (arrow). (G) Wild type; Wg is restricted to the DP. (H) Ubx>Lines; Wg (red) was detected in the pouch (short arrow) and hinge (long arrows) in both layers of the disc epithelium but not in the notum. In wild type, (I) Dll and (K)Sal are induced in response to Wg and Dpp signaling, respectively. In Ubx>Lines, (J) Dll (red), Zfh2 (green) and(L) Sal were induced in both layers of these discs. Scale bar: 100μm in B-D,F-L; 200 μm in A,E-K.

Fig. 4.

The DV, AP and PD patterning systems are elaborated in the absence of the PE. Analysis of Ubx>Lines discs with molecular markers for the PE and for the AP, DV and PD patterning systems. (A,E,G,I,K)Wild type; (B-D,F,H,J,L) Ubx>Lines; maximal intensity projections. Arrowheads in D,H,J,L indicate the plane of z-sections.(A) Wild type; Ubx (red) and Bowl (green) are expressed in the PE in nested domains. Ubx occupies a broad central domain. Bowl occupies a broader domain. (B-D) Ubx>Lines; expression of the peripodial markers Bowl (B) and Ubx (C) is absent; arrow in B indicates a leg where Bowl accumulation is not affected. (D) Lines was broadly nuclear.(E) Wild type; Vg (red) marks the pouch; Zfh2 (green) marks the hinge.(F) Ubx>Lines; Vg and Zfh2 were expressed in adjacent domains in both layers of the wing disc. The Zfh2-negative notum formed a tiny rudiment (arrow). (G) Wild type; Wg is restricted to the DP. (H) Ubx>Lines; Wg (red) was detected in the pouch (short arrow) and hinge (long arrows) in both layers of the disc epithelium but not in the notum. In wild type, (I) Dll and (K)Sal are induced in response to Wg and Dpp signaling, respectively. In Ubx>Lines, (J) Dll (red), Zfh2 (green) and(L) Sal were induced in both layers of these discs. Scale bar: 100μm in B-D,F-L; 200 μm in A,E-K.

Clonal expression of lines transforms the PE into DP and permits induction of secondary wings

We also induced expression of lines in FLP-out clones to determine whether lines eliminates or transforms the PE into DP. If lines transforms the PE into DP, then it should be possible to recover large lines-expressing clones in wing rudiments that lack PE. If, however, lines overexpression eliminates the PE then it should be possible to recover wing rudiments in which the lines-expressing clones were lost together with the PE. Ectopic expression of linesduring the early first instar gave rise to two general classes of wing abnormalities. In the most severe class, the PE was replaced with a mirror image duplication of the pseudostratified CE, which correlated with the expression of the DP markers Nub and Tsh in both layers of the wing disc(z-section in Fig. 5D). These discs were spherical and as small as the Ubx>Lines discs described above. The clones that gave rise to this phenotype formed large confluent patches that occupied roughly 56% of the entire wing surface (n=7, s.d.=17%). As wing rudiments lacking large patches of lines-expressing clones were not recovered(n=58 rudimentary discs examined), we were forced to conclude that the presumptive peripodial cells survived in experimental disc and assumed an inappropriate DP fate. In the second class, the discs formed secondary wing fields (Fig. 5E,F; arrows in`merged' images point to secondary wings; small arrowheads in red channel point to the two wing margins). The notum appeared intact in some discs(Fig. 5E) but reduced or missing in others (Fig. 5F,asterisk in red channel indicates the loss of Wg expression in the notum). The clones that gave rise to this phenotype adopted DP fate (z-section in Fig. 5F), and occupied ∼26%of the surface area of the entire wing (n=9, s.d.=5%) and roughly 50%of the surface area of the secondary wing fields. To further clarify the origin of clones that permitted induction of secondary wings, we expressed UAS-Lines with Ptc-GAL4 in both the DP and the PE (marked by apposing arrowheads) along the AP compartment boundary. The Ptc>Lines-expressing cells in the PE adopted DP fate and permitted the induction of secondary wing (Fig. 5H). We conclude that smaller patches of lines-expressing clones generated in the PE adopted DP fate, and subsequently acquired the competence to respond to wing inducing signals. Clones that did not disrupt wing development localized to the DP even when they formed large confluent patches (Fig. 5C). Taken together, we interpret these results as evidence that lines can reprogram the PE to adopt DP fate during early stages of wing development.

