Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium

SUMMARY

Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system to understand how such asymmetries are generated and how they contribute to the formation of complex anatomical structures. 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, while 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 critical roles for this major asymmetry in wing growth and patterning.

INTRODUCTION

The subdivision of fields of cells into progressively smaller domains is critical for the formation of complex anatomical structures. One of the earliest events in wing development is the subdivision of the wing primordium into two apposing surfaces known as the peripodial epithelium (PE) and the disc proper (DP) (Cohen, 1993; Fristrom, 1993; Milner, 1984). Despite the central role of this event in generating two fields of cells with distinct morphologies and developmental potentials, the mechanism that establishes this major subdivision and its role in wing development remain poorly understood. The formation of the wing appendage depends on the establishment of the anterio-posterior (AP), dorso-ventral (DV) and proximo-distal (PD) axes within the DP (reviewed in 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 by the secreted Wg ligand and the Epidermal Growth Factor (EGF) receptor ligand, Vein. Wg is induced in a wedge of ventro-anterior cells (Couso et al., 1993; Klein and Arias, 1998; Ng et al., 1996; Williams et al., 1993), and induces the expression of the Pdm1 gene 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 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 zfh-2 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, 1993; Milner, 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 certain 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 in) (Gibson and Schubiger, 2001). Lineage analysis implied that peripodial cells stream laterally to populate the DP (McClure and Schubiger, 2005; Pallavi and Shashidhara, 2003). Despite these intriguing results, the precise contribution of the PE to the growth and patterning of the wing appendage remain poorly understood largely because the mechanisms that specify PE fate are not known.

The 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 Fig1L 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 C, 2006; Hao et al., 2003). While 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 C, 2006; Hao et al., 2003; Hatini et al., 2005).

Figure 1. Bowl accumulates in the PE and is absent from the DP.

Bowl (red); wg-LacZ or odd-lacZ (green). Optical sections at the plane of the PE (A, C, E & G) and DP (B, D, F & H). (B) 48 AEL; wg-LacZ was detected in the DP in a wedge of ventral cells (arrow). (D) 72h AEL; (F) 96h AEL; wg was upregulated along the DV compartment boundary (arrows in D and F). (A, C and E) During these developmental stages, Bowl accumulated broadly in the PE, and was absent from the DP (B, D, F) except for the lateral margins of the notum (arrowheads in D and F). (G-H) 120–144h 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-J)) Everting discs. (I) 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 point the lateral margins of the notum, asterisk to ventro-anterior hinge). (K) Adult wings; odd-lacZ was detected in ventro-proximal hinge nuclei. (L) The regulatory interactions connecting drm,lines and bowl.

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. Instead, the PE supports the growth of the DP and the formation of the notum and prevents the induction of secondary wing fields. Our findings reveal a mechanism that generates a major asymmetry across the wing primordium and the contribution of this asymmetry to wing growth and patterning.

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, 1F, arrows). Bowl, however, was detected in the PE (Fig 1C and 1E) and in the lateral margins of the notum (arrowheads in Fig1D and 1F). 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 and 1H). However, Bowl was downregulated in the medial region of the PE and absent from its posterior margin. In everting discs, 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 and 2B, 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, while in the DP where Bowl was absent Lines was nuclear and active.

Figure 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 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 and E-G point to 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 24–48h AEL in A-D and at 72–96h AEL in E-G.

Lines inhibits Bowl accumulation in the DP

We also expressed a Flag-Bowl transgene in FLP-out clones to determine if 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& 2F, respectively). Reciprocally, Bowl was destabilized in the PE in clones expressing a “strong” lines transgene (UAS-Lines 9.2; 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 also found that Bowl levels were unaffected in odd mutant clones. However, we were unable to test sob function since sob mutant alleles are not available. 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 proteins necessary to stabilize Bowl in the PE.

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 overexpressed the “strong” lines transgene 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. 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’, 3C and 3D’-D”’) was replaced with the thickened pseudostratified CE that forms the DP (Fig 3B’ and 3E’-E’”). Coincidentally, the expression of the peripodial markers Bowl, Ubx and Eya was lost in these discs (Fig 4B and C and 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 Figs 3A to 3B; Experimental discs: 39 mm², N=7, SD=18 mm²; Wild type: 147 mm², N=7, SD=21 mm²). These discs survived past the larval stages and elongated during metamorphosis (Fig S1) but failed to contribute to adult structures. We obtained similar phenotypes by driving expression of UAS-Lines with C311-GAL4 and Tsh-GAL4 (Fig S2).

Figure 3. Overexpression of lines in the PE replaces the PE with DP.

(A, C-D) Wild type; (B, E, F-G) Ubx-GAL4 UAS-Lines (Ubx>Lines); Arrowheads in A, B, D and E point to plane of Z-section in A’, B’, D’-D’” and E’-E’”, respectively. (A-B) Armadillo; cellular outlines are detected in apical sections; bright dots reveal adherence junctions in Z-sections. (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) Nub (red) is restricted to the DP; Dapi (blue). (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) dRice (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.

