The role of the T-box gene optomotor-blind in patterning the Drosophila wing

Developmental Biology 268 (2004) 481 – 492 www.elsevier.com/locate/ydbio

The role of the T-box gene optomotor-blind in patterning the Drosophila wing ´ lamo Rodrı´guez, Javier Terriente Felix, and Fernando J. Dı´az-Benjumea * David del A Centro de Biologı´a Molecular-C.S.I.C, Universidad Auto´noma de Madrid-Cantoblanco, Madrid E-28049, Spain Received for publication 10 September 2003, revised 19 December 2003, accepted 9 January 2004

Abstract The development of the Drosophila wing is governed by the action of two morphogens encoded by the genes decapentaplegic (dpp; a member of the BMP gene family) and wingless (wg; a member of the WNT gene family), which promote cell proliferation and pattern the wing. Along the anterior/posterior (A/P) axis, the precise expression of decapentaplegic and its receptors is required for the transcriptional regulation of specific target genes. In the present work, we analyze the function of the T-box gene optomotor-blind (omb), a decapentaplegic target gene. The wings of optomotor-blind mutants have two apparently opposite phenotypes: the central wing is severely reduced and shows massive cell death, mainly in the distal-most wing, and the lateral wing shows extra cell proliferation. Here, we present genetic evidence that optomotor-blind is required to establish the graded expression of the decapentaplegic type I receptor encoded by the gene thick veins (tkv) to repress the expression of the gene master of thickveins and also to activate the expression of spalt (sal) and vestigial (vg), two decapentaplegic target genes. optomotor-blind plays a role in wing development downstream of decapentaplegic by controlling the expression of its receptor thick veins and by mediating the activation of target genes required for the correct development of the wing. The lack of optomotor-blind produces massive cell death in its expression domain, which leads to the mis-activation of the Notch pathway and the overproliferation of lateral wing cells. D 2004 Elsevier Inc. All rights reserved. Keywords: Drosophila; T-box; optomotor-blind; decapentaplegic; thickveins; master of thickveins; spalt; vestigial

Introduction Members of the T-box family of transcription factors have been identified in many different vertebrate and invertebrate organisms. In vertebrates, T-box genes play essential roles in early development, including specification of the mesoderm, and heart and limb morphogenesis (reviewed in Wilson and Conlon, 2002). Tbx genes share a DNA-binding domain of 200 amino acids (Pflugfelder et al., 1992a), the T-box, and, with the exception of Tbx2 and Tbx3, they are transcriptional activators. It is not clear what functions T-box genes have, and few target genes have been identified (reviewed in Tada and Smith, 2001). In Drosophila, at least eight different T-box genes have been identified (Brook and Cohen, 1996; Kispert et al., 1994; Pflugfelder et al., 1992b; Porsch et al., 1998; Reim et al., 2003; Statho-

* Corresponding author. Fax: +34-91-497-4799. E-mail address: [email protected] (F.J. Dı´az-Benjumea). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.01.005

poulos et al., 2002). optomotor-blind (omb), the best-characterized T-box gene in the fly, has been implicated in the development of the optic lobe (Pflugfelder and Heisenberg, 1995) and, in addition, strong loss-of-function omb alleles die as pharate adults and show severe defects in distal wing patterning. It has been suggested that omb may play a role in proximal/distal (P/D) patterning (Grimm and Pflugfelder, 1996). The Drosophila wing is patterned by the action of two secreted molecules, encoded by the genes decapentaplegic (dpp) and wingless (wg). These two genes are expressed at the borders of the anterior/posterior (A/P) and the dorsal/ ventral (D/V) compartments, respectively, which subdivide the wing primordium into two orthogonal axes (reviewed in Brook et al., 1996). Dpp/BMP signals through binding to a receptor complex composed of two serine – threonine kinase components: the type I receptor encoded by the gene thick veins (tkv), and the type II receptor encoded by the gene punt (put). Dpp regulates the expression of many target genes in a concentration-dependent manner. Among them,

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spalt (sal), omb, Daughters against dpp (Dad), and vestigial (vg) are activated at different distances from the A/P compartment border, and brinker (brk) is repressed (reviewed in Tabata, 2001). The concentration of Dpp is maximal at the A/P border and fades away laterally. The distribution of receptor Tkv plays a crucial role in shaping the Dpp activity gradient. tkv expression is negatively regulated by two different mechanisms: on one hand, Dpp signaling itself, which allows Dpp to diffuse further away from its source (Lecuit and Cohen, 1998); on the other hand, the putative transcription factor encoded by the gene master of thickveins (mtv), which is expressed under the control of Hedgehog (Hh) signaling and is maximally expressed at the center of the wing (Funakoshi et al., 2001). This complex regulatory network shapes the Dpp activity gradient. In this report, we have studied the function of omb in wing development. The results presented indicate that omb is required to mediate several Dpp functions: omb is required for the activation of sal and vg, and for the repression of tkv and mtv. We also present evidence that repression of tkv by Mtv requires Omb. So Omb plays major roles downstream of Dpp, both in shaping its activity gradient and in mediating the activation of target genes.

