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The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila
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Developmental Biology 257 (2003) 117–126
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The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila Kazumasa Kubota,a,b Satoshi Goto,a,c and Shigeo Hayashia,c,d,* b
a Genetic Strain Research Center, 1111 Yata Mishima, Shizuoka-ken 411-8540, Japan Department of Molecular Craniofacial Embryology, Graduate School of Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan c The Graduate University for Advanced Studies, 1111 Yata Mishima, Shizuoka-ken 411-8540 Japan d RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
Received for publication 15 July 2002, revised 17 January 2003, accepted 21 January 2003
Abstract Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal– distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal–proximal, distal, and ventral–proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Embryogenesis; Imaginal disc; Cell fate maintenance; Proximal– distal patterning; wingless; decapentaplegic
Introduction Wings and legs are two types of appendages located at the dorsal and ventral sides of the insect body wall. Although they are anatomically distinct structures, they share a common developmental origin, the embryonic limb primordium (Wieschaus and Gehring, 1976; Cohen et al., 1993). Limb primordial cells are first detected at stage 11 (staging according to Campos-Ortega and Hartenstein, 1997) as cells expressing the homeobox gene Distal-less (Dll; Cohen et al., 1989). Cell migration separates those cells into two types of primordia. The wing disc primordium invaginates to form sac-like wing imaginal discs in the dorsolateral position, and the leg primordium stays at the * Corresponding author. Fax: ⫹81-78-306-3183. E-mail address: [email protected] (S. Hayashi).
ventrolateral position. Separation of leg discs into proximal and distal domains is the first sign of proximodistal (PD) axis specification, and this subdivision is maintained throughout the leg development. PD subdivision in the leg disc is first manifested in stage 14 when the cells expressing Esg surround the distal leg cells expressing Distal-less in a circular pattern (Goto and Hayashi, 1997a, 1999). Proximal leg cells also express Homothorax (Hth), which plays a crucial role in PD patterning in later stages (Abu-Shaar and Mann, 1998; Goto and Hayashi, 1999). Cell division does not appear to play significant roles in the early stage of PD patterning, since rapid cell proliferation in imaginal discs does not take place before second larval instar. It is more likely that the embryonic imaginal disc is patterned mainly by cell specification. Intercellular signals in this patterning process are the focus of this paper. The secreted protein Wingless (Wg) is a homolog of the
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Fig. 1. Markers for proximal and distal leg domains. Second and third leg discs at stage 15 are shown. (A) Expression of Dll (blue, A⬘) and Esg (green, A⬙). About a dozen cells coexpress both markers. (B) Expression of Hth (red, B⬘) and Esg (green, B⬙). All Esg-positive cells express Hth at a detectable level. Scale bars, 50 m. Fig. 2. Relationship between the expression pattern of wg, dpp, and markers for the leg and wing disc cells. Lateral views of first to third thoracic segments are shown, except for (C), which shows left half of a ventral view. Anterior facing left. Arrowheads indicate the leg disc, and a dagger in (A) indicates the limb primordium. Arrow indicates the wing disc primordium. (A, B) Expression of wg-LacZ (red) and Dll (green) in the limb primordium at early stage 11 (A) and in the leg disc at late stage 12 (B). Dll is initially expressed in the common limb precursor cells (A). (C) At stage 15 when the wing disc (out of focus) has separated from the leg disc, expression of Esg (green) in the leg disc overlaps with the wg stripe (red), except for the dorsal proximal part of the leg disc. (D) Wing discs labeled with Vg (green) do not overlap with the expression of wg-LacZ at early stage 12. (E) dpp mRNA (red) and Esg (green) in the leg discs at stage 14. (F) Cells responding to Dpp signal at stage 13 are marked with anti-pMad (green). Scale bar, 50 m (A). (B–F) The same magnification as (A).
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Fig. 3. Effects of altered Wg signaling on the leg and wing disc formation. (A–F, I, J) Development of leg and wing discs was followed by the expression of Dll mRNA (red), Dll protein (inset), and Esg protein (green). (G, H) Proximal leg discs and the epidermal cells are marked with anti-Hth antibody (green). Arrowheads indicate leg discs, and arrows indicate wing discs. Broken line is the ventral midline. (A, C) Ventral view. (B, D, E–J) Lateral view. (A, B) Wild type embryos at stages 11 (A) and 15 (B). (C, D) armH8.6 mutant embryos at stage 11 show normal expression of Dll mRNA (C); however, at stage 15, Dll and Esg expression in the leg disc is severely reduced (D). (E) Expression of a dominant-negative form of TCF (TCF.DN) by Dll-Gal4 results in a reduction of the leg disc marker expression. Esg expression in the dorsal side of the leg disc, in particular, was most severely affected. (F) A Dll-GAL4 embryo carrying UAS-Daxin shows a similar phenotype. (G, H) Hth is not expressed in the distal leg region of wild type embryos (G), but is upregulated in armH8.6 mutant
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vertebrate protooncogene Wnt1 and mediates various inductive interactions in Drosophila development (Nusse and Varmus, 1992). Previous studies demonstrated that Wg is required for induction of imaginal discs during embryogenesis (Cohen et al., 1993; Simcox et al., 1989). Wg is expressed in stripes along the anterior–posterior (AP) compartment boundaries of the embryonic ectoderm and induces Dll expression, which marks the limb primordium at early stage 11 (Cohen et al., 1993). Dll is repressed at the dorsal and the ventral side of the embryo, respectively, by Decapentaplegic (Dpp) and epidermal growth factor receptor (EGFR) activity (Goto and Hayashi, 1997b). At late stage 11, the two signals start the second phase of action by being activated in the limb primordial cells (Kubota et al., 2000). Differential activation of the Dpp and EGFR creates asymmetry in the limb primordium along the dorsoventral (DV) axis and specifies the wing and leg primordium (Kubota et al., 2000). Studies on larval leg disc development have revealed pivotal roles of the PD subdivision of the leg disc in PD axis patterning. The distal domain corresponds to the tarsus, tibia, femur, and trochanter and is under the influence of Wg and Dpp, whose combined activity promotes expression of Dll (Lecuit and Cohen, 1997). The proximal leg domain corresponds to the coxa, and a part of the body wall, and is identified by the expression of Homothorax (Hth) and nuclear localization of Extradenticle (Exd). These homeodomain proteins form a complex that promote proximal leg development and prevent distal leg development by repressing cellular response to Wg/Dpp (Abu-Shaar and Mann, 1998; Gonzalez et al., 1998). Loss of the distal leg structures due to the hedgehog (hh) mutation leaves the proximal domain apparently intact (Gonzalez and Morata, 1996), suggesting that the proximal leg domain is set as a default in the larval leg disc. One crucial question remains unanswered: how is proximal leg identity established in the embryonic leg primordium? In this work, we investigated the roles of Wg signaling in the formation of leg discs during embryogenesis. Unlike the previous work reporting that Wg plays no role in the late stage of embryonic leg disc development (Cohen et al., 1993), we show that Wg and Armadillo (Arm), the general transducer of Wnt signaling, are required to maintain the fate of both proximal and distal leg cells. We also show that Dll restricts the expression of the proximal marker Esg. In addition, ectopic activation of wg in wing discs suppresses its formation. These results demonstrate that late roles of Wg signaling in embryonic leg discs are to establish PD axis and to suppress wing development.
embryos (H). (I, J) Ectopic activation of Wg signaling in the wing disc inhibits wing disc formation. When compared with the wild type control (B), Esg-positive cells in the wing and haltere discs are reduced in embryos expressing wg (I) or armS10 (J) induced by Dll-Gal4. The pattern of leg discs was nearly normal. Scale bar (H), 50 m.