Figure 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 and L point to plane of Z-sections. (A) Wild type; Ubx (red) and Bowl (green) are detected 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) were absent; Arrow in B points to a leg where Bowl accumulation is not affected. (D) Lines was broadly nuclear. (E) Wild type; Vg (red) marks the pouch; Zfh-2 (green) marks the hinge. (F) Ubx>Lines; Vg and Zfh-2 were expressed in adjacent domains in both layers of the wing disc. The Zfh-2 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. (I, K) Wild type. (I) Dll (red), and (K) Sal are induced in response to Wg and Dpp signaling, respectively. (J) Dll, Zfh-2 (green) and (L) Sal were induced in both layers of these discs. Scale bar=100µm in B-D and F, H, J and L and 200µm in A, E, G, I and K.

Organ growth largely depends on a balance between cell proliferation and cell death. Therefore, to determine the basis for the reduction in wing size, we examined the expression of Phospho-Histone H3 (PH3) and activated Caspase-3 (dRice) 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 to 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 Zfh-2 expression (Whitworth and Russell, 2003) to determine if the pouch, the hinge or the notum were affected. In wild type, Vg is restricted to the distal pouch, while Zfh-2 is restricted to the hinge (Fig 4E). Nub is detected in the pouch and in part of the dorsal hinge (Fig 3D, 3D” and 3D’”). In experimental discs, Nub (Fig 3E, 3E”-3E’”), Vg, and Zfh-2 (Fig 4F and 4J) 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 Zfh-2 (arrowhead in Fig 4F) indicating that the notum, which is Zfh-2 negative, was nearly lost. The reduction in notal growth could partially account for the reduction in overall wing size.

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 (Fig S3) 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 traverses 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 (Fig S3). Dll and Sal were also detected in both layers of these discs (Fig 4J and 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 critical role in promoting the growth of the notum.

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 if 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 lines during the early first instar gave rise to two general classes of wing abnormalities. In the most severe class (Class I, Fig 5D), 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, SD=17%). Since wing rudiments lacking large patches of lines expressing clones were not recovered (N=58 rudimentary discs examined) we were forced to conclude that peripodial cells survived in experimental discs and assumed an inappropriate DP fate. In the second class (Class II), 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 denotes 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 roughly 26% of the surface area of the entire wing (N=9, SD=5%) and roughly 50% of the surface area of the secondary wing fields. To 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 in Fig 5G-H) along the AP compartment boundary. The Ptc>Lines expressing cells in the PE adopted DP fate and permitted the induction of secondary wings (Figure 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). We therefore conclude that lines can reprogram the PE to adopt DP fate during early stages of wing development.

Figure 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. (G) Ptc and (H) Ptc>Lines expressing cells marked with GFP. 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 point to 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 point to secondary wings, small arrowheads point to Wg expression along the wing margin). These clones localized to the central region of the secondary wings. (F) In a subset of discs, the notum was reduced or absent. Asterisk denotes the loss of Wg expression in the presumptive notum. Large arrowheads point to plane of Z-section shown in insets. (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); Aristaless (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.

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 points to 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.

Figure 6. lines promotes cell survival in the DP, while bowl promotes cell survival in the PE and the specification of PE fate.

(A, D) Control clones, (B-C) lines mutant FLP/FRT clones, (E) drm expressing clones, (F) bowl mutant MARCM clones, and (H) bowlRNAi expressing 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. 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 points to a bowl mutant clone near the disc stalk. (G) Quantitative analysis of the recovery of wild type, bowl and lines mutant clones generated at the first (L1) and second (L2) instars. To calculate the % recovery, we pooled clones and separately the corresponding wild type twin spots from several discs. Large confluent patches of bowlRNAi expressing clones completely transform the PE into DP (not shown), while (H) smaller patches permitted the induction of secondary wings.

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, lines mutant clones survived poorly in the DP (Fig 6C), and were much smaller than respective wild type twin spots. Similar to the lines mutant 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, while lines promotes cell survival in the DP.

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

While the bowl mutant clones generated at the first instar survived poorly, bowlRNAi-expressing clones readily survived and phenocpied the phenotypes induced by ectopic expression of lines. These clones gave rise to small and spherical discs in which the PE was replaced with the DP (Class I), or to discs containing supernumerary wing fields (Class II) depending on the size and position of the clones (Fig 6H and data not shown). These results indicate that bowl acts reciprocally to lines to specify PE fate and to inhibit DP fate. We speculate that the residual activity of bowl that remained in bowlRNAi and in lines expressing clones described above allowed the clones to survive and adopt an alternative cell fate. We therefore conclude that bowl promotes cell survival in the PE and specifies PE fate at the early first instar. We note that the bowlRNAi transgene may weakly downregulate odd and sob. Thus, it is formally possible that bowl acts either alone or cooperates with odd, sob or both genes to specify PE fate and to inhibit DP fate.