Materials and methods

Genetic mosaic analysis The genotypes of the different mosaic analyses shown are: (A) for clones analyzed in adult wings: omb3198f 36a FRT18A/w FRT18A; hs-FLP122/+ (B) for clones analyzed in discs: omb3198f 36a FRT18A/Ubi-GFP FRT18A; hs-FLP122/+ omb3198f 36a FRT18A/Ubi-GFP FRT18A; mtv-lacZ/+; hsFLP122/+ omb3198f 36a FRT18A/Ubi-GFP FRT18A; sal-lacZ/+; hsFLP122/+ omb3198f 36a FRT18A/Ubi-GFP FRT18A; tkv-lacZ/+; hsFLP122/+ omb3198f 36a FRT18A/Ubi-GFP FRT18A; vgQE-lacZ/+; hsFLP122/+, omb3198f 36a FRT18A/Ubi-GFP FRT18A; hs-FLP122/ pucE69-lacZ omb3198f 36a FRT18A/Ubi-GFP FRT18A; hs-FLP122/dpplacZ Larvae were heat-shocked at 36 F 6, 60 F 6, or 96 F 6 h AEL for 1 h in a water bath at 37jC. (C) for clones of ectopic expression: y w FLP122; Ac5C > y+ > Gal4 UAS-GFP Sp tkv-lacZ; UAS-puc UAS-omb. Larvae were heat-shocked at 36 F 12 h AEL for 12 min in a water bath at 34.5jC.

Fly strains and genetics Wing mounting All the experiments shown were carried out with the allele omb3198, but similar results were obtained with the allele ombD4. omb3198 is a lack-of-function allele produced by a point mutation that introduces a stop codon and generates a truncated protein (Poeck et al., 1993). ombbi (Banga et al., 1986) and omb-Gal4 (Lecuit et al., 1996) are two weak alleles. The fly stocks used were: vallecas (wild type), aprK568-lacZ (Cohen et al., 1992), brkX47-lacZ (Campbell and Tomlinson, 1999), caps02937-lacZ (Shishido et al., 1998), Dad1883-lacZ (Tsuneizumi et al., 1997), dpp-lacZ (Blackman et al., 1991), mtvk00702-lacZ (Funakoshi et al., 2001), ombP1-lacZ (Sun et al., 1995), salrN350-lacZ G, sal10.2S/C-lacZ (Ku¨hnlein et al., 1997), tkv16713-lacZ (George and Terracol, 1997), trns064117-lacZ (Salzberg et al., 1997), vgQE-lacZ (Kim et al., 1996), dppblk-Gal4 (Wilder and Perrimon, 1995), vgBE-Gal4 (Morimura et al., 1996), nubAC62-Gal4 (gift of M. Calleja, CBM-CSIC Madrid, Spain), UAS-GFP (Davis et al., 1995), UAS-omb (Grimm and Pflugfelder, 1996), pucE69-lacZ and UAS-puc (Martı´nBlanco et al., 1998), UAS-p35 (Hay et al., 1994), Ac5C > y+ > Gal4 (Ito et al., 1997) and Nts1 (Schallenberger and Mohler, 1978). The omb3198 brkX47 and Nts omb3198 lines were generated by meiotic recombination. Nts1 omb3198 larvae were grown at permissive temperature (17jC), shifted to the restrictive temperature (29jC) at the early third instar larval stage (approximately 6 days AEL), and dissected for inmunostaining 36 h later.

Flies were stored in a 1:3 glycerol/ethanol solution, then dissected and washed with 70% and 100% ethanol, and mounted in Sandeural. Immunohistochemistry Imaginal discs were fixed and stained by the standard techniques for confocal microscopy. The specific antibodies used were: rabbit anti-h-galactosidase (#55976; Cappel), mouse anti-cleaved caspase-3 (#9661; Cell Signaling), rat anti-Brk (provided by G. Morata); rat anti-Ci, mouse anti-En, and mouse anti-p-Mad (provided by I. Guerrero); rabbit anti-Sal (provided by R. Barrios); rabbit anti-Vg (provided by S. Carroll) and mouse anti-Wg (#4D4; DSHB). Standard protocols were followed for Xgal staining.