Reciprocal roles for lines and bowl in promoting cell survival in the DP and the PE

To investigate the contribution of bowl to wing development, we induced marked bowl mutant clones and examined clone recovery relative to wild-type twin spots generated by the same mitotic recombination event. Control clones and their twin clones survived in both the PE and the DP(Fig. 6A; PE nuclei are spread out; DP nuclei are densely packed). Most of the bowl mutant clones generated at the early first instar survived in the DP. However, only 50% of clones survived in the PE mostly near the disc stalk(Fig. 6F, arrow indicates a stalk clone) indicating that the bowl mutant clones were either dying or sorting out from the PE at early stages. bowl mutant clones generated at the second instar survived in the PE(Fig. 6G) and adopted PE fate,suggesting that the maintenance of PE fate depends on additional mechanisms.

Fig. 5.

Ectopic lines expression can completely transform the PE into DP, induce formation of secondary wings, and disrupt notal growth. (A,B)Wild type. (C-H) lines expressing clones (green) induced at the early first instar. Dapi (blue), Tsh (yellow), Wg, Nub or Al (red). (A) Tsh is excluded and Nub is restricted to the pouch and distal hinge. (B) Wg demarcates the pouch, the proximal and the distal hinge, and the medial region of the notum. (C) Large patches of lines-expressing cells in the DP had no effect on wing development. (D) Large patches of lines-expressing clones in the PE completely transformed the PE into DP. Arrowheads indicate the z-section shown in insets. Tsh and Nub were symmetrically expressed in both layers of the disc. (E,F)Smaller clones led to formation of secondary wings (arrows in right panels indicate secondary wings, arrowheads indicate Wg expression along the wing margin). These clones localized to the central region of the secondary wings. Large arrowheads indicate plane of z-section shown in insets. (F) In a subset of discs, the notum was reduced or absent. Asterisk indicates the loss of Wg expression in the presumptive notum. (G) Ptc-GAL4 UAS-GFP; GFP expression was detected along the AP compartment boundary in the DP and in the PE (marked by apposing arrows); Al marks the anterior part of the pouch. (H) Ptc-GAL4 UAS-GFP UAS-Lines; the Ptc-expressing cells in the PE adopted DP fate and permitted formation of secondary wings. Al and GFP were co-expressed in a subset of pouch cells.

Fig. 5.

Ectopic lines expression can completely transform the PE into DP, induce formation of secondary wings, and disrupt notal growth. (A,B)Wild type. (C-H) lines expressing clones (green) induced at the early first instar. Dapi (blue), Tsh (yellow), Wg, Nub or Al (red). (A) Tsh is excluded and Nub is restricted to the pouch and distal hinge. (B) Wg demarcates the pouch, the proximal and the distal hinge, and the medial region of the notum. (C) Large patches of lines-expressing cells in the DP had no effect on wing development. (D) Large patches of lines-expressing clones in the PE completely transformed the PE into DP. Arrowheads indicate the z-section shown in insets. Tsh and Nub were symmetrically expressed in both layers of the disc. (E,F)Smaller clones led to formation of secondary wings (arrows in right panels indicate secondary wings, arrowheads indicate Wg expression along the wing margin). These clones localized to the central region of the secondary wings. Large arrowheads indicate plane of z-section shown in insets. (F) In a subset of discs, the notum was reduced or absent. Asterisk indicates the loss of Wg expression in the presumptive notum. (G) Ptc-GAL4 UAS-GFP; GFP expression was detected along the AP compartment boundary in the DP and in the PE (marked by apposing arrows); Al marks the anterior part of the pouch. (H) Ptc-GAL4 UAS-GFP UAS-Lines; the Ptc-expressing cells in the PE adopted DP fate and permitted formation of secondary wings. Al and GFP were co-expressed in a subset of pouch cells.

