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. Author manuscript; available in PMC: 2012 Jul 15.
Published in final edited form as: Dev Biol. 2011 May 7;355(2):336–348. doi: 10.1016/j.ydbio.2011.04.032

Segment-specific regulation of the Drosophila AP-2 gene during leg and antennal development

Youngwook Ahn 1,a,b,*, Jizhong Zou 1,a,c, Pamela J Mitchell 1,*
PMCID: PMC3118927  NIHMSID: NIHMS295415  PMID: 21575621

Abstract

Segmentation involves subdivision of a developing body part into multiple repetitive units during embryogenesis. In Drosophila and other insects, embryonic segmentation is regulated by genes expressed in the same domain of every segment. Less is known about the molecular basis for segmentation of individual body parts occurring at later developmental stages. The Drosophila transcription factor AP-2 gene, dAP-2, is required for outgrowth of leg and antennal segments and is expressed in every segment boundary within the larval imaginal discs. To investigate the molecular mechanisms generating the segmentally repetitive pattern of dAP-2 expression, we performed transgenic reporter analyses and isolated multiple cis-regulatory elements that can individually or cooperatively recapitulate endogenous dAP-2 expression in different segments of the appendages. We further analyzed an enhancer specific for the proximal femur region which corresponds to the distal-most expression domain of homothorax (hth) in the leg imaginal discs. Hth is known to be responsible for the nuclear localization and, hence, function of the Hox cofactor, Extradenticle (Exd). We show that both Hth and Exd are required for dAP-2 expression in the femur and that a conserved Exd/Hox binding site is essential for enhancer activity. Our loss- and gain-off-function studies further support direct regulation of dAP-2 by Hox proteins and suggest that Hox proteins function redundantly in dAP-2 regulation. Our study reveals that discrete segment-specific enhancers underlie the seemingly simple repetitive expression of dAP-2 and provides evidence for direct regulation of leg segmentation by regional combinations of the proximodistal patterning genes.

Keywords: transcription factor AP-2, leg, antenna, segmentation, gap genes, Homothorax, Hox, Extradenticle, Drosophila, enhancer, Antennapedia

Introduction

Adult legs of Drosophila consist of five segments along the proximodistal (PD) axis. From proximal to distal, these are the coxa, trochanter, femur, tibia, and tarsus. The tarsus is divided into five subfragments, tarsal1–5, and pretarsus (claw). The anlagen of adult legs, the leg imaginal discs, develop inside the larval body and are derived from precursor cells set aside from thoracic ectoderm during embryogenesis. At the end of the third larval instar, the leg disc is a sac-like structure of mono-layered epithelial cells with a series of concentric folds. During the early pupal stage, the leg disc everts and elongates so that the center of the disc becomes the distal-most part (future pretarsus) of the adult leg, and more peripheral folds become progressively more proximal structures (reviewed by Kojima, 2004).

In early stages of Drosophila leg development, two secreted signals, Decapentaplegic (Dpp) and Wingless (Wg), play an important role in patterning the dorsoventral (DV) axis (Brook and Cohen, 1996). Dpp and Wg also regulate the expression of the PD patterning genes Distal-less (Dll), dachshund (dac), and homothorax (hth) (Campbell et al., 1993; Diaz-Benjumea et al., 1994; Estella and Mann, 2008; Estella et al., 2008). Initially, Dll is expressed in the central region (future distal segments) and hth in the periphery (future proximal segments) of leg imaginal discs. At early third instar, expression of dac begins in a ring of cells between the Dll and Hth domains. By mid-third instar stage, the distal part of the dac expression domain overlaps with the Dll domain. In leg discs from late-third instar larvae, a narrow domain of cells expressing hth, Dll, and dac arises at the junction between the dac and hth expression domains. This region corresponds to the future trochanter-femur joint (proximal end of the future femur and distal end of the future trochanter). Thus, by the end of larval development, each leg disc is subdivided into five domains based on expression of hth, dac, and Dll. These three PD axis specifying genes are often referred to as the ‘leg gap genes’ because their loss-of-function mutations result in the loss or shortening of the specific leg segments where they are expressed (Abu-Shaar and Mann, 1998; Cohen et al., 1989; Gorfinkiel et al., 1997; Lecuit and Cohen, 1997; Mardon et al., 1994; Wu and Cohen, 1999).

Hth is required for nuclear localization of the homeodomain transcription factor Extradenticle (Exd) (Noro et al., 2006; Pai et al., 1998; Rieckhof et al., 1997). Similar to Hth, Exd is required for development of proximal leg segments (Gonzalez-Crespo et al., 1998; Gonzalez-Crespo and Morata, 1995; Rauskolb et al., 1995). As a Hox cofactor, Exd dimerizes with Hox proteins and cooperatively binds to unique bipartite recognition sites in cis-regulatory regions of specific target genes (Chan et al., 1994; Ryoo and Mann, 1999; van Dijk and Murre, 1994). Hox genes encode homeodomain transcription factors that play a role in determining segmental identity along the antero-posterior axis (Botas, 1993; Krumlauf, 1994; McGinnis and Krumlauf, 1992).