Results omb expression in the wing disc The expression of omb in the wing disc is activated by the combined action of the Wg and Dpp signaling pathways (Grimm and Pflugfelder, 1996) and fills a central sector in the wing imaginal disc from middle second instar larval

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stage (Fig. 1A). At this stage, omb expression covers the whole presumptive wing domain but, as development progresses and cell proliferation increases the number of wing cells, omb expression becomes restricted to the central-most domain and is not found in the lateral wing. This can be observed by examining omb expression in wing discs immunostained with an antibody against Wg. Wg is expressed in the wing margin and in two rings that surround the presumptive wing hinge and delimit the wing region in the disc (Figs. 1B, C) (Couso et al., 1993). The level of omb expression is higher in the distal-most region of the wing, where the expression patterns of wg and dpp intersect, and fades away proximally. Indeed, the omb expression has three domains that correspond to the three domains where the expression of wg intersects with the dpp expression domain: the wing margin, the inner ring, and the outer ring. This can be easily appreciated in pupal wings (Fig. 1D). When either the dpp or the wg signaling pathways are compromised, omb expression is lost (Grimm and Pflugfelder, 1996; Lecuit et al., 1996; Nellen et al., 1996). JNK-mediated cell death in omb mutant discs In omb mutants, wing tissue is dramatically reduced. The phenotypes of different omb allelic combinations can be ordered in a sequence that follows the gradient of omb expression, showing a maximum requirement in the region

Fig. 1. Pattern of expression of omb in the wing disc. Double staining for omb-lacZ (green) and wingless antibody (red) in wing discs of (A) middle second instar larvae (60 F 6 h AEL), (B) early third instar larvae (80 F 6 h AEL), (C) late third instar larvae (120 F 6 h AEL), and (D) 24-h old pupae. The rings of wg expression define the limits of the wing presumptive domain. White bars indicate the relative sizes of the discs. In all discs, anterior is to the left and dorsal is to the top. In all wings, proximal is to the left and anterior is to the top.

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of the highest level of expression (Figs. 2A – C). Flies hemizygous for omb3198, the strongest available allele, die as pharate adults, and when they are extracted from the pupae, a complete deletion of the omb expression domain is revealed (Fig. 2C). In many cases, we also observe, in addition to the deletion of the central wing, huge overgrowths that usually do not evaginate but remain internal (data not shown). This overproliferation can also be observed in mature wing discs (see below). It has been reported that the omb wing phenotype is caused by JNKdependent apoptosis (Adachi-Yamada et al., 1999). We have monitored the activity of the JNK pathway throughout larval development by looking at the expression of puckered (puc), a dual-specificity phosphatase which is a target of the JNK pathway (Martı´n-Blanco et al., 1998). In wild-type wing discs, puc expression is restricted to a small domain in the presumptive notum from early second to late third instar larvae (Fig. 2D inset). In omb wing discs of second instar larvae, puc is expressed in a patch of cells in the center of the presumptive wing (Fig. 2D). In early third instars, puc expression is expanded and shows the highest level in the region that corresponds to the wing margin and the anterior – posterior compartment boundary, as revealed by codetection of wg or engrailed (en), which label the wing margin and the posterior compartment, respectively (data not shown). This expression is maintained throughout larval development (Figs. 2E, F). To confirm these results, we examined the expression of puc in clones of cells mutant for omb3198 (Fig. 2G). In clones located in the center of the wing (closer to the A/P compartment boundary), puc-lacZ is expressed in most of the clone cells (30 clones). Lateral clones (far away from the A/P boundary) either do not express puc or do so only in the most centrally located cells (35 clones). We obtained similar results when we detected apoptosis with an antibody that recognizes the activated form of caspase-3 (Thornberry and Lazebnik, 1998), a key component of the apoptotic pathway (data not shown). omb mutant discs of mature larvae are enlarged in the D/ V axis, compared with wild-type discs. These overgrowths, which mainly affect the ventral compartment, are similar to the overgrowths produced by the ectopic activation of the N pathway within the wing blade (Dı´az-Benjumea and Cohen, 1995) (Doherty et al., 1996). The activation of the N pathway in the wing margin requires the expression of the dorsal selector gene apterous (ap) and can be monitorized by the expression of wg. We examined the expression of ap and wg in omb3198 discs and detected that both ap and wg are mis-expressed in the ventral compartment of the wing (Figs. 2H, I). Since cell death in the D/V border could cause the loss of stability of this region (AdachiYamada and O’Connor, 2002; Delanoue et al., 2002), we wanted to suppress the overgrowth by mis-expressing puc, which would act as a repressor of JNK-mediated apoptosis. Indeed, when puc was ubiquitously expressed in the wing (omb3198; nub-Gal4/+; UAS-puc/+), the overgrowth was suppressed and wg expression reverted to wild-type (Fig.

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Fig. 2. Phenotypes and cell death in omb mutant wing discs. (A) Wild-type (inset) and ombbi/omb3198 adult wing. (B) ombGal4/omb3198. (C) omb3198. (D – F) puclacZ expression in wild-type third instar wing disc (D inset), and in omb3198 wing discs of late second (72 F 6 h AEL; D), middle third (96 F 6 h AEL; E) and mature third (120 F 6 h AEL; F) instar larvae. In wild-type discs, puc expression is detected only in the proximal-most region of the notum. In omb3198 discs, puc is detected in the wing, mainly in the compartment boundaries. Red and black bars indicate the relative sizes of adult wings and wing discs, respectively. (G) omb3198 clones detected by the lack of GFP (green) and stained with puc-lacZ (red). (H) wg expression in omb3198 wing disc. Arrowheads in H – J indicate the D/V compartment boundary. The overgrowth happens in ventral cells and wg is mis-expressed in ventral cells. (I) ap-lacZ expression in omb3198 wing discs at 120 F 6 h AEL. ap is mis-expressed in ventral cells. (J) wg expression in omb3198; nub-Gal4/UAS-puc wing disc. (K) omb3198; nubGal4/UAS-puc adult wing. Note that the overgrowth is suppressed.