We also induced expression of bowlRNAi transgene in FLP-out clones at first larval instar to determine if reduced levels of bowlaffected the survival or the fate of the PE. Clonal expression of bowlRNAi gave rise to two general classes of wing abnormalities that phenocopied the phenotypes induced by ectopic expression of lines. Large patches of clones led to the formation of small and spherical discs in which the PE was replaced with DP. Smaller clones permitted the induction of secondary wings (data not shown). The bowlRNAi transgene can also knockdown the expression level of three other genes, odd, sob and not1, indicating that bowl, either alone or together with odd or sob or both genes, specifies PE fate and inhibits DP fate.

We also examined the recovery of lines mutant clones induced at the first instar. lines mutant and wild-type twin spots survived in the PE (Fig. 6B). However,mostly wild-type twin spots survived in the DP(Fig. 6C), and were much smaller than respective wild-type twin spots. Similar to the linesmutant clones, drm-expressing clones generated at the first instar survived in the PE but not in the DP (Fig. 6E). We therefore conclude that at early stages of wing development bowl promotes cell survival in the PE, whereas lines promotes cell survival in the DP. The quantitative analysis is presented in Fig. 6G.

Fig. 6.

lines and bowl promote cell survival in the DP and the PE, respectively. (A,D) Control clones, (B,C) linesmutant FLP/FRT clones, (E) drm expressing clones and (F) bowl mutant MARCM clones. Dapi, blue in A-D,F. (A) Control and wild-type twin clones induced at the first instar survived in both the PE and DP. Optical section is at the level of the PE but DP clones are also visible.(B,C) lines mutant clones and twin spots survived in the PE (B), but mostly twin spots survived in the DP (C). (D) Control clones survived in both the DP and the PE. (E) drm-expressing clones were recovered in the PE but not in the DP. Wg expression (red) in the DP is visible. (F) bowl mutant clones survived in the DP but poorly in the PE. Arrowhead indicates a bowl mutant clone near the disc stalk. (G) Quantitative analysis of the recovery of wild-type, bowl and lines mutant clones. To calculate the the percentage recovery, we pooled the number of clones and separately the corresponding wild-type twin spots from several discs. L1, first instar; L2,second instar.

Fig. 6.

lines and bowl promote cell survival in the DP and the PE, respectively. (A,D) Control clones, (B,C) linesmutant FLP/FRT clones, (E) drm expressing clones and (F) bowl mutant MARCM clones. Dapi, blue in A-D,F. (A) Control and wild-type twin clones induced at the first instar survived in both the PE and DP. Optical section is at the level of the PE but DP clones are also visible.(B,C) lines mutant clones and twin spots survived in the PE (B), but mostly twin spots survived in the DP (C). (D) Control clones survived in both the DP and the PE. (E) drm-expressing clones were recovered in the PE but not in the DP. Wg expression (red) in the DP is visible. (F) bowl mutant clones survived in the DP but poorly in the PE. Arrowhead indicates a bowl mutant clone near the disc stalk. (G) Quantitative analysis of the recovery of wild-type, bowl and lines mutant clones. To calculate the the percentage recovery, we pooled the number of clones and separately the corresponding wild-type twin spots from several discs. L1, first instar; L2,second instar.

The loss of lines function in the DP transforms DP into PE fate

lines mutant clones generated at the second instar survived poorly in the DP relative to wild-type twin spots(Fig. 6G). The surviving clones, however, extruded basally from the DP(Fig. 7). Only clones that were generated in the PE, the lateral margins of the notum and the ventro-anterior hinge intermingled freely with their wild-type neighbors(Fig. 6B; data not shown). Occasionally, clones that originated in the DP grew to a large size. We examined the positional identity of these clones with molecular markers. We found that the clones lost expression of the DP specific proteins Nub and Vg(Fig. 7A,B, respectively) and ectopically expressed the PE specific proteins Ubx and Eya(Fig. 7C,D). Ubx is restricted to the posterior compartment, and was detected in the DP only in lines mutant clones generated in the posterior compartment(McClure and Schubiger, 2005; Pallavi and Shashidhara,2003). Tsh and Hth, which are expressed in the PE as well as in the notum and hinge, but are excluded from the pouch, were also ectopically expressed in the clones (Fig. 7E-F). We conclude that lines is necessary to specify DP fate and to inhibit the specification of PE fate at early stages of wing development.