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Materials and methods Fly strains The following fly strains were used: DllP-LacZ (D11l(2)01092; Spradling et al., 1995); DllMP; D11-GAL4 (Calleja et al., 1996); wg-LacZ (Kassis et al., 1992); wgIL114 (Baker, 1987); UAS-wg (Lawrence et al., 1995); UAS-armS10 (Pai et al., 1997), UAS-DTCFdeltaN (van de Wetering et al., 1997), armYD35 and armH8.6 (Peifer, 1995) ; UAS-Daxin (Hamada et al., 1999); UAS-dad (Tsuneizumi et al., 1997); UAS-esg (Fuse et al., 1994); and tkv8 and UAS-dpp from Bloomington Stock Center. See FLYBASE (FlyBase, 2003; http://flybase.org/) for more information. The UAS-Dll strain was made by inserting a Dll cDNA into the pUAST vector (Brand and Perrimon, 1993) and injecting the resultant plasmid by the germ line transformation technique. The cDNA was isolated by RT-PCR and spanned from 4 bp upstream of the initiator codon to ⫹45 bp downstream of the terminator codon. An insertion into the third chromosome induced secondary axis formation, when expressed in the larval leg discs, and this strain was used in subsequent analyses. Temperature shift experiments Temperature shift experiments were performed as follows: embryos from wgIL114/CyO-ftzlacZ stock were collected for 1.5 h at 18°C and were cultured at 18°C until stages 12, 13, and 14, and then the temperature was shifted to 29°C. Embryos at stage 15 or 16 were fixed and stained with the wing and leg disc markers and with anti- galactosidase to identify genotypes.
here expression patterns of proximal and distal leg marker genes at a cellular resolution. At stage 11, the early expression of D11 expression marks entire limb primordium that gives rise to both wing and leg discs (Cohen et al., 1993; Goto and Hayashi, 1997b). After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc (Cohen et al., 1993). Double labeling of stage 15 leg discs revealed that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg (Fig. 1A), suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium. It was shown that in the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage (Abu-Shaar and Mann, 1998). Double labeling with antibodies against Esg and Hth revealed that the Esg expression overlaps with Hth expression (Fig. 1B). Hereafter, we used Esg as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region. The relationship between Wg expression and limb primordia
Results
We next compared the expression domain of wg and the position of wing and leg primordia. Wg expression in the trunk ectoderm started as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlapped with wg stripes, as revealed by the wg-lacZ reporter (Fig. 2A; Cohen et al., 1993). At late stage 11, wg-lacZ stripes broke up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein became limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes (Fig. 2B; Couso et al., 1993). The ventromedial wg stripe also overlapped with proximal leg cells that were labeled with anti-Esg at stage 15 (Fig. 2C). The ventral half of proximal leg cells was nearly completely included within the ventral wg stripes. The dorsal half of leg cells was also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia were first recognizable at stage 12 as cells expressing Vestigial (Vg), they did not overlap with the stripe of wg (Fig. 2D). Dorsal cell migration further separated wing primordia from the source of Wg at stage 15 (Fig. 2C; Cohen et al., 1993). The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development.
Markers for proximal and distal leg domains
Requirement for Wg signaling in leg disc formation
The expression patterns of Dll enhancer trap (DllP-LacZ) and Esg were reported previously (Goto and Hayashi, 1997b). For the sake of discussion that follows, we describe
It was previously shown that Wg is required for the induction of the thoracic limb primordium (Cohen et al., 1993) and other imaginal discs (Simcox et al., 1989). To
Histochemical analyses In situ hybridization was performed with digoxigenin-labeled antisense RNA probes of Dll (Panganiban et al., 1994) and dpp (Padgett et al., 1987). Anti-Dll (Panganiban et al. 1994), anti-Hth (Pai et al., 1998), anti-Esg (Fuse et al., 1994), anti-Vg (Williams et al., 1991), anti-pMad (Kubota et al., 2000), and anti--galactosidase (Cappell) were used for immunostaining, which was sometimes amplified with a TSA indirect kit (NEN). Antibody binding was detected with Cy2or Cy3-conjugated secondary antibodies or streptavidine (Amersham) and observed by a confocal microscope (LSM410; Carl Zeiss). Homozygous mutant embryos were identified by the absence of LacZ expression from balancer chromosomes.
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investigate a late role of Wg signaling, we studied the functions of intracellular signal transducers of Wg. The Drosophila homologue of -catenin encoded by armadillo (arm) plays dual roles, one as a mediator of Wg signaling by regulating transcription of various target genes, and the other as a component of Cadherin-dependent cell adhesion (Peifer, 1995). We analyzed two alleles of arm, one being null allele armYD35, and the other armH8.6, which is specifically defective in Wg signaling (Peifer, 1995). In the embryos hemizygous for either of the allele, Dll was expressed normally at stage 11 (Fig. 3C, and data not shown), suggesting that the maternally supplied arm product is sufficient for induction of the limb primordium. Dll expression decayed shortly after this stage (data not shown), and by stage 15, both protein and mRNA expression remained only in a few cells. Furthermore, the proximal leg markers Esg was completely absent (Fig. 3D, and data not shown). On the other hand, formation of the wing disc was normal; suggesting that the late function of Wg signaling is dispensable for wing formation. Since both armYD35 and armH8.6 showed the same phenotype, the function of Arm in Wg signaling, but not in cell adhesion, is required for leg disc development. To confirm whether Wg signaling is required cell autonomously for leg disc development, we expressed the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) in the limb primordia. TCF is an HMG domain-containing DNA binding protein that interacts with Arm to regulate transcription (van de Wetering et al., 1997). Daxin is a regulator of Arm degradation and negatively regulates Wg signaling (Hamada et al., 1999). We used the Dll-Gal4 driver (Calleja et al., 1996), which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs (Kubota et al., 2000). In Dll-GALA embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced (Fig. 3E and F). Expression of Esg was preferentially reduced in the dorsal side (Fig. 3E). The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells (Fig. 3D–F). In arm mutants, Hth-expressing cells expanded to the distal domain (Fig. 3H). This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. On the other hand, disc-specific reduction of Wg signaling did not affect wing disc formation (Fig. 3E and F), although the DllGal4 driver is active in the wing primordium (see Fig. 5B). We conclude that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation.
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Proximal leg development is defective in wg mutants The requirement for Arm in the leg disc development was unexpected from the result of Cohen et al. (1993). The authors reported that wgIL114 (wgts) embryos transferred from a permissive to a restrictive temperature after the limb primordium is fully induced showed a normal pattern of Dll mRNA expression. They concluded that wg is not required for leg disc patterning in late embryogenesis. We decided to revisit the phenotype of wgts with use of additional disc markers. Temperature shift experiments were performed as shown in Fig. 4A. wgts embryos carrying DllP-LacZ were allowed to develop at a permissive temperature until stage 12 (Fig. 4D), stage 13 (Fig. 4F), and stage 14 (Fig. 4H), and the cultures were then transferred to the restrictive temperature. Embryos were fixed at stage 15 (Fig. 4E and G) or 16 (Fig 4I). Embryos were double labeled with anti-Esg and anti-galactosidase to mark wing and leg disc cells (Fig. 4B–I). At the permissive temperature, DllP-LacZ expressing cells were encircled by cells expressing Esg as early as stage 14 (Fig. 4H) and stage 15 (Fig. 4B), suggesting that the PD domains in the leg disc are already established by stage 14. The pattern of Esg expression wgts embryos at the permissive temperature was indistinguishable from the control embryos (Fig. 4B). In wgts embryos, we observed a loss of Esg expression. The severity in the phenotype increased when the temperature was shifted at an earlier stage. Temperature shift at stage 12 (Fig. 4D) caused a near complete loss of the Esg expression (Fig. 4E), while the temperature shift at stage 13 or 14 caused a partial loss of the dorsal part of the Esg expression (Fig. 4G and I). These results suggest that Wg is required for the cell fate maintenance of the proximal leg expressing Esg. On the other hand, we were unable to detect significant change in the expression of DllP-LacZ expression (Fig. 4E, G, and I), and Dll mRNA (data not shown), as previously reported (Cohen et al., 1993). The preferential loss of dorsoproximal cells in wgts mutants suggests that dorsal and ventral halves of proximal leg cells have different requirements for Wg. The higher requirement for Wg by dorsal–proximal cells is consistent with the location of those cells away from the stripe of Wg (Fig. 2C). Perhaps a larger amount of active Wg must be produced to reach dorsal–proximal cells to induce esg. Wing discs are present in wgts embryos at the restrictive temperature (Fig. 4E, G, and I), indicating that, while induction of the limb primordium took place normally, PD patterning in the leg disc was specifically impaired. More importantly, circular arrangement of proximal and distal cells was established by stage 14 (Fig. 4H), and this pattern decayed after inactivation of wg activity (Fig. 4I). We therefore concluded that the continuous input of Wg activity is necessary to maintain the proximal– distal pattern in the leg disc.