2J), but the loss of tissue in the adult wing was not restored (Fig. 2K). Wild-type control wings in which puc was ubiquitously expressed (nub-Gal4/+; UAS-puc/+) do not show any phenotype. omb controls the graded expression of tkv and mtv genes in the wing To assess the developmental basis of omb mutant phenotypes, we examined the pattern of expression of genes that are expressed in the omb domain. tkv is expressed in a pattern complementary to the omb expression domain (Brummel et al., 1994) (Lecuit and Cohen, 1998) (Haerry et al., 1998). Fig. 3A (inset) shows a wild-type wing disc from early third instar larvae double stained for omb and tkv. From this stage until the end of larval development, omb and tkv are expressed in the wing disc in complementary patterns (Fig. 3A). Thus, tkv expression is maximal in the lateral wing and fades away centrally. It has been proposed that the expression of tkv is negatively regulated by dpp

(Lecuit and Cohen, 1998), but this does not agree with its expression pattern since both genes are co-expressed in the notum. We examined whether Omb might mediate this negative regulation. Certainly, in omb3198 discs, tkv expression is expanded in the central wing from the early third instar stage until the end of larval development (Fig. 3B and inset), with only a stripe of cells on the anterior side of the A/P compartment boundary showing a weaker expression (see below). This result suggests that the control of tkv expression requires Omb. We confirmed this by examining the expression of tkv in omb3198 clones (Figs. 3C, D). In these clones, tkv is cell-autonomously overexpressed within the clone cells, raising the level of expression in the lateralmost regions. We also examined tkv expression in cells that mis-express omb. To this end, we made use of the flip-out UAS/Gal4 system (Pignoni and Zipursky, 1997) to produce clones of genetically marked cells expressing omb. These clones are lethal and when we mis-expressed omb in the wing disc with different Gal4 lines, we detected puc expression, indicating that the JNK pathway mediates this

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Fig. 3. omb controls the expression of tkv and mtv. (A) Double staining for omb (green; omb-Gal4/UASGFP) and tkv (red; tkv-lacZ) in early (80 F 6 h AEL; inset) and mature wild-type third instar larvae. The patterns of expression are complementary. (B) tkv-lacZ expression in early (80 F 6 h AEL; inset) and mature (red) omb3198 third instar wing discs. tkv expression is expanded and covers the entire wing region. A stripe of cells on the anterior side of the A/P compartment boundary shows a weaker expression (arrow). en expression is shown (green; anti-En antibody). (C, D) omb3198 clones revealed by the lack of GFP showing the expression of tkv-lacZ (red). The level of tkv expression increases within the clone cells. The magnified regions to the right of the main image show each channel independently and correspond to the areas delimited by the white squares. (E) omb and puc-ectopic expressing clones labeled with GFP (green) labeling the expression of tkv-lacZ (red). tkv is repressed within the clone. (F) mtv-lacZ expression in wild-type (inset) and omb3198 wing disc. In omb mutant discs, mtv expression covers the whole of the wing domain. (G, H) omb3198 clones revealed by the lack of GFP and showing the expression of mtv-lacZ (red). mtv is overexpressed within the clone cells. Note that twin spots (bright green) with two omb+ copies show a level of mtv expression lower than the surrounding cells (light green), which have a single omb+ copy.

lethality (data not shown). To avoid the lethality, we coexpress omb and puc. These clones although survive under these conditions are small in size, and tkv expression is cellautonomously lost within the clone cells (Fig. 3E). We conclude that omb is a critical component of the mechanisms that control tkv expression in the wing, and because omb mis-expression is able to repress tkv expression in places where Dpp is not signaling (lateral-most wing), we conclude that Omb mediate the proposed repression of tkv by Dpp. The Drosophila gene mtv encodes a novel transcription factor required for wing development, axon guidance in the visual system, and larval behavior (Funakoshi et al., 2001; Senti et al., 2000; Yang et al., 2000). In the central domain of the wing disc, mtv acts as a repressor of tkv expression, although the lateral-most cells express both genes (Funakoshi et al., 2001). mtv is expressed, under the control of the hedgehog (hh) signaling pathway, in a stripe of anterior cells at the edge of the anterior – posterior compartment