After the initial PD axis pre-pattern is established by the leg gap genes, the outgrowth of leg segments and local development of joints requires a set of genes collectively called leg segmentation genes (reviewed by Kojima, 2004). The leg segmentation gene group is principally comprised of components of the Notch signaling pathway that is essential for leg outgrowth and tarsal joint development in Drosophila (Bishop et al., 1999; de Celis et al., 1998; Hao et al., 2003; Rauskolb, 2001; Rauskolb and Irvine, 1999). Leg segmentation genes are characteristically expressed in segmentally repeated patterns along the PD axis of the nascent limb. The patterns are seen as multiple concentric rings in late third instar leg discs. The molecular mechanism underlying the repetitive pattern of leg segmentation genes remains largely unknown. However, early studies on the regulation of the Notch ligands, Serrate and Delta, and a modulator of Notch signaling, fringe, have led to the hypothesis that distinct combinations of the PD patterning genes individually regulate each reiterated ring of leg segmentation gene expression (Bachmann and Knust, 1998; Rauskolb, 2001).

In this study, we analyzed cis-regulatory elements of the dAP-2 gene to gain further insight into the mechanisms of segmental gene expression during appendage development. dAP-2 is the sole Drosophila homolog of the AP-2 family of transcription factor genes in mammals (Bauer et al., 1998; Eckert et al., 2005; Monge and Mitchell, 1998). During late larval development, dAP-2 is expressed in leg and antennal imaginal discs as concentric rings representing presumptive segment boundaries (Kerber et al., 2001; Monge et al., 2001). It has been known that dAP-2 is required cell-autonomously for tarsal joint formation, and non-cell-autonomously for survival of inter-joint cells and for outgrowth of the legs and antenna (Kerber et al., 2001; Monge et al., 2001). Previous studies have shown that AP-2 expression in presumptive tarsal joints requires Notch signaling, and that, once activated, AP-2 negatively regulates Serrate and Delta (Ciechanska et al., 2007; Kerber et al., 2001). In contrast, dAP-2 expression in the other leg segments (coxa, trochanter, femur and tibia) is not Notch-dependent (Zou et al., unpublished data).

Through comprehensive, transgenic reporter analyses, we identified multiple cis-regulatory elements that mediate dAP-2 expression in different leg and antennal segments. Further characterization of one of the identified enhancers demonstrates that dAP-2 is directly regulated by Exd and Hox proteins in the proximal femur in a segment-specific manner. Our data indicate that AP-2 has evolved multiple enhancers that function independently to activate gene expression at particular positions along the limb PD axis. Notably, this regulatory strategy is reminiscent of that used to establish the seven domains of even-skipped expression during segmentation of the embryonic antero-posterior axis in Drosophila (Harding et al., 1989; Howard and Davidson, 2004).

Materials and methods

Fly strains

The yw stock was used for generation of transgenic flies. Germ-line transformation was done using the yw;Δ2–3 Sb/TM6 Ubx strain. A Drosophila pseudoobscura strain MV-25 was used to prepare genomic DNA. The following fly strains were also used: Dll-lacZ/CyO (Bloomington Stock #10981), dac-lacZ/CyO (#12047), dac1/CyO (#4273), hth-lacZ/TM3 Sb (#11670), odd-skipped-lacZ/CyO (#11111), Antp73b/TM3 Sb (#1000165), dpp-GAL4 (#1553), UAS-dAP-2 (Monge et al., 2001), UAS-Scr (#4273), UAS-Ubx (#911) and Dll3 (Emerald and Cohen, 2004).

Clonal analysis

Stocks used for clonal analysis were FRT19A exd1/FM7 GFP (Gonzalez-Crespo et al., 1998), FRT19A arm-lacZ;ey-FLP (Tsuji et al., 2000), FRT82B hthC1/TM6B Tb (Wu and Cohen, 1999), hs-FLP;FRT82B arm-lacZ (Wu and Cohen, 1999), FRT82B AntpNs+RC3/TM6B Tb (Emerald and Cohen, 2004).

exd, hth, Antp mutant clones were generated in larvae of genotypes: FRT19A exd1/FRT19A arm-lacZ;ey-FLP/+, hs-FLP/+;FRT82B hthC1/FRT82B arm-lacZ, and hs-FLP/+;FRT82B AntpNs+RC3/FRT82B arm-lacZ, respectively. The hth and Antp mutant clones were induced by incubating vials in 37°C water bath for 1 hour at 48–72 hours after egg laying.