Fig. 4. Temporal requirement for Wg activity by leg disc cells. (A) Temperature shift protocol. Embryos were shifted from the permissive to the restrictive temperature at stages 12, 13, or 14. (B–I) Lateral views of embryos showing the first to third thoracic segments. Distal leg disc cells were marked with DllP-LacZ (red). Proximal leg disc cells and wing disc cells were marked with Esg (green). Anterior is left. Arrowheads indicate the leg disc. (B) A stage 15 wgIL114 embryo cultured continuously at the permissive temperature. Expression patterns of DllPLacZ and Esg were indistinguishable from wild type control (compare with Fig. 3B). (C) Same as (A), showing only the expression of Esg. (D, F, H) wgIL114 embryos were allowed to develop at 18°C until stages 12 (D), 13 (F), or 14 (H). (E, G, I) wgIL114 embryos were cultured at the restrictive temperature and fixed at stage 15 (E, G) or 16 (I). While the number of the proximal (Esgpositive) leg disc cells decreased (arrows), distal leg disc and the wing disc were normal. The number of dorsal–proximal leg disc cells in (E) was most severely reduced. Scale bar, 50 m (C).
Fig. 5. Role of Dll in the circular patterning of the leg disc. Anterior is left. Arrowheads indicate the leg disc cells. (A, C–F) Distal and proximal leg cells at stage 15 were labeled with antibodies against Dll and Esg as indicated in each panel. (A) A Dll null mutant embryo shows a disruption of the circular pattern of the Esg expression in the leg disc. Compare this figure with Fig. 3B as a control. (B) Expression pattern of Dll-GAL4 revealed by UAS-LacZ reporter at stage 15. Dll-GAL4 construct is expressed in both the wing and the leg disc cells. (C, D) An embryo overexpressing Dll driven by Dll-GAL4. Expression of Dll was expanded to the proximal leg domain, and Esg expression was reduced in the leg disc. Dll protein was accumulated to a high level in wing discs, but Esg expression was not affected. (E, F) Dll expression was not eliminated by overexpression of Esg by Dll-GAL4. Compare this figure with Fig. 1A as a control. Scale bar, 50 m (A).
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Contribution of Dpp in PD patterning of the leg
Role of Wg signaling on wing disc formation
Although Wg is expressed in ventrolateral stripes, formation of distal leg cells is limited to the dorsal tip of the wg stripe (Fig. 2B), suggesting the presence of a second localized signal restricting Dll expression. Previous works have shown that dpp is expressed prominently in the dorsal side of the limb primordium at late stage 11 (Goto and Hayashi, 1997b; Kubota et al., 2000). Subsequently, intense dpp expression moves in a dorsal direction, forming a longitudinal stripe overlapping the wing primordium (Cohen et al., 1993; Goto and Hayashi, 1997b). Close inspection of embryos revealed an additional expression of dpp, running along the DV direction in stripes that reach the center of the leg discs (Fig. 2E). To examine the activation pattern of Dpp signaling, we monitored Dpp-dependent signaling events using an antibody recognizing the phosphorylated form of MAD (pMAD; described as pSSVS in Kubota et al., 2000). pMAD was expressed in broad stripes that appear to include dpp-expressing cells, and the ventral edge of pMad expression abuts the dorsal edge of wg-lacZ expression (Fig. 2F, compared with E). These observations indicate that, from their inception, leg discs are subdivided into dorsal and ventral domains, each marked with high expression of pMAD or wg, respectively. We previously showed that, upon a loss of Dpp type I receptor Thick veins (Tkv), the expression of Dll RNA decays after stage 15 (Kubota et al., 2000). This result was confirmed here by the use of anti-Dll antibody (data not shown). It was also shown that reduction of Dpp signaling by overexpression of Dad, an inhibitory Smad, by Dll-Gal4 severely reduced Dll and Esg expression (Kubota et al., 2000). These results indicate that Dpp signaling in the dorsal side of the leg disc is required for both proximal and distal leg cell fate. The complementary pattern of Wg and Dpp expression in larval leg disc is maintained by mutual repression (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffmann, 1996; Theisen et al., 1996). We asked whether a similar mechanism is used to regulate Wg and Dpp expression of embryonic leg discs. dpp expression in the lateral ectoderm did not change in arm mutants, and no change of Wg expression was observed upon overexpression of Dad (data not shown). In addition, Dll-GAL4 embryos expressing Wg or armS10 showed normal expression of dpp mRNA, and embryos with overexpression of Dpp by DllGAL4 showed no significant change in the pattern of Wg expression (data not shown). Since these treatments are sufficient to alter the fates of wing and leg disc cells (Goto and Hayashi, 1997b; Kubota et al., 2000), we conclude that once the leg disc is specified, the domain of Wg and dpp expression cannot be altered by change of Wg and dpp signaling within the physiological range. The results suggest that the domains of Wg and Dpp expression within the leg disc are set up independently of each other.
Does Wg signaling have any function in the wing disc formation? There appears to be no requirement for the Wg signal in wing discs, because wg is not expressed there from stage 12 (Fig. 2D) to stage 15, and reduction of wg activity after specification of wing discs caused no change in the wing disc formation (Figs. 3 and 4). In contrast, ectopic activation of the Wg signal in the wing disc by expression of Wg or stabilized Arm (armS10) driven by Dll-Gal4 caused a reduction in the number of wing disc cells (Fig. 3I and J), suggesting that the Wg signal must be downregulated in lateral ectoderm to allow wing disc development. Restriction of proximal domain by Dll The circular pattern of Esg expression in the leg disc at stage 15 is a hallmark of proximal– distal subdivision in the leg. The next question was how the pattern of Dll and Esg expression, each requiring Wg and Dpp signals, is established. We wanted to determine whether Dll regulates expression of Esg. A null mutation of Dll allowed the expression of Esg at the center of the leg disc (Fig. 5A). Cell counting demonstrated that the number of Esg-positive cells did not change significantly upon loss of Dll. We next asked what effect Dll overexpression has on Esg expression. DllGal4 allowed Dll to be expressed in the proximal domain of the leg disc (Fig. 5C). In such embryos, a reduction of Esg expression was observed in proximal leg discs, where ectopic expression of Dll was high (Fig. 5D). On the other hand, ectopic Dll did not affect Esg expression wing discs, suggesting that the inhibitory effect of Dll on Esg is contextdependent. These results suggest that Dll specifically limits Esg expression in leg discs. We also wanted to know whether Esg regulates Dll expression. However, misexpression of Esg by Dll-Gal4 caused only a marginal reduction of Dll expression (Fig. 5E and F; compare this figure with Fig. 1A as a control), and loss of esg caused no change in the pattern of Dll expression (data not shown). Taken together, these results suggest that Dll is capable of restricting the proximal leg domain.
Discussion Specification of animal appendages from a two-dimensional field of the embryonic ectoderm is one major question in embryology. This process in Drosophila involves multiple steps: induction of limb primordial cells in the lateral ectoderm followed by separation of those cells by cell migration into two clusters. The dorsal cells become the wing disc, and ventral cells become the leg disc. The PD axis in the leg disc is apparent at the onset of its formation, suggesting that its formation is an immediate consequence of cell interaction in the embryonic ectoderm. We show here that Wg signaling plays a key role in this process by helping
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to recruit two types of cells that constitute the proximal and distal domains. Establishment of PD axis We have shown that the cell fate maintenance of proximal leg requires continuous signaling by Wg (Fig. 4). The requirement for arm and wg was higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe (Fig. 2C), we propose that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal–proximal cells require a higher level of Wg to be produced to reach their position. Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp that is expressed in stripes abutting the Wg stripe. The requirement for Dpp by distal leg development was previously shown (Goto and Hayashi, 1997b; Kubota et al., 2000). Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by distal gene, as we have shown here by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, repression of proximal genes by Dll in larval leg discs was previously shown (Gonzalez et al., 1998; Abu-Shaar and Mann, 1998). The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc were shown to have distinct cell-adhesive properties from those in the Dll-expressing distal domain (Goto and Hayashi, 1999; Wu and Cohen, 1999), and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc. With the results presented here, we tested a distal organizer model (Campbell and Tomlinson, 1995; Fig. 6A ). The model proposes that a distal organizer placed in the field of developing appendage signals surrounding cells to acquire proximal cell fate. If this signal acts in all directions, circular arrangement of proximal cells can be achieved (Fig. 6A). Although a recent work on postembryonic leg devel-
Fig. 6. Two models of leg specification. (A) Distal organizer (red circle) is specified in the embryonic ectoderm and instructs surrounding cells to acquire proximal cell fate (Campbell and Tomlinson, 1995). (B) Dorsal– proximal and ventral–proximal cells (green crescents) and distal cells are specified separately. These cells (red circles) are assembled to form a circular pattern. Central figure is a schematic drawing of a leg imaginal disc at stage 15 showing the relationship between Wg and Dpp expression domains. Red cells are Dll-expressing distal cells. Green cells are Esgexpressing proximal cells.