boundary, this expression fades away anteriorly. mtv is also expressed in the lateral-most cells (Fig. 3F inset). To determine whether Omb represses mtv expression, we examined the expression of mtv in omb mutant wing discs. In these discs, mtv expression covers the whole presumptive wing domain (Fig. 3F). We confirmed this result by making clones of omb3198 homozygous cells. In these clones, mtv is overexpressed. Strikingly, the twin clones, that have two wild-type copies of omb, show a reduction in the level of mtv expression when compared with the surrounding omb heterozygous cells (Figs. 3G, H). We conclude that Omb controls mtv by repressing its expression in the wing. Our results suggest that two different factors control the expression of mtv in the wing. One factor depends on Hh signaling and is not affected by Omb (Funakoshi et al., 2001), and the other factor drives ubiquitous expression and is repressed by Omb in central wing. In omb mutant discs and clones, mtv expression is expanded all over the posterior compartment of the wing pouch and shows an uniform

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staining in anterior cells. Although mtv is mis-expressed in omb mutant discs, this has no effect on the expression of tkv, which on the contrary is also mis-expressed. We only observed in some discs a weak depletion of tkv expression in a stripe of cells adjacent to the anterior – posterior boundary (Fig. 3B, arrow). This is probably because of the higher level of mtv expression in these cells. Taken together, these results suggest that the repression of tkv expression by mtv requires Omb. Consistent with this hypothesis, mtv and tkv are co-expressed in lateral cells and in the notum (where omb is not expressed) but omb mis-expression in these domains is sufficient to repress tkv expression (Fig. 3E). The expression of vestigial and spalt requires Omb The vg gene encodes a nuclear protein and is considered to be the selector gene for wing fate. It is expressed throughout the wing under the control of two different enhancers that are expressed in complementary domains (Kim et al., 1996; Williams et al., 1994). The Boundary Enhancer (vgBE) is activated in the D/V boundary under the control of the N signaling pathway (Kim et al., 1996). The Quadrant Enhancer (vgQE) is activated in the rest of the wing cells and requires several inputs, including Wg and Dpp (Certel et al., 2000; Kim et al., 1997). We have examined whether omb is required for the activation of vgQE. Although vgQE is expressed in late third instar larvae in a domain broader than the omb domain (Fig. 4A inset), in early third instar discs the initial activation of vgQE occurs in cells within the omb domain (Fig. 4A). We observed that vgQE-lacZ expression is lost in omb mutant wing discs (Fig. 4B). We also found that in genetically marked omb3198 clones, vgQE expression was cell-autonomously lost (Figs. 4C, D), even in lateral clones outside of the omb domain. This indicates an early requirement for omb in the initial activation of vgQE. It has been reported that vg lack-offunction clones are lethal. This would appear to be incompatible with large omb mutant clones that do not express vg. We found that a fraction of central omb clones is indeed lost. This can be revealed by the presence of isolated twin clones (bright green in Fig. 4E). At the same time, vg was not completely lost in omb clones, and low levels were detected in some cells with anti-Vg antibody (data not shown). In addition, vgBE, which drives vg expression in the D/V boundary, is not affected in omb3198 wing discs (data not shown). Hence, we conclude that the lack of vg may be one of the causes of the poor survival of the omb clones in the center of the wing. The domain of vgQE expression is broader than the domain of omb. This suggests that although omb is required for the initial activation of vgQE, it might not be required for its maintenance. To test this prediction, we examined vgQE expression in omb3198 clones generated at a later stage of development (96 F 12 h after egg lay, or AEL). In these clones, the expression of vgQE-lacZ was not affected (Fig. 4F).

We next examined the expression of the gene sal. sal expression in the presumptive wing blade is first detected in early third instar discs. In late third instars, sal is expressed in a band of cells centered on the dpp stripe (de Celis et al., 1996) (Fig. 4G inset). In omb3198 discs, sal-lacZ expression was lost in the wing pouch, although its expression in the notum was not affected (Fig. 4G). We obtained the same results both using anti-Sal antibody (Barrio et al., 1996) or a transgene construct that drives a lacZ reporter gene in the wing domain (sal10.2S/C-lacZ) (Ku¨hnlein et al., 1997). We also examined sal expression in omb3198 clones generated in first instar larvae (36 F 6 h AEL). sal expression was lost in most of the clone cells but low levels of expression remained in some cells (Figs. 4H, I). It has been reported that sal mutant clones within the sal expression domain were recovered as vesicles that segregate from the disc epithelium (Mila´n et al., 2002). This observation is consistent with the presence of vesicles (33 clones) in omb mutant clones in the center of the wing when analyzed in adult flies (Fig. 4J). In late third instar wing discs, sal represses the expression of the LRR transmembrane proteins encoded by the genes capricious (caps) and tartan (trn) (Mila´n et al., 2002). Thus, in wild-type discs, caps and trn are expressed in two lateral domains complementary to the sal domain (Fig. 4K inset). We found that in omb mutant discs, the expression of caps and trn cover the whole wing pouch (Fig. 4K and data not shown). We conclude that omb is required to mediate the activation of vgQE and sal, two Dpp target genes. We have presented evidence implicating omb in the control of the expression of a set of genes. An alternative explanation could be that the massive cell death observed in omb mutant discs is the cause but not the consequence of the observed alterations in gene expression. To test this, we therefore examined the expression of tkv, sal, and vgQE in omb discs in which cell death was suppressed by misexpressing puc (omb3198; nub-Gal4/+; UAS-puc/+). In these discs, the expression of tkv, sal, and vgQE was the same as observed in omb3198 discs (Figs. 4L – N), with tkv being expressed in the entire wing primordium with lower levels at the stripe of mtv expression, and with sal and vgQE expression lost. We obtained the same results by misexpressing the apoptosis suppressor p35 (Hay et al., 1994) (data not shown). Dpp signaling is not impaired in omb mutant discs Our results indicate that omb function is required in wing development to control the expression of four different genes: tkv, mtv, vg, and sal. omb is required to repress tkv and mtv, and to activate vg and sal. It has been reported that tkv, vg, and sal are Dpp target genes. This suggests that Omb, as a nuclear factor, would be directly involved in the interpretation of Dpp signaling in the nucleus. Another possibility is that in omb discs, Dpp signaling is impaired in a nonspecific way. To discriminate between these two