lines maintains distal pouch identities and inhibits proximal hinge identities at later stages of wing development

lines could be needed either continuously or transiently in the DP to specify DP fate and to inhibit PE fate. Moreover, lines could play yet another role at later stages of wing development. To address these issues,we examined the behavior of lines mutant clones and drm-expressing clones that were generated at the mid to late third instar following the formation of the PE and the DP. Most of the clones generated in the DP and the posterior lateral margin of the hinge minimized contact with their wild-type neighbors and formed round vesicles that extruded basally (Fig. 8A-A″,B′,C-H). We examined the positional identity of these clones with molecular markers to determine whether the clones assumed an alternative cell fate. The peripodial markers Ubx and Eya were not detected in the clones, indicating the clones did not assume PE fate (data not shown). However, the Tsh, Hth, Wg and Zfh2 proteins, which localize to the hinge and control hinge formation, were ectopically expressed in the clones(Fig. 8C-E; data not shown). Expression of these markers was lower near the AP compartment boundary and increased at a distance from this boundary, suggesting that Dpp signaling antagonized this fate transformation in a graded manner, as previously proposed (Azpiazu and Morata,2000; Casares and Mann,2000). Hinge-specific markers were also induced in linesRNAi clones generated in the pouch, but were either downregulated or lost in linesRNAi clones lacking bowlfunction, indicating that the lines clonal phenotypes were due to the stabilization of Bowl (see Fig. S4 in the supplementary material). nub, which localizes to both the pouch and the distal hinge, was maintained in these clones (Fig. 8F). Reciprocally, the pouch specific proteins Dll and Sal were lost in lines mutant clones generated in the pouch in both distal and proximal positions (Fig. 8G,H,respectively). Overall, these results indicate that lines and bowl are required at early stages to specify the DP and the PE,suggesting that the maintenance of these fates depends on additional mechanisms. However, they reveal a later role for lines in maintaining distal pouch identities and in inhibiting the specification of proximal hinge identities.

DISCUSSION

The generation of molecular asymmetries across the wing primordium is central to the control of wing growth and patterning. Early during development, the two surfaces of the flattened wing imaginal disc assume distinct DP and PE fates, and hence distinct morphologies and developmental roles. Our data defines, for the first time, a mechanism that establishes this asymmetry. We show that lines and bowl act as a binary switch to specify DP and PE fates, respectively. By blocking the specification of the PE we were further able to explore the earliest requirement of the PE to wing growth and patterning.

Fig. 7.

Loss of lines function in the DP results in transformation of DP into PE fate.lines mutant clones generated at the second instar and stained for peripodial, hinge and pouch markers. (B,D-F) FLP/FRT and (A,C) MARCM clones; arrows point to magnified regions shown in right insets. Arrowheads in C′-C″ and D′-D″ indicate planes of z-sections shown in C‴-C″″ and D‴,respectively. Images in A-B,E-F are projections. lines mutant clones occasionally overproliferated and protruded from the disc epithelium.(A,B) DP markers Nub (A) and Vg (B) were lost in these clones. The clone marked with an arrowhead in A,A′ is below the Nub domain and lost Nub expression. (C-D‴) Reciprocally, the PE markers Ubx (C)and Eya (D) were ectopically expressed in these clones. Ubx was detected only in posterior DP clones. (C‴,C″″,D‴) z-sections reveal basal extrusion of clones from the DP. (E)Tsh and (F) Hth, which mark the PE, the notum and the hinge, were ectopically expressed in these clones.

Fig. 7.