opment (Lecuit and Cohen, 1997) does not support this model, it is still one of the best possible models for embryonic leg specification. We demonstrated that dorsal and ventral halves of proximal leg cells have different requirements for Wg. The way Wg acts to organize proximal cell differentiation is not consistent with the distal organizer model. Rather, the results support a second model (Fig. 6B) where dorsoproximal, distal, and ventral–proximal cells are specified separately and assembled to form a circular pattern. Function of Wg and EGFR signals in leg development The preferential loss of proximal leg cells upon partial loss of Wg signaling is very similar to the phenotype of EGFR mutant embryos we have previously reported (Kubota et al., 2000), suggesting that both Wg and EGFR contribute to differentiation of proximal leg cells. We noted, however, a difference in the temporal requirement for the two signals. EGFR signaling is activated transiently at the time of disc specification, and the requirement of its activity is limited to a short period around this stage (Kubota et al., 2000). It was proposed that EGFR acts within limb primordial cells to promote leg development (Kubota et al., 2000). We have shown here that Wg is persistently expressed at the ventral part of the leg disc and maintains the fate of proximal and distal leg cells (Figs. 2– 4). These findings indicate that the leg disc development is initiated by transient activation of EGFR, and its cell fate is maintained by the persistent activity of Wg. Inhibition of wing disc development This and our previous works have demonstrated that Wg and EGFR signaling are not active in wing discs and are not
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required for wing formation after stage 11 (Figs. 2– 4; Kubota et al., 2000). On the other hand, ectopic activation of either of the signals in the limb primordium suppresses the wing disc development (Fig. 3I and J; Kubota et al., 2000), suggesting that downregulation of Wg and EGFR signals have a permissive role in the wing development. These signals are reactivated in postembryonic stages to organize the wing disc. The downregulation of Wg and EGFR signals in a prospective wing disc is accomplished by two mechanisms, one by limiting activation of the two signals to the ventral side of the embryo, and the other by allowing wing primordium to migrate to the dorsal direction away from the source of the inhibitory signals. Thus, cell migration serves as a novel mechanism to restrict the effect of diffusible signaling molecules.
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question as to which of the mechanisms is used in other primitive hemimetabolous insects, where the specification and growth of the leg occur simultaneously. Acknowledgments We thank Grace Panganiban, Henry Sun, Sean Carroll, Steve Cohen, Makio Tokunaga, Tadashi Uemura, Makoto Nakamura, and the Bloomington Stock Center for gifts of antibodies, cDNAs, and/or fly strains. We are grateful to Yasushi Hiromi for critically reading an early version of the manuscript. This work was supported by grants from JSPS (Research for the Future) (to S.H.) and from the Ministry of Education, Science, Sports and Culture (to S.G.).
Differences in the mechanism of leg patterning in the embryo and larva
References
Although the analogous pattern of Wg and Dpp expression plays essential roles in PD patterning in embryonic and larval leg development (this work; Goto and Hayashi, 1997b; Lecuit and Cohen, 1997), we noted significant differences. In embryonic leg discs, expression of both proximal and distal leg markers was lost in mutants of Wg signaling (Fig. 3) or Dpp signaling (Goto and Hayashi, 1997b). Therefore, Wg and Dpp contribute to both proximal and distal leg development in the embryo. In the larvae, reduction of Wg and Dpp expression due to the loss of hh function causes a loss of the distal domain, but no effect on the proximal gene expression was observed (Gonzalez and Morata, 1996), suggesting that Wg and Dpp play little or no role in the development of proximal domain. The inability of Wg or Dpp to participate in the proximal leg patterning in the larvae is due to, at least in part, the function of Hth to block activation of target genes for Wg and Dpp (Abu-Shaar and Mann, 1998). In the embryo, however, Hth does not block expression of esg, a target gene for Wg, as demonstrated by coexpression of Esg and Hth (Fig. 1B). Therefore, proximal domains of embryonic and larval leg discs are different in the way Hth regulates target genes for Wg. This difference may reflect distinct stages of leg development in the embryo, where proximal leg and epidermal cells are continuous, as defined by Hth expression (Fig. 1B), and in the larvae, where they are separated by the peripodial membrane. The complementary pattern of Wg and Dpp expression in the larval leg disc is maintained by mutual repression (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffmann, 1996; Theisen et al., 1996). We were unable to detect any evidence for mutual repression of Wg and Dpp in embryonic leg discs. Perhaps the complementary expression pattern of Wg and Dpp in the embryonic leg disc is under the control of the mechanism regulating the global dorsoventral pattern of the embryo. We have shown here that specific mechanisms are involved in embryonic development as opposed to larval leg development in Drosophila. This finding gives rise to the
Abu-Shaar, M., Mann, R.S., 1998. Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development. Development 125, 3821–3830. Baker, N.E., 1987. Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos. EMBO J. 6, 1765–1773. Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401– 415. Brook, W.J., Cohen, S.M., 1996. Antagonistic interactions between wingless and decapentaplegic responsible for dorsal–ventral pattern in the Drosophila leg. Science 273, 1373–1377. Calleja, M., Moreno, E., Pelaz, S., Morata, G., 1996. Visualization of gene expression in living adult Drosophila. Science 274, 252–255. Campbell, G., Tomlinson, A., 1995. Initiation of the proximodistal axis in insect legs. Development 121, 619 – 628. Campos-Ortega, J.A., Hartenstein, V., 1997. The Embryonic Development of Drosophila melanogaster, 2nd ed. Springer Verlag, Berlin. Cohen, B., Simcox, A.A., Cohen, S.M., 1993. Allocation of the thoracic imaginal primordia in the Drosophila embryo. Development 117, 597– 608. Cohen, S.M., Bro¨ nner, G., Ku¨ ttner, F., Ju¨ rgens, G., Ja¨ ckle, H., 1989. Distal-less encodes a homoeodomain protein required for limb development in Drosophila. Nature 338, 432– 434. Couso, J.P., Bate, M., Martıˆnez-Arias, A., 1993. A wingless-dependent polar coordinate system in Drosophila imaginal disc. Science 259, 484 – 489. FlyBase Consortium, The, 2003. The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Research 31, 172–175 (http://flybase.org/). Fuse, N., Hirose, S., Hayashi, S., 1994. Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot. Genes & Dev. 8, 2270 –2281. Gonzalez, C.S., Abu, S.M., Torres, M., Martinez, A.C., Mann, R.S., Morata, G., 1998. Antagonism between extradenticle function and Hedgehog signalling in the developing limb. Nature 394, 196 –200. Gonzalez, C.S., Morata, G., 1996. Genetic evidence for the subdivision of the arthropod limb into coxopodite and telopodite. Development 122, 3921–3928. Goto, S., Hayashi, S., 1997a. Cell migration within the embryonic limb primordium of Drosophila revealed by a novel fluorescent method to visualize mRNA and protein. Dev. Genes Evol. 207, 194 –198. Goto, S., Hayashi, S., 1997b. Specification of the embryonic limb primordium by graded activity of Decapentaplegic. Development 124, 125– 132.