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 (Figure 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, and 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 and 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.

Figure 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” point to planes of Z-sections shown in C”’-C”“ and D”‘, respectively. Images in A-B and E-F are projections. lines mutant clones occasionally overproliferated and protruded from the disc epithelium (A, E-F). Wing 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. Reciprocally, the peripodial markers Ubx (C) and Eya (D) were ectopically expressed in these clones. Ubx was only detected in posterior DP clones. (C”’-C”“, D”’) Z-sections reveal basal extrusion of clones from the disc proper. (E) Tsh and (F) Hth, which mark the PE, the notum and the hinge, were ectopically expressed in these clones.

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 questions, 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”, C-H & Fig 8B’, respectively). We examined the positional identity of these clones with molecular markers to determine if 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 Zfh-2 proteins, which localize to the hinge and control hinge formation, were ectopically expressed in the clones (Fig 8C, D and E, respectively, and 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 antagonizes this fate transformation in a graded manner as was 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 bowl function, indicating that the lines clonal phenotypes were due to the stabilization of Bowl (Fig S4). 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 and H, respectively). Overall, these results indicate that lines and bowl are required at the early first instar to specify the DP and the PE, respectively, 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.

Figure 8. lines specifies distal pouch fate and inhibits proximal hinge fate at the third instar.

(A, C-H) Late third instar lines mutant clones. (B) drm-expressing clones. Arrowheads in A and B point to planes of Z-sections shown in A’-A” and B’-B”. Arrows in C-H point to magnified regions shown in insets. (A) Bowl (red), GFP (green). (A) lines mutant clones and (B) drm-expressing clones extruded basally (arrows in A’ and B’). (C) Tsh, (D) Hth, and (E) Wg, whose expression 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) 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.

DISCUSSION

The generation of molecular asymmetries across undifferentiated fields of cells is central to embryonic development. Early during development, the two surfaces of the flattened wing primordium assume distinct DP and PE fates and hence distinct morphologies and developmental roles. Our findings elucidate, 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 able to provide definitive evidence that the PE controls the scope and overall growth of the wing field and the formation of the notum.

The establishment of molecular asymmetries across the wing primordium

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 ectoderm (Cohen et al., 1991; Cohen, 1990). Bowl accumulates in the posterior en-expressing cells in the embryonic ectoderm (Hatini et al., 2005), suggesting that the wing primordium also inherits the PE/DP subdivisions from preexisting asymmetries across the embryonic surface ectoderm.

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

lines and odd-skipped genes act as a binary 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; Hohansen et al., 200; 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 bowl function in the PE transforms the PE into DP. Reciprocally, the removal of lines function from the DP transforms the DP into PE. Our data further suggests that lines exerts its function by controlling the stability of the Bowl protein. 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, 3E, 4B-D, 4F-L), and the notum was dramatically reduced in size (Fig 4F). 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 PE is neither required for the establishment or the maintenance of wg or dpp expression, nor for the long-range signaling activities of these morphogens. (Fig 4J, 4L). Instead, our findings suggest that the PE acts in parallel to the AP, DV and PD patterning systems, in part, by promoting cell survival in the DP. The PE may also contribute progenitor cells to the growing DP (McClure and Schubiger, 2005; Pallavi and Shashidhara, 2003) and the loss this progenitor cell population may account for the severe reduction in notal growth.

Our results argue that lines and bowl function as field-specific selector genes to specify the survival, identity and behavior of the DP and the PE of the wing primordium, respectively (reviewed in Mann and Morata, 2000). bowl may specify the squamous epithelial morphology, the responsiveness of the PE to wing inducing signals, and the expression of signals that support the growth of the DP. By destabilizing Bowl in the DP, lines may specify the pseudostratified columnar morphology of the DP, its responsiveness to wing inducing signals and its interaction with the PE. Our studies define a new system in which to dissect these developmental pathways.

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 ywhs-FLP; FRT42D Ubi-GFP and ywhs-FLP; Ubi-GFP FRT40A (B. Edgar) were used to induce FLP/FRT clones and ywhs-FLP Tub-GAL4 UAS-GFP-6Xmyc-NLS; FRT42D Tub-Gal80 hs-CD2, y⁺ (gift of G. Struhl) to induced MARCM clones. The lines^G2 (Bokor and DiNardo, 1996), lines^2f (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}odd^rk111 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-Bowl RNAi (VDRC #3774 and #3775), UAS-LinesRNAi (VDRC #16801), were expressed in clones using ywhs-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 were described elsewhere (Hatini et. al 2005). Primary antibodies used: 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-Zfh-2 (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-active 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 were described elsewhere (Patel et al., 1989).