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Fig. 4. omb is required to activate the expression of vgQE and sal. (A) Double staining for omb-Gal4/UAS-GFP (green) and vgQE-lacZ (red) in early (80 F 6 h AEL) and late (120 F 6 h AEL; inset) third instar larvae wing discs. vgQE expression is first seen as four small groups of cells included within the omb expression domain. Later in development, it expands laterally out of the region of omb-expressing cells. (B) vgQE-lacZ expression in wild-type (inset) and omb3198 wing discs. No expression is detected in omb3198 wing discs. (C, D) omb3198 clones revealed by the lack of GFP showing vgQE-lacZ expression (red). vgQE expression is lost within the clone cells. (E) omb3198 clones labeled by the lack of GFP showing the twin spots (bright green). omb clones in the center of the wing are underrepresented, indicating cell death. (F) omb3198 clones as in C and D bud induced at 96 F 12 h AEL. vgQE-lacZ expression is not affected. (G) sal-lacZ expression in wild-type (inset) and omb3198 wing discs. sal expression is lost in the wing domain. (H, I) omb3198 clones labeled by the lack of GFP showing sal-lacZ expression (red). sal expression is lost or reduced. (J) Adult wing showing a vesicle formed by a f 36a omb3198 clone (arrow). A high magnification of this clone is shown at the bottom. Note the f trichomes. (K) trn-lacZ expression in wild-type (inset) and omb3198 wing disc. trn expression is expanded and covers the center of the wing. (L – N) Red staining shows tkv-lacZ (K), anti-Sal antibody (L), and anti-Vg antibody (M) expression in omb3198; nub-Gal4/UAS-GFP/UAS-puc wing discs. The nub expression domain is shown in green.

hypotheses, we examined the level of activity of Dpp signaling. The levels of Dpp signaling can be monitored in situ by detecting the level of the phophorylated form of Mad (p-Mad) with a specific antibody (Tanimoto et al., 2000). In wild-type discs, p-Mad is expressed in a complex pattern (Fig. 5A inset) with two peaks on either side of the mtv expression stripe in the center of the wing (Fig. 3F inset). p-Mad expression fades away laterally. This pattern reflects a combination of the levels of expression of Dpp ligand and the receptor Tkv (Funakoshi et al., 2001; Teleman et al., 2001). In omb3198 wing discs, we observed a broad stripe of high levels of p-Mad staining in the center of the disc (Fig. 5A). We confirmed this result by examining the level of p-Mad in omb3198 clones. In these clones, the

level of p-Mad is clearly increased within the clone cells when compared with the surrounding cells (Figs. 5B, C). This is probably a direct consequence of the high level of tkv expression observed in omb discs and clones and indicates that Dpp signaling is not impaired in omb discs. Another way to test the activity of Dpp signaling is to examine the expression of the gene brinker (brk). brk encodes a novel protein with features of a transcriptional repressor; Dpp negatively regulates its expression (Campbell and Tomlinson, 1999; Jazwinska et al., 1999; Minami et al., 1999). Brk antagonizes Dpp signaling by directly binding target genes, such as omb (Sivasankaran et al., 2000) (Mu¨ller et al., 2003). Although the two lateral domains of brk expression expand towards the A/P com-