Loss of lines function in the DP results in transformation of DP into PE fate.lines mutant clones generated at the second instar and stained for peripodial, hinge and pouch markers. (B,D-F) FLP/FRT and (A,C) MARCM clones; arrows point to magnified regions shown in right insets. Arrowheads in C′-C″ and D′-D″ indicate planes of z-sections shown in C‴-C″″ and D‴,respectively. Images in A-B,E-F are projections. lines mutant clones occasionally overproliferated and protruded from the disc epithelium.(A,B) DP markers Nub (A) and Vg (B) were lost in these clones. The clone marked with an arrowhead in A,A′ is below the Nub domain and lost Nub expression. (C-D‴) Reciprocally, the PE markers Ubx (C)and Eya (D) were ectopically expressed in these clones. Ubx was detected only in posterior DP clones. (C‴,C″″,D‴) z-sections reveal basal extrusion of clones from the DP. (E)Tsh and (F) Hth, which mark the PE, the notum and the hinge, were ectopically expressed in these clones.

The establishment of molecular asymmetries across the wing imaginal disc

The wing PE can be identified molecularly and morphologically as a thin epithelial sheet overlying the thickened DP epithelium(Baena-Lopez et al., 2003; Cho et al., 2000; Gibson and Schubiger, 2000; McClure and Schubiger, 2005; Pallavi and Shashidhara,2003). Our mapping studies show that the distribution of Bowl and Lines correlates with the establishment of this asymmetry (Figs 1, 2). The wing primordium inherits its subdivision into en-expressing cells that form the posterior compartment and adjacent anterior compartment cells from the embryonic epidermis (Cohen et al.,1991; Cohen,1990). Bowl accumulates in the posterior en-expressing cells in the embryonic epidermis (Hatini et al., 2005), suggesting that the wing primordium also inherits the PE/DP subdivisions from pre-existing asymmetries across this tissue.

Reciprocal roles for lines and bowl in specifying alternative cell fates across the wing imaginal disc

lines and odd-skipped genes act as a switch to specify alternative cell fates across fields of cells(Fig. 1L). bowl and lines specify the alternative 1°-3° and 4° cell fate across the dorsal embryonic epidermis(Bokor and DiNardo, 1996; Hatini et al., 2000; Hatini et al., 2005). bowl and lines also specify alternative cell fates in the developing gut, leg and eye imaginal discs(Iwaki et al., 2001; Johansen et al., 2003; Bras-Pereira et al., 2006; de Celis Ibeas and Bray, 2003; Hatini et al., 2005). The asymmetric distributions of Bowl and odd-skipped genes, and the reciprocal distribution of Lines in the wing primordium are also used to specify the alternative PE and DP fates. Indeed, our functional studies show that ectopic lines expression or the inhibition of bowlfunction in the PE transforms PE into DP fate. Reciprocally, the removal of lines function from the DP transforms DP into PE fate. Our data further suggest that lines exerts its function by controlling the stability of the Bowl protein (see Fig. S4 in the supplementary material). Thus, lines and bowl act as a switch to specify alternative DP and PE fates across the wing primordium. The distribution of Lines and Bowl correlates with the subdivision of the wing primordium into a thin squamous and a thickened columnar epithelial sheet. The activation of EGF receptor and Wg signaling in the DP may specify the formation of a columnar epithelial morphology (Baena-Lopez et al.,2003). The pathways that specify the squamous morphology of the PE downstream to bowl remain to be elucidated.

The subdivision of the wing imaginal disc into DP and PE is critical for wing growth