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Goto, S., Hayashi, S., 1999. Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning. Development 126, 3407–3413. Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M., Nagai, S., Suzuki, A., Fujita, F., Shibuya, H., Toyoshima, K., Ueno, N., Akiyama, T., 1999. Negative regulation of Wingless signaling by D-axin, a Drosophila homolog of axin. Science 283, 1739 – 42. Jiang, J., Struhl, G., 1996. Complementary and mutually exclusive activities of decapentaplegic and wingless organize axial patterning during Drosophila leg development. Cell 86, 401– 409. Kassis, J.A., Noll, E., VanSickle, E.P., Odenwald, W.F., Perrimon, N., 1992. Altering the insertional specificity of a Drosophila transposable element. Proc. Natl. Acad. Sci. USA 89, 1919 –1923. Kubota, K., Goto, S., Eto, K., Hayashi, S., 2000. EGF receptor attenuates Dpp signaling and helps to distinguish the wing and leg cell fates in Drosophila. Development 127, 3769 –3776. Lawrence, P.A., Bodmer, R., Vincent, J.P., 1995. Segmental patterning of heart precursors in Drosophila. Development 121, 4303– 4308. Lecuit, T., Cohen, S.M., 1997. Proximal– distal axis formation in the Drosophila leg. Nature 388, 139 –145. Nusse, R., Varmus, H.E., 1992. Wnt genes. Cell 69, 1073–1087. Padgett, R.W., St. John, R.D., Gelbart, W.M., 1987. A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature 325, 81– 84. Pai, C.Y., Kuo, T.S., Jaw, T.J., Kurant, E., Chen, C.T., Bessarab, D.A., Salzberg, A., Sun, Y.H., 1998. The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, extradenticle, and suppresses eye development in Drosophila. Genes Dev. 12, 435– 446. Pai, L.-M., Orsulic, S., Bejsovec, A., Peifer, M., 1997. Negative regulation of Armadillo, a Wingless effector in Drosophila. Development 124, 2255–2266. Panganiban, G., Nagy, L., Carroll, S.B., 1994. The role of the Distal-less gene in the development and evolution of insect limbs. Curr. Biol. 4, 671– 675.
Peifer, M., 1995. Cell adhesion and signal transduction: the Armadillo connection. Trends Cell Biol. 5, 224 –229. Penton, A., Hoffmann, F.M., 1996. Decapentaplegic restricts the domain of wingless during Drosophila limb patterning. Nature 382, 162–164. Simcox, A.A., Roberts, I.J.H., Hersperger, E., Gribbin, M.C., 1989. Imaginal discs can be recovered from cultured embryos mutant for the segment-polarity genes engrailed, naked and patched but not from wingless. Development 107, 715–722. Spradling, A.C., Stern, D.M., Kiss, I., Roote, J., Laverty, T., Rubin, G.M., 1995. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc. Natl. Acad. Sci. USA 92, 10824 –10830. Theisen, H., Haerry, T.E., O’Connor, M.B., Marsh, J.L., 1996. Developmental territories created by mutual antagonism between Wingless and Decapentaplegic. Development 122, 3939 –3948. Tsuneizumi, K., Nakayama, T., Kamoshida, Y., Kornberg, T.B., Christian, J.L., Tabata, T., 1997. Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 386, 627– 631. van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A., Peifer, M., Mortin, M., Clevers, H., 1997. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88, 789 –799. Wieschaus, E., Gehring, W., 1976. Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster. Dev. Biol. 50, 249 – 263. Williams, J.A., Bell, J.B., Carroll, S.B., 1991. Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5, 2481–2495. Wu, J., Cohen, S.M., 1999. Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less. Development, 109 –117.
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www.elsevier.com/locate/ydbio
The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila Kazumasa Kubota,a,b Satoshi Goto,a,c and Shigeo Hayashia,c,d,* b
a Genetic Strain Research Center, 1111 Yata Mishima, Shizuoka-ken 411-8540, Japan Department of Molecular Craniofacial Embryology, Graduate School of Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan c The Graduate University for Advanced Studies, 1111 Yata Mishima, Shizuoka-ken 411-8540 Japan d RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
Received for publication 15 July 2002, revised 17 January 2003, accepted 21 January 2003
Abstract Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal– distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal–proximal, distal, and ventral–proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Embryogenesis; Imaginal disc; Cell fate maintenance; Proximal– distal patterning; wingless; decapentaplegic
Introduction Wings and legs are two types of appendages located at the dorsal and ventral sides of the insect body wall. Although they are anatomically distinct structures, they share a common developmental origin, the embryonic limb primordium (Wieschaus and Gehring, 1976; Cohen et al., 1993). Limb primordial cells are first detected at stage 11 (staging according to Campos-Ortega and Hartenstein, 1997) as cells expressing the homeobox gene Distal-less (Dll; Cohen et al., 1989). Cell migration separates those cells into two types of primordia. The wing disc primordium invaginates to form sac-like wing imaginal discs in the dorsolateral position, and the leg primordium stays at the * Corresponding author. Fax: ⫹81-78-306-3183. E-mail address: [email protected] (S. Hayashi).
ventrolateral position. Separation of leg discs into proximal and distal domains is the first sign of proximodistal (PD) axis specification, and this subdivision is maintained throughout the leg development. PD subdivision in the leg disc is first manifested in stage 14 when the cells expressing Esg surround the distal leg cells expressing Distal-less in a circular pattern (Goto and Hayashi, 1997a, 1999). Proximal leg cells also express Homothorax (Hth), which plays a crucial role in PD patterning in later stages (Abu-Shaar and Mann, 1998; Goto and Hayashi, 1999). Cell division does not appear to play significant roles in the early stage of PD patterning, since rapid cell proliferation in imaginal discs does not take place before second larval instar. It is more likely that the embryonic imaginal disc is patterned mainly by cell specification. Intercellular signals in this patterning process are the focus of this paper. The secreted protein Wingless (Wg) is a homolog of the
0012-1606/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0012-1606(03)00062-9
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Fig. 1. Markers for proximal and distal leg domains. Second and third leg discs at stage 15 are shown. (A) Expression of Dll (blue, A⬘) and Esg (green, A⬙). About a dozen cells coexpress both markers. (B) Expression of Hth (red, B⬘) and Esg (green, B⬙). All Esg-positive cells express Hth at a detectable level. Scale bars, 50 m. Fig. 2. Relationship between the expression pattern of wg, dpp, and markers for the leg and wing disc cells. Lateral views of first to third thoracic segments are shown, except for (C), which shows left half of a ventral view. Anterior facing left. Arrowheads indicate the leg disc, and a dagger in (A) indicates the limb primordium. Arrow indicates the wing disc primordium. (A, B) Expression of wg-LacZ (red) and Dll (green) in the limb primordium at early stage 11 (A) and in the leg disc at late stage 12 (B). Dll is initially expressed in the common limb precursor cells (A). (C) At stage 15 when the wing disc (out of focus) has separated from the leg disc, expression of Esg (green) in the leg disc overlaps with the wg stripe (red), except for the dorsal proximal part of the leg disc. (D) Wing discs labeled with Vg (green) do not overlap with the expression of wg-LacZ at early stage 12. (E) dpp mRNA (red) and Esg (green) in the leg discs at stage 14. (F) Cells responding to Dpp signal at stage 13 are marked with anti-pMad (green). Scale bar, 50 m (A). (B–F) The same magnification as (A).