Transgenic reporter analysis

For the in vivo reporter analyses, multiple DNA fragments covering a ~32 kb genomic region in and around the dAP-2 gene were cloned into P-element transformation vectors containing a copy of the lacZ gene. Two dAP-2 genomic DNA fragments, KE and E7, were derived from a Drosophila BAC clone, and the others were derived from multiple overlapping lambda phage clones. D. psuedoobscura EB (DPEB) was amplified by genomic PCR using the primer set: 5’-GCAGGATCCCAAATGCTGGAAATGCGTG and 5’-GCAGGATCCACAAAGCCATGACGAAGC.

In order to test the regulatory potential of genomic fragments in the context of the endogenous promoters of dAP-2, the Pelican vector was engineered to have the dAP-2 exon1a promoter (−473 to +31) to generate pAlacZ. In brief, the promoter sequence was amplified by genomic PCR using the primer sets; 5’-GTGGGATCCGTATCGCACTCGCATCTCG and 5’-GGTAGATCTTTGCGCAGCCACCAGACGTAG. The promoter sequence was then cloned into the BamHI site of Pelican in fusion with the lacZ gene. The Pelican vector contains insulator sequences which reduce position effects on reporter gene expression (Barolo et al., 2000). For the E6-GAL4 construct, the E6 fragment was first cloned into a pGaTB (Brand and Perrimon, 1993) derivative engineered to have the exon1a promoter fused to the GAL4 gene. The resulting construct was then cloned into a P-element transformation vector, pCasper (Thummel et al., 1988).

For the deletion analysis of EB, fragments were amplified by PCR using combinations of forward and reverse primers (Suppl. Table S1) and subcloned into the EcoRI and BamHI sites of pAlacZ.

To make an internal deletion of the region between positions 556–593 in EB, a 5’ 555 bp fragment and a 3’ 1.4 kb fragment were amplified by PCR using the following primer sets: 5’-CACGATTTCGAGGCAGAGAG and 5’-GGATCCTAAGGGAAGCCATCAATCAG for the 555 bp fragment, and 5’-GGATCCGTTCCAATTTACAATGTGC and 5’-GGATCCTCAGTCTCTTCCTTCG for the 1.4 kb fragment. The EcoRI/BamHI-digested 555 bp fragment was first subcloned into pAlacZ followed by the BamHI-digested 1.4 kb fragment to generate pAlacZ-EBΔ556–593.

Site-directed mutagenesis of the Exd/Hox binding site in EB was performed by a PCR-based method (Statagene).

The resulting constructs were prepared using a Qiagen Midi prep column, and injected into embryos by standard P-element transformation procedures.

X-gal staining and Immunostaining

X-gal staining was performed to assay β-galactosidase (β-gal) activity in the lacZ reporter transgenic flies. Inverted anterior halves of late third instar larvae were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) with 0.1% TritonX-100 (PBT) for 10 minutes, washed three times with PBT, and incubated in X-gal staining solution (PBS, 1mM MgCl2, 5mM K4Fe(CN)6·3H2O, 5 mM K3Fe(CN)6, 0.3% TritonX-100) with 1 mg X-gal/mL at 30°C. Discs were stained for appropriate times up to 16 hours. After washing in PBT, discs were dissected and mounted in 90% glycerol for microscopic analysis. For the X-gal staining of pupal legs, pupae were taken out of the pupal case before fixation.

For immunostaining, leg discs were dissected and processed following standard procedures (Therianos et al., 1995), and incubated overnight at 4°C in the following primary antibodies: mouse anti-β-gal (Promega), mouse anti-GFP (Molecular Probes), rabbit anti-dAP-2 (Monge et al., 2001), and rabbit anti-β-gal (Cappel). For immunofluorescence, the following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes), and Cy3 goat anti-rabbit IgG (Molecular Probes). Discs were dissected and cover-slipped in 90% glycerol for confocal microscopy. All images are from a single optical section.

Electrophoretic mobility shift assay (EMSA)

His-tagged recombinant proteins were expressed in E. coli BL21 cells and purified with Ni-NTA agarose (Qiagen) according to the manufacturer’s protocol. pET14b-exd (Rieckhof et al., 1997) and pQE31-Scr (Ryoo and Mann, 1999) have been described. pETM11-Antp was a gift from R. Mann. For pET14b-Ubx, the coding sequence for Ubx from amino acid 56 to carboxyl terminus was amplified by PCR using pQE9-Ubx (Ryoo and Mann, 1999) as a template, and cloned into pET14b (Novagen).