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Fig. 5. Effects of the lack of omb in Dpp signaling. (A) Wild-type (inset) and omb3198 wing discs stained with p-Mad antibody. In omb319 discs, p-Mad labeling covers a broad region in the center of the disc. (B, C) omb3198 clones labeled by the lack of GFP showing p-Mad staining (red). omb mutant cells show a more intense p-Mad labeling than surrounding omb3198/+ cells. (D) brkX47-lacZ expression in wild-type (inset) and omb3198 wing discs. (E) dpp-lacZ expression in wild-type (inset) and omb3198 wing discs. (F, G) omb3198 clones labeled by the lack of GFP and showing dpp-lacZ expression (red). Anterior clones that involve dpp-expressing cells show an expansion of the dpp expression domain (F). Posterior clones do not show any effect (G). (+/ ) +/omb3198; ( / ) omb3198/ omb3198. (H) Wild-type (inset) and omb3198 wing discs stained with anti-Ci antibody. In omb discs, the stripe of high-level ci expression in the center of the discs is expanded anterior-wards.

partment boundary in omb3198 discs (Fig. 5D and inset), they do not converge to cover the central region of the disc. This result is consistent with the elevated levels of Tkv in the center of the disc, trapping all free Dpp ligand and reducing Dpp levels in the lateral cells. We next examined the expression of dpp. In omb3198 discs, the stripe of dpp expression in the center of the disc is widened in the omb expression domain (Fig. 5E and inset). This is also observed in omb clones. Although dpp is not ectopically activated in posterior omb clones, when the clone involved anterior cells within the dpp expression domain, its expression is expanded several cell diameters within the clone cells (Figs. 5F, G). We consider that this result could be a consequence of cell death. A similar expansion in the dpp expression domain has been reported in discs in which cell death has been induced by different methods (Alves et al., 1998; Brook et al., 1993). The expression of dpp becomes wild type in omb discs in which cell death was suppressed by mis-expressing puc (omb3198; nub-Gal4/dpp-lacZ; UAS-puc/+) (data not shown). These results would suggest an expansion in the diffusion range of Hedgehog (Hh). Indeed, we observed a widening in the

pattern of expression of the activated form of Cubitus interruptus (Ci), which defines the range of Hh signaling (Fig. 5H and inset). Taken together, these results suggest that cell death in the center of the disc causes the activation of dpp expression in a broader domain.

Discussion Dpp is a long-range morphogen that acts in the development of the wing through the activation of a set of target genes in a concentration-dependent manner (Tabata, 2001). Little is known about the function and the mechanisms of action of these target genes. In the present work, we have carried out a developmental analysis of the Dpp target gene omb. Here, we have shown that omb is expressed from early larval stages in a pattern that covers the whole central domain of the wing. The effects of the lack of omb are already detectable in second instar larvae as the activation of JNK-mediated apoptosis. As development progresses, the lack of omb becomes more evident as a groove in the center of the presumptive wing accompanied by massive cell

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death, mainly in the distal-most region of the disc. Adult wings that develop from these discs show a huge deletion of the distal part of the wing and, in some cases, overproliferation of the lateral-most domains. We have shown that omb is required to control the expression of the Dpp-type I receptor encoded by tkv. Previous reports have suggested that Dpp signaling represses the expression of tkv. Our results indicate that this repression is mediated by Omb, a finding that is consistent with the complementary patterns of expression of these genes in the wing. tkv expression is also repressed by mtv, which is expressed in anterior cells under the control of the Hh signaling pathway, and also in two lateral domains where it co-expresses with tkv. Our results indicate that Omb repress mtv expression in the central wing, away from the domain where mtv is being activated by Hh, which suggests that mtv expression is controlled by two different factors, one ubiquitous factor repressible by Omb, and other one that is activated by Hh signaling. The tight correlation found between the levels of mtv expression and the number of copies of omb supports this hypothesis. Thus, in omb mutant wing discs, both tkv and mtv are derepressed. These results also indicate that although omb expression is sufficient to repress tkv expression, mtv requires omb to repress tkv expression. Thus, in omb discs and in lateral regions of wild-type discs, where omb is not expressed, both tkv and mtv co-express. In summary, our data indicate that omb is a principal component of a complex regulatory network that controls the expression of tkv, and through this the activity and the shape of the Dpp gradient. We have found that Omb is required for the expression of two Dpp target genes: sal and vg. We first confirmed that Dpp/BMP pathway is active in omb discs. Thus, the patterns of expression of p-Mad, brk, and Dad (data not shown) suggest that Dpp pathway is signaling. Nevertheless, in spite of this and the overexpression of the tkv receptor, we have shown that the Dpp-dependent sal and the vg expressions are missing in omb mutant discs and clones. An independent line of evidence for the requirement for omb in the activation of sal comes from the pattern of expression of cap and trn, two genes that are negatively regulated by sal and whose expression pattern in omb mutant discs is expanded, covering the whole wing. It is important to point out that whereas sal expression is always affected in omb clones induced at different times of larval development, vg expression is not affected in clones induced at 96 F 12 h AEL. This result indicates that two different mechanisms initiates and maintains vg quadrant enhancer expression. In summary, we have presented evidence that suggests an important role for omb in controlling of the expression of several Dpp target genes (Fig. 6). An important distinction between the function of omb as a repressor of tkv and mtv expression and its function as a co-activator of sal and vg expression is that the first one does not require a simultaneous activation of Dpp signaling. This conclusion arises from the ability of omb-expressing