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Fig. 3. Effects of altered Wg signaling on the leg and wing disc formation. (A–F, I, J) Development of leg and wing discs was followed by the expression of Dll mRNA (red), Dll protein (inset), and Esg protein (green). (G, H) Proximal leg discs and the epidermal cells are marked with anti-Hth antibody (green). Arrowheads indicate leg discs, and arrows indicate wing discs. Broken line is the ventral midline. (A, C) Ventral view. (B, D, E–J) Lateral view. (A, B) Wild type embryos at stages 11 (A) and 15 (B). (C, D) armH8.6 mutant embryos at stage 11 show normal expression of Dll mRNA (C); however, at stage 15, Dll and Esg expression in the leg disc is severely reduced (D). (E) Expression of a dominant-negative form of TCF (TCF.DN) by Dll-Gal4 results in a reduction of the leg disc marker expression. Esg expression in the dorsal side of the leg disc, in particular, was most severely affected. (F) A Dll-GAL4 embryo carrying UAS-Daxin shows a similar phenotype. (G, H) Hth is not expressed in the distal leg region of wild type embryos (G), but is upregulated in armH8.6 mutant
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vertebrate protooncogene Wnt1 and mediates various inductive interactions in Drosophila development (Nusse and Varmus, 1992). Previous studies demonstrated that Wg is required for induction of imaginal discs during embryogenesis (Cohen et al., 1993; Simcox et al., 1989). Wg is expressed in stripes along the anterior–posterior (AP) compartment boundaries of the embryonic ectoderm and induces Dll expression, which marks the limb primordium at early stage 11 (Cohen et al., 1993). Dll is repressed at the dorsal and the ventral side of the embryo, respectively, by Decapentaplegic (Dpp) and epidermal growth factor receptor (EGFR) activity (Goto and Hayashi, 1997b). At late stage 11, the two signals start the second phase of action by being activated in the limb primordial cells (Kubota et al., 2000). Differential activation of the Dpp and EGFR creates asymmetry in the limb primordium along the dorsoventral (DV) axis and specifies the wing and leg primordium (Kubota et al., 2000). Studies on larval leg disc development have revealed pivotal roles of the PD subdivision of the leg disc in PD axis patterning. The distal domain corresponds to the tarsus, tibia, femur, and trochanter and is under the influence of Wg and Dpp, whose combined activity promotes expression of Dll (Lecuit and Cohen, 1997). The proximal leg domain corresponds to the coxa, and a part of the body wall, and is identified by the expression of Homothorax (Hth) and nuclear localization of Extradenticle (Exd). These homeodomain proteins form a complex that promote proximal leg development and prevent distal leg development by repressing cellular response to Wg/Dpp (Abu-Shaar and Mann, 1998; Gonzalez et al., 1998). Loss of the distal leg structures due to the hedgehog (hh) mutation leaves the proximal domain apparently intact (Gonzalez and Morata, 1996), suggesting that the proximal leg domain is set as a default in the larval leg disc. One crucial question remains unanswered: how is proximal leg identity established in the embryonic leg primordium? In this work, we investigated the roles of Wg signaling in the formation of leg discs during embryogenesis. Unlike the previous work reporting that Wg plays no role in the late stage of embryonic leg disc development (Cohen et al., 1993), we show that Wg and Armadillo (Arm), the general transducer of Wnt signaling, are required to maintain the fate of both proximal and distal leg cells. We also show that Dll restricts the expression of the proximal marker Esg. In addition, ectopic activation of wg in wing discs suppresses its formation. These results demonstrate that late roles of Wg signaling in embryonic leg discs are to establish PD axis and to suppress wing development.
embryos (H). (I, J) Ectopic activation of Wg signaling in the wing disc inhibits wing disc formation. When compared with the wild type control (B), Esg-positive cells in the wing and haltere discs are reduced in embryos expressing wg (I) or armS10 (J) induced by Dll-Gal4. The pattern of leg discs was nearly normal. Scale bar (H), 50 m.
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Materials and methods Fly strains The following fly strains were used: DllP-LacZ (D11l(2)01092; Spradling et al., 1995); DllMP; D11-GAL4 (Calleja et al., 1996); wg-LacZ (Kassis et al., 1992); wgIL114 (Baker, 1987); UAS-wg (Lawrence et al., 1995); UAS-armS10 (Pai et al., 1997), UAS-DTCFdeltaN (van de Wetering et al., 1997), armYD35 and armH8.6 (Peifer, 1995) ; UAS-Daxin (Hamada et al., 1999); UAS-dad (Tsuneizumi et al., 1997); UAS-esg (Fuse et al., 1994); and tkv8 and UAS-dpp from Bloomington Stock Center. See FLYBASE (FlyBase, 2003; http://flybase.org/) for more information. The UAS-Dll strain was made by inserting a Dll cDNA into the pUAST vector (Brand and Perrimon, 1993) and injecting the resultant plasmid by the germ line transformation technique. The cDNA was isolated by RT-PCR and spanned from 4 bp upstream of the initiator codon to ⫹45 bp downstream of the terminator codon. An insertion into the third chromosome induced secondary axis formation, when expressed in the larval leg discs, and this strain was used in subsequent analyses. Temperature shift experiments Temperature shift experiments were performed as follows: embryos from wgIL114/CyO-ftzlacZ stock were collected for 1.5 h at 18°C and were cultured at 18°C until stages 12, 13, and 14, and then the temperature was shifted to 29°C. Embryos at stage 15 or 16 were fixed and stained with the wing and leg disc markers and with anti- galactosidase to identify genotypes.
here expression patterns of proximal and distal leg marker genes at a cellular resolution. At stage 11, the early expression of D11 expression marks entire limb primordium that gives rise to both wing and leg discs (Cohen et al., 1993; Goto and Hayashi, 1997b). After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc (Cohen et al., 1993). Double labeling of stage 15 leg discs revealed that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg (Fig. 1A), suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium. It was shown that in the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage (Abu-Shaar and Mann, 1998). Double labeling with antibodies against Esg and Hth revealed that the Esg expression overlaps with Hth expression (Fig. 1B). Hereafter, we used Esg as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region. The relationship between Wg expression and limb primordia
Results
We next compared the expression domain of wg and the position of wing and leg primordia. Wg expression in the trunk ectoderm started as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlapped with wg stripes, as revealed by the wg-lacZ reporter (Fig. 2A; Cohen et al., 1993). At late stage 11, wg-lacZ stripes broke up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein became limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes (Fig. 2B; Couso et al., 1993). The ventromedial wg stripe also overlapped with proximal leg cells that were labeled with anti-Esg at stage 15 (Fig. 2C). The ventral half of proximal leg cells was nearly completely included within the ventral wg stripes. The dorsal half of leg cells was also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia were first recognizable at stage 12 as cells expressing Vestigial (Vg), they did not overlap with the stripe of wg (Fig. 2D). Dorsal cell migration further separated wing primordia from the source of Wg at stage 15 (Fig. 2C; Cohen et al., 1993). The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development.
Markers for proximal and distal leg domains
Requirement for Wg signaling in leg disc formation
The expression patterns of Dll enhancer trap (DllP-LacZ) and Esg were reported previously (Goto and Hayashi, 1997b). For the sake of discussion that follows, we describe
It was previously shown that Wg is required for the induction of the thoracic limb primordium (Cohen et al., 1993) and other imaginal discs (Simcox et al., 1989). To
Histochemical analyses In situ hybridization was performed with digoxigenin-labeled antisense RNA probes of Dll (Panganiban et al., 1994) and dpp (Padgett et al., 1987). Anti-Dll (Panganiban et al. 1994), anti-Hth (Pai et al., 1998), anti-Esg (Fuse et al., 1994), anti-Vg (Williams et al., 1991), anti-pMad (Kubota et al., 2000), and anti--galactosidase (Cappell) were used for immunostaining, which was sometimes amplified with a TSA indirect kit (NEN). Antibody binding was detected with Cy2or Cy3-conjugated secondary antibodies or streptavidine (Amersham) and observed by a confocal microscope (LSM410; Carl Zeiss). Homozygous mutant embryos were identified by the absence of LacZ expression from balancer chromosomes.
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investigate a late role of Wg signaling, we studied the functions of intracellular signal transducers of Wg. The Drosophila homologue of -catenin encoded by armadillo (arm) plays dual roles, one as a mediator of Wg signaling by regulating transcription of various target genes, and the other as a component of Cadherin-dependent cell adhesion (Peifer, 1995). We analyzed two alleles of arm, one being null allele armYD35, and the other armH8.6, which is specifically defective in Wg signaling (Peifer, 1995). In the embryos hemizygous for either of the allele, Dll was expressed normally at stage 11 (Fig. 3C, and data not shown), suggesting that the maternally supplied arm product is sufficient for induction of the limb primordium. Dll expression decayed shortly after this stage (data not shown), and by stage 15, both protein and mRNA expression remained only in a few cells. Furthermore, the proximal leg markers Esg was completely absent (Fig. 3D, and data not shown). On the other hand, formation of the wing disc was normal; suggesting that the late function of Wg signaling is dispensable for wing formation. Since both armYD35 and armH8.6 showed the same phenotype, the function of Arm in Wg signaling, but not in cell adhesion, is required for leg disc development. To confirm whether Wg signaling is required cell autonomously for leg disc development, we expressed the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) in the limb primordia. TCF is an HMG domain-containing DNA binding protein that interacts with Arm to regulate transcription (van de Wetering et al., 1997). Daxin is a regulator of Arm degradation and negatively regulates Wg signaling (Hamada et al., 1999). We used the Dll-Gal4 driver (Calleja et al., 1996), which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs (Kubota et al., 2000). In Dll-GALA embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced (Fig. 3E and F). Expression of Esg was preferentially reduced in the dorsal side (Fig. 3E). The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells (Fig. 3D–F). In arm mutants, Hth-expressing cells expanded to the distal domain (Fig. 3H). This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. On the other hand, disc-specific reduction of Wg signaling did not affect wing disc formation (Fig. 3E and F), although the DllGal4 driver is active in the wing primordium (see Fig. 5B). We conclude that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation.