EMSA was performed in a buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2, 6% glycerol, 200 µg of BSA per ml, and 50 µg of poly(dI-dC)poly(dI-dC) per ml. In 30 µl reactions, each Hox protein was tested at two concentrations: 50 ng and 100 ng for Scr and Ubx, and 30 ng or 60 ng for Antp. The amount of Exd used was 60 ng. After approximately 1 ng of probes end-labeled with 32P was added to each reaction, the reaction was incubated for 30 minutes at room temperature, and then run on a 5% polyacrylamide gel for 3 hours. The probes used were fkh[250con] (5’-GATCTCAATGTCAAGATTTATGGCCAGCTGTGGGACGA/ 5’-CCTCGTCCCACAGCTGGCCATAAATCTTGACATTGAGA), PrFWT (5’-TGATGGCTTCCCTTATGATTTATTGAATAATTTCTTTA/5’-TATAAAGAAATTATTCAATAAATCATAAGGGAAGCCAT), PrFEXD (5’-TGATGGCTTCCCTTATAGTTTATTGAATAATTTCTTTA/5’-TATAAAGAAATTATTCAATAAACTATAAGGGAAGCCAT), and PrFHOX (5’-TGATGGCTTCCCTTATGATTTTTTGAATAATTTCTTTA/5’-TATAAAGAAATTATTCAAAAAATCATAAGGGAAGCCAT).

Results

Isolation of dAP-2 enhancer elements

We utilized a comparative genomics approach to identify cis-elements essential for dAP-2 expression in leg discs since these elements are more likely to be conserved between distantly related Drosophila species during evolution (Richards et al., 2005). A VISTA plot depicting the alignment of ~32 kb of D. melanogaster genomic DNA containing the AP-2 locus to the homologous region of the D. pseudoobscura genome was obtained from the VISTA Browser web site (http://pipeline.lbl.gov/cgi-bin/gateway2?bg=dm2&selector=vista) and is shown in Fig. 1A. This plot reveals that highly conserved regions are scattered throughout the AP-2 locus. Therefore, we tested the regulatory potential of partially overlapping genomic fragments from this 32 kb region of D. melanogaster dAP-2 locus using in vivo lacZ reporter analysis (Fig. 1A). We used a P-element transformation vector containing a dAP-2 promoter fused to lacZ (See Materials and Methods). All the genomic fragments except B6d and BG were tested in the context of the exon1a promoter because it is the promoter predominantly active in larval leg discs (Zou et al., unpublished data). B6d was tested using a transformation vector containing an hsp70 promoter fused to lacZ (Monge et al., 2001) and BG was directly fused to lacZ because it includes the predicted exon1b promoter.

Fig. 1. Isolation of dAP-2 enhancers for leg and antennal disc expression.

Fig. 1

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(A) VISTA plot comparing the ~32 kb dAP-2 genomic sequences of D. melanogaster with those of D. pseudoobscura. The y-axis represents sequence identity (50%–100%). Highly conserved regions are colored according to the annotation as exons (dark blue), UTRs (light blue) or non-coding sequences (pink). Partially overlapping bars below the plot represent D. melanogaster genomic fragments tested by lacZ reporter transgenes (see text).

(B–M) lacZ expression driven by each genomic fragment in leg discs. pAlacZ without an insert showed basal expression in distal leg discs (B).

(N–S) lacZ expression driven by selected genomic fragments in antennal discs. Leg and antennal discs were oriented with dorsal to the top.

Multiple independent transgenic lines were established for each reporter construct (Suppl. Table S2). Each construct produced very similar patterns of activity in different lines with variation observed only in expression levels. X-gal staining of third instar (L3) leg discs from the established transgenic lines identified leg enhancer activity in the four BamHI or EcoRI genomic fragments, E7, B6d, B4 and E6 (Figs. 1B–M). E7 and B6d both drove lacZ expression in presumptive tarsal joints suggesting that the overlapping region contains the distal enhancer activity (Figs. 1D,E). The enhancer activity for the intermediate and proximal leg segments appeared to reside in E6, which is positioned between the two alternative promoters. E6 drove reporter expression in multiple proximal and intermediate rings and generated a diffused distal pattern (Fig. 1I). E6 overlaps with B4 in its sub-fragment EB, and both B4 and EB displayed activity in two proximal rings with stronger activity observed in the dorsal region (Figs. 1G–H). Another sub-fragment of E6, B3, drove expression in an incomplete proximal ring with strongest activity in the lateral region. B3 also led to a non-specific pattern in the intermediate and distal leg disc (Fig. 1J). Although there are distinct differences in both size and morphology between different leg discs (prothoracic, mesothoracic, and metathoracic), we did not observe significant differences in reporter activity among the different leg discs.

Antennal versus leg enhancers

The Drosophila antenna and leg have been considered to be homologous structures as shown by leg