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Fig. 6. A diagram summarizing the function of omb in wing development. (A) Hh is secreted from posterior cells and signals to anterior cells activating the expression of dpp and mtv in a stripe of cells. The combined action of Dpp and Wg, which is expressed in the dorsoventral compartment boundary, activates the expression of omb. (B) Omb acts as a transcriptional repressor of the expression of tkv in the center of the wing and also of a ubiquitous factor that drives the expression of mtv, but does not affect the mtv expression in anterior cells that is controlled by the Hh signaling. Omb also acts as a nuclear cofactor for the activation of sal and the vg quadrant enhancer by the Dpp signal transduction pathway. So Omb plays a double role in Dpp signaling: one, by controlling the expression of its receptor, and two, being required for the expression of sal and vg.

clones to repress tkv expression in lateral brk-expressing cells (Fig. 3E), and for the tight control that omb exerts on mtv expression (Figs. 3G, H). Conversely, the activation of both sal and vg expressions requires, in addition to Omb, Dpp signaling. This is suggested by the facts that brk Mad double-mutant clones express high levels of omb but only low levels of sal (Jazwinska et al., 1999), and that brk mutant clones in the lateral wing also express high levels of omb but only low levels of vg in part of the clone (Campbell and Tomlinson, 1999). These results suggest that Omb acts as a cofactor in the activation of several Dpp target genes. It has been shown that T-box proteins can act either as a transcriptional activator or repressor, although very few cases have been reported where the same gene has both functions. The DNA binding domain of the T-box proteins comprises approximately 180 amino acid and shares approximately 70% of homology among T-box family members. Several reports have shown that T-box proteins recognize similar sequences, suggesting that association with putative cofactors, rather the DNA binding domain, is the determinant of specificity. This seems to be the case for the dual-specificity T-box transcription factor Mga, which contains two separate DNA-binding motifs. Mga

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can act either as transcriptional activator or repressor in a cofactor-dependent manner (Hurlin et al., 1999). Apoptosis and overgrowth in omb mutant discs The results of the omb clonal analyses show that cell death is not restricted to the D/V compartment boundary; cells away from the wing margin also undergo apoptosis. Although it could seem contradictory, the massive cell death found in omb discs has the opposite effect in lateral cells, producing overproliferation. We think that this result is a consequence of the mis-expression of the dorsal selector gene apterous in ventral cells, leading to the activation of the N pathway, which promotes cell proliferation. The effect of cell death on the loss of stability of the D/V compartment boundary has been reported (Adachi-Yamada and O’Connor, 2002; Delanoue et al., 2002). Nevertheless, we consider that these are secondary effects that have nothing to do with the real function of omb in wing development. This is confirmed by the observation that the suppression of cell death by blocking the JNK pathway suppresses the overproliferation but does not restore the wild-type expression of tkv, mtv, sal, and vg. The role of omb in proximal/distal patterning Pattern formation in the vertebrate limb requires cell interactions between different cell layers: the ectoderm and the mesenchyme (reviewed in Capdevila and Izpisu´a Belmonet, 2001). In Drosophila, a single layer of epidermal cells forms the imaginal discs, therefore, cell interactions take place in a two-dimension field. There is a set of genes that are activated by the combined action of Wg and Dpp, and it has been reported that a cooperative activity exists between these pathways (Letamendia et al., 2001). In the leg disc, Wg/Dpp target genes are expressed in concentric circles that define P/D sectors in the adult leg (Galindo et al., 2002). In the wing, the expression of omb, one of the Wg/Dpp target genes, is proximally/distally graded with a maximum in the distal-most tip of the wing. We have now shown that Omb shapes, by repression, the expression of tkv in a complementary pattern. This implies that the Dpp activity gradient is not only shaped in the A/P axis as a result of how Dpp spreads in the tissue, but also in the P/D axis as a result of the graded distribution of its receptor. This view is supported by the phenotypes of different allelic combinations of either omb or dpp, which can be ordered in a phenotypic series to reveal a major requirement at the distal end, and a proximal expansion of the phenotype when the loss-of-function becomes more severe.

Acknowledgments We thank T. Adachi-Yamada, R. Barrios, K. Basler, M. Calleja, G. Campbell, S. Campuzano, S. Carroll, S. Cohen,

I. Guerrero, A. Laughon, E. Martı´n-Blanco, M. Nakamura, T. Tabata, and the Bloomington Stock Center for fly stocks; R. Barrios, S. Carroll, I. Guerrero, and G. Morata for antibodies. I. Guerrero for constructive discussions and W. Brook for critical reading of the manuscript. This work was supported by a grant from the Ministerio de Ciencia y Tecnologı´a and an institutional grant from Ramo´n Areces Foundation to Centro de Biologı´a Molecular-Severo Ochoa.

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