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Proximal leg development is defective in wg mutants The requirement for Arm in the leg disc development was unexpected from the result of Cohen et al. (1993). The authors reported that wgIL114 (wgts) embryos transferred from a permissive to a restrictive temperature after the limb primordium is fully induced showed a normal pattern of Dll mRNA expression. They concluded that wg is not required for leg disc patterning in late embryogenesis. We decided to revisit the phenotype of wgts with use of additional disc markers. Temperature shift experiments were performed as shown in Fig. 4A. wgts embryos carrying DllP-LacZ were allowed to develop at a permissive temperature until stage 12 (Fig. 4D), stage 13 (Fig. 4F), and stage 14 (Fig. 4H), and the cultures were then transferred to the restrictive temperature. Embryos were fixed at stage 15 (Fig. 4E and G) or 16 (Fig 4I). Embryos were double labeled with anti-Esg and anti-galactosidase to mark wing and leg disc cells (Fig. 4B–I). At the permissive temperature, DllP-LacZ expressing cells were encircled by cells expressing Esg as early as stage 14 (Fig. 4H) and stage 15 (Fig. 4B), suggesting that the PD domains in the leg disc are already established by stage 14. The pattern of Esg expression wgts embryos at the permissive temperature was indistinguishable from the control embryos (Fig. 4B). In wgts embryos, we observed a loss of Esg expression. The severity in the phenotype increased when the temperature was shifted at an earlier stage. Temperature shift at stage 12 (Fig. 4D) caused a near complete loss of the Esg expression (Fig. 4E), while the temperature shift at stage 13 or 14 caused a partial loss of the dorsal part of the Esg expression (Fig. 4G and I). These results suggest that Wg is required for the cell fate maintenance of the proximal leg expressing Esg. On the other hand, we were unable to detect significant change in the expression of DllP-LacZ expression (Fig. 4E, G, and I), and Dll mRNA (data not shown), as previously reported (Cohen et al., 1993). The preferential loss of dorsoproximal cells in wgts mutants suggests that dorsal and ventral halves of proximal leg cells have different requirements for Wg. The higher requirement for Wg by dorsal–proximal cells is consistent with the location of those cells away from the stripe of Wg (Fig. 2C). Perhaps a larger amount of active Wg must be produced to reach dorsal–proximal cells to induce esg. Wing discs are present in wgts embryos at the restrictive temperature (Fig. 4E, G, and I), indicating that, while induction of the limb primordium took place normally, PD patterning in the leg disc was specifically impaired. More importantly, circular arrangement of proximal and distal cells was established by stage 14 (Fig. 4H), and this pattern decayed after inactivation of wg activity (Fig. 4I). We therefore concluded that the continuous input of Wg activity is necessary to maintain the proximal– distal pattern in the leg disc.
Fig. 4. Temporal requirement for Wg activity by leg disc cells. (A) Temperature shift protocol. Embryos were shifted from the permissive to the restrictive temperature at stages 12, 13, or 14. (B–I) Lateral views of embryos showing the first to third thoracic segments. Distal leg disc cells were marked with DllP-LacZ (red). Proximal leg disc cells and wing disc cells were marked with Esg (green). Anterior is left. Arrowheads indicate the leg disc. (B) A stage 15 wgIL114 embryo cultured continuously at the permissive temperature. Expression patterns of DllPLacZ and Esg were indistinguishable from wild type control (compare with Fig. 3B). (C) Same as (A), showing only the expression of Esg. (D, F, H) wgIL114 embryos were allowed to develop at 18°C until stages 12 (D), 13 (F), or 14 (H). (E, G, I) wgIL114 embryos were cultured at the restrictive temperature and fixed at stage 15 (E, G) or 16 (I). While the number of the proximal (Esgpositive) leg disc cells decreased (arrows), distal leg disc and the wing disc were normal. The number of dorsal–proximal leg disc cells in (E) was most severely reduced. Scale bar, 50 m (C).
Fig. 5. Role of Dll in the circular patterning of the leg disc. Anterior is left. Arrowheads indicate the leg disc cells. (A, C–F) Distal and proximal leg cells at stage 15 were labeled with antibodies against Dll and Esg as indicated in each panel. (A) A Dll null mutant embryo shows a disruption of the circular pattern of the Esg expression in the leg disc. Compare this figure with Fig. 3B as a control. (B) Expression pattern of Dll-GAL4 revealed by UAS-LacZ reporter at stage 15. Dll-GAL4 construct is expressed in both the wing and the leg disc cells. (C, D) An embryo overexpressing Dll driven by Dll-GAL4. Expression of Dll was expanded to the proximal leg domain, and Esg expression was reduced in the leg disc. Dll protein was accumulated to a high level in wing discs, but Esg expression was not affected. (E, F) Dll expression was not eliminated by overexpression of Esg by Dll-GAL4. Compare this figure with Fig. 1A as a control. Scale bar, 50 m (A).
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Contribution of Dpp in PD patterning of the leg
Role of Wg signaling on wing disc formation
Although Wg is expressed in ventrolateral stripes, formation of distal leg cells is limited to the dorsal tip of the wg stripe (Fig. 2B), suggesting the presence of a second localized signal restricting Dll expression. Previous works have shown that dpp is expressed prominently in the dorsal side of the limb primordium at late stage 11 (Goto and Hayashi, 1997b; Kubota et al., 2000). Subsequently, intense dpp expression moves in a dorsal direction, forming a longitudinal stripe overlapping the wing primordium (Cohen et al., 1993; Goto and Hayashi, 1997b). Close inspection of embryos revealed an additional expression of dpp, running along the DV direction in stripes that reach the center of the leg discs (Fig. 2E). To examine the activation pattern of Dpp signaling, we monitored Dpp-dependent signaling events using an antibody recognizing the phosphorylated form of MAD (pMAD; described as pSSVS in Kubota et al., 2000). pMAD was expressed in broad stripes that appear to include dpp-expressing cells, and the ventral edge of pMad expression abuts the dorsal edge of wg-lacZ expression (Fig. 2F, compared with E). These observations indicate that, from their inception, leg discs are subdivided into dorsal and ventral domains, each marked with high expression of pMAD or wg, respectively. We previously showed that, upon a loss of Dpp type I receptor Thick veins (Tkv), the expression of Dll RNA decays after stage 15 (Kubota et al., 2000). This result was confirmed here by the use of anti-Dll antibody (data not shown). It was also shown that reduction of Dpp signaling by overexpression of Dad, an inhibitory Smad, by Dll-Gal4 severely reduced Dll and Esg expression (Kubota et al., 2000). These results indicate that Dpp signaling in the dorsal side of the leg disc is required for both proximal and distal leg cell fate. The complementary pattern of Wg and Dpp expression in larval leg disc is maintained by mutual repression (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffmann, 1996; Theisen et al., 1996). We asked whether a similar mechanism is used to regulate Wg and Dpp expression of embryonic leg discs. dpp expression in the lateral ectoderm did not change in arm mutants, and no change of Wg expression was observed upon overexpression of Dad (data not shown). In addition, Dll-GAL4 embryos expressing Wg or armS10 showed normal expression of dpp mRNA, and embryos with overexpression of Dpp by DllGAL4 showed no significant change in the pattern of Wg expression (data not shown). Since these treatments are sufficient to alter the fates of wing and leg disc cells (Goto and Hayashi, 1997b; Kubota et al., 2000), we conclude that once the leg disc is specified, the domain of Wg and dpp expression cannot be altered by change of Wg and dpp signaling within the physiological range. The results suggest that the domains of Wg and Dpp expression within the leg disc are set up independently of each other.