Previous studies that relied on surgical and genetic ablations of the PE,and on inhibition of certain signaling pathways within the PE, suggested important roles for the PE in disc growth and patterning(Baena-Lopez et al., 2003; Cho et al., 2000; Gibson and Schubiger, 2000; McClure and Schubiger, 2005; Pallavi and Shashidhara,2003). We were now able to examine wing development in discs lacking PE. These discs were significantly smaller than wild type(Fig. 3B,E; Fig. 4B-D,F-L), and the notum was dramatically reduced in size relative to the pouch and hinge(Fig. 4F). Progenitor cells that originate in the PE may stream laterally to populate the growing notum(McClure and Schubiger, 2005; Pallavi and Shashidhara,2003), and the loss of this progenitor cell population may account for the severe reduction in notal growth. The reduction in wing growth could have resulted from the disruption of Wg or Dpp signaling activities, as these morphogens control cell survival and cell proliferation in the wing(Burke and Basler, 1996; Gibson and Perrimon, 2005; Giraldez and Cohen, 2003; Johnston and Sanders, 2003; Martin-Castellanos and Edgar,2002; Moreno et al.,2002; Neumann and Cohen,1996; Shen and Dahmann,2005). Indeed, a block to Dpp or Wg signaling results in formation of tiny wing rudiments (Adachi-Yamada et al., 1999; Bryant,1978; Couso et al.,1993; Morata and Lawrence,1977; Sharma and Chopra,1976). However, the expression of Wg and Dpp and their target genes was normal in these discs, indicating that the reduction in wing growth was not a consequence of the loss of wg or dpp expression,or of signaling activities (Fig. 4J,L). Our findings instead suggest that the PE acts in parallel to the AP, DV and PD patterning systems, in part, by promoting cell survival in the DP, and in part by promoting the growth of the notum.

Fig. 8.

lines specifies distal pouch fates and inhibits proximal hinge fate at the third instar. (A,C-H) Late third instar lines mutant FLP/FRT clones. (B) drm-expressing clones. Arrowheads in A and B indicate planes of z-sections shown in A′,A″,B′,B″. Arrows in C-H indicate magnified regions shown in insets. (A) Bowl (red) and GFP (green). (A-A″) lines mutant clones and (B-B″) drm-expressing clones extruded basally (arrows in A′ and B′). (C-C″)Tsh, (D-D″) Hth and (E-E″) Wg, the expression of which overlaps in the hinge, were ectopically expressed in these clones. The expression of these proteins increased at a distance from the AP compartment boundary. (F-F″) Expression of Nub, which localizes to both the pouch and distal hinge was unaffected. (G-H″) The expression of the wing specific proteins (G) Dll and (H) Sal was lost from the clones.

Fig. 8.

lines specifies distal pouch fates and inhibits proximal hinge fate at the third instar. (A,C-H) Late third instar lines mutant FLP/FRT clones. (B) drm-expressing clones. Arrowheads in A and B indicate planes of z-sections shown in A′,A″,B′,B″. Arrows in C-H indicate magnified regions shown in insets. (A) Bowl (red) and GFP (green). (A-A″) lines mutant clones and (B-B″) drm-expressing clones extruded basally (arrows in A′ and B′). (C-C″)Tsh, (D-D″) Hth and (E-E″) Wg, the expression of which overlaps in the hinge, were ectopically expressed in these clones. The expression of these proteins increased at a distance from the AP compartment boundary. (F-F″) Expression of Nub, which localizes to both the pouch and distal hinge was unaffected. (G-H″) The expression of the wing specific proteins (G) Dll and (H) Sal was lost from the clones.

The results presented in this study argue that lines and bowl function as field-specific selector genes to specify the identity and the behavior of the DP and the PE of the wing primordium,respectively. Selector genes control cell fate, cell affinity and the competence to send and respond to patterning signals in a cell autonomous manner (reviewed by Mann and Morata,2000). lines and bowl act cell autonomously to control the fate, affinity and the interaction between the PE and the DP. As a putative transcription factor, Bowl may regulate a developmental program to control the identity and the behavior of the PE. By inhibiting Bowl accumulation, lines may allow the execution of an alternative developmental program in the DP. Our studies define a new system to identify,in a systematic way, these developmental programs.

Acknowledgements

We thank S. DiNardo for his support and crucial insight at the early stages of this work; S. Bray, S. Cohen, S. Carroll, G. Campbell, M. Lundell, R. Mann,G. Struhl, H. Sun and R. White for providing reagents; and K. Commons, P. Jou,L. Zeng, E. Kula and N. Neuman for critically reading the manuscript. We thank the Bloomington Stock Center, the Vienna Drosophila Research Center(VDRC) and the Developmental Studies Hybridoma Bank for fly stocks and antibodies, respectively. This work was supported by the National Institute of Health grants (GM068069) to V.H. and (T32HD007403) to D.N.

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