Does Wg signaling have any function in the wing disc formation? There appears to be no requirement for the Wg signal in wing discs, because wg is not expressed there from stage 12 (Fig. 2D) to stage 15, and reduction of wg activity after specification of wing discs caused no change in the wing disc formation (Figs. 3 and 4). In contrast, ectopic activation of the Wg signal in the wing disc by expression of Wg or stabilized Arm (armS10) driven by Dll-Gal4 caused a reduction in the number of wing disc cells (Fig. 3I and J), suggesting that the Wg signal must be downregulated in lateral ectoderm to allow wing disc development. Restriction of proximal domain by Dll The circular pattern of Esg expression in the leg disc at stage 15 is a hallmark of proximal– distal subdivision in the leg. The next question was how the pattern of Dll and Esg expression, each requiring Wg and Dpp signals, is established. We wanted to determine whether Dll regulates expression of Esg. A null mutation of Dll allowed the expression of Esg at the center of the leg disc (Fig. 5A). Cell counting demonstrated that the number of Esg-positive cells did not change significantly upon loss of Dll. We next asked what effect Dll overexpression has on Esg expression. DllGal4 allowed Dll to be expressed in the proximal domain of the leg disc (Fig. 5C). In such embryos, a reduction of Esg expression was observed in proximal leg discs, where ectopic expression of Dll was high (Fig. 5D). On the other hand, ectopic Dll did not affect Esg expression wing discs, suggesting that the inhibitory effect of Dll on Esg is contextdependent. These results suggest that Dll specifically limits Esg expression in leg discs. We also wanted to know whether Esg regulates Dll expression. However, misexpression of Esg by Dll-Gal4 caused only a marginal reduction of Dll expression (Fig. 5E and F; compare this figure with Fig. 1A as a control), and loss of esg caused no change in the pattern of Dll expression (data not shown). Taken together, these results suggest that Dll is capable of restricting the proximal leg domain.
Discussion Specification of animal appendages from a two-dimensional field of the embryonic ectoderm is one major question in embryology. This process in Drosophila involves multiple steps: induction of limb primordial cells in the lateral ectoderm followed by separation of those cells by cell migration into two clusters. The dorsal cells become the wing disc, and ventral cells become the leg disc. The PD axis in the leg disc is apparent at the onset of its formation, suggesting that its formation is an immediate consequence of cell interaction in the embryonic ectoderm. We show here that Wg signaling plays a key role in this process by helping
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to recruit two types of cells that constitute the proximal and distal domains. Establishment of PD axis We have shown that the cell fate maintenance of proximal leg requires continuous signaling by Wg (Fig. 4). The requirement for arm and wg was higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe (Fig. 2C), we propose that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal–proximal cells require a higher level of Wg to be produced to reach their position. Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp that is expressed in stripes abutting the Wg stripe. The requirement for Dpp by distal leg development was previously shown (Goto and Hayashi, 1997b; Kubota et al., 2000). Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by distal gene, as we have shown here by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, repression of proximal genes by Dll in larval leg discs was previously shown (Gonzalez et al., 1998; Abu-Shaar and Mann, 1998). The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc were shown to have distinct cell-adhesive properties from those in the Dll-expressing distal domain (Goto and Hayashi, 1999; Wu and Cohen, 1999), and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc. With the results presented here, we tested a distal organizer model (Campbell and Tomlinson, 1995; Fig. 6A ). The model proposes that a distal organizer placed in the field of developing appendage signals surrounding cells to acquire proximal cell fate. If this signal acts in all directions, circular arrangement of proximal cells can be achieved (Fig. 6A). Although a recent work on postembryonic leg devel-
Fig. 6. Two models of leg specification. (A) Distal organizer (red circle) is specified in the embryonic ectoderm and instructs surrounding cells to acquire proximal cell fate (Campbell and Tomlinson, 1995). (B) Dorsal– proximal and ventral–proximal cells (green crescents) and distal cells are specified separately. These cells (red circles) are assembled to form a circular pattern. Central figure is a schematic drawing of a leg imaginal disc at stage 15 showing the relationship between Wg and Dpp expression domains. Red cells are Dll-expressing distal cells. Green cells are Esgexpressing proximal cells.
opment (Lecuit and Cohen, 1997) does not support this model, it is still one of the best possible models for embryonic leg specification. We demonstrated that dorsal and ventral halves of proximal leg cells have different requirements for Wg. The way Wg acts to organize proximal cell differentiation is not consistent with the distal organizer model. Rather, the results support a second model (Fig. 6B) where dorsoproximal, distal, and ventral–proximal cells are specified separately and assembled to form a circular pattern. Function of Wg and EGFR signals in leg development The preferential loss of proximal leg cells upon partial loss of Wg signaling is very similar to the phenotype of EGFR mutant embryos we have previously reported (Kubota et al., 2000), suggesting that both Wg and EGFR contribute to differentiation of proximal leg cells. We noted, however, a difference in the temporal requirement for the two signals. EGFR signaling is activated transiently at the time of disc specification, and the requirement of its activity is limited to a short period around this stage (Kubota et al., 2000). It was proposed that EGFR acts within limb primordial cells to promote leg development (Kubota et al., 2000). We have shown here that Wg is persistently expressed at the ventral part of the leg disc and maintains the fate of proximal and distal leg cells (Figs. 2– 4). These findings indicate that the leg disc development is initiated by transient activation of EGFR, and its cell fate is maintained by the persistent activity of Wg. Inhibition of wing disc development This and our previous works have demonstrated that Wg and EGFR signaling are not active in wing discs and are not
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required for wing formation after stage 11 (Figs. 2– 4; Kubota et al., 2000). On the other hand, ectopic activation of either of the signals in the limb primordium suppresses the wing disc development (Fig. 3I and J; Kubota et al., 2000), suggesting that downregulation of Wg and EGFR signals have a permissive role in the wing development. These signals are reactivated in postembryonic stages to organize the wing disc. The downregulation of Wg and EGFR signals in a prospective wing disc is accomplished by two mechanisms, one by limiting activation of the two signals to the ventral side of the embryo, and the other by allowing wing primordium to migrate to the dorsal direction away from the source of the inhibitory signals. Thus, cell migration serves as a novel mechanism to restrict the effect of diffusible signaling molecules.
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question as to which of the mechanisms is used in other primitive hemimetabolous insects, where the specification and growth of the leg occur simultaneously. Acknowledgments We thank Grace Panganiban, Henry Sun, Sean Carroll, Steve Cohen, Makio Tokunaga, Tadashi Uemura, Makoto Nakamura, and the Bloomington Stock Center for gifts of antibodies, cDNAs, and/or fly strains. We are grateful to Yasushi Hiromi for critically reading an early version of the manuscript. This work was supported by grants from JSPS (Research for the Future) (to S.H.) and from the Ministry of Education, Science, Sports and Culture (to S.G.).
Differences in the mechanism of leg patterning in the embryo and larva
References
Although the analogous pattern of Wg and Dpp expression plays essential roles in PD patterning in embryonic and larval leg development (this work; Goto and Hayashi, 1997b; Lecuit and Cohen, 1997), we noted significant differences. In embryonic leg discs, expression of both proximal and distal leg markers was lost in mutants of Wg signaling (Fig. 3) or Dpp signaling (Goto and Hayashi, 1997b). Therefore, Wg and Dpp contribute to both proximal and distal leg development in the embryo. In the larvae, reduction of Wg and Dpp expression due to the loss of hh function causes a loss of the distal domain, but no effect on the proximal gene expression was observed (Gonzalez and Morata, 1996), suggesting that Wg and Dpp play little or no role in the development of proximal domain. The inability of Wg or Dpp to participate in the proximal leg patterning in the larvae is due to, at least in part, the function of Hth to block activation of target genes for Wg and Dpp (Abu-Shaar and Mann, 1998). In the embryo, however, Hth does not block expression of esg, a target gene for Wg, as demonstrated by coexpression of Esg and Hth (Fig. 1B). Therefore, proximal domains of embryonic and larval leg discs are different in the way Hth regulates target genes for Wg. This difference may reflect distinct stages of leg development in the embryo, where proximal leg and epidermal cells are continuous, as defined by Hth expression (Fig. 1B), and in the larvae, where they are separated by the peripodial membrane. The complementary pattern of Wg and Dpp expression in the larval leg disc is maintained by mutual repression (Brook and Cohen, 1996; Jiang and Struhl, 1996; Penton and Hoffmann, 1996; Theisen et al., 1996). We were unable to detect any evidence for mutual repression of Wg and Dpp in embryonic leg discs. Perhaps the complementary expression pattern of Wg and Dpp in the embryonic leg disc is under the control of the mechanism regulating the global dorsoventral pattern of the embryo. We have shown here that specific mechanisms are involved in embryonic development as opposed to larval leg development in Drosophila. This finding gives rise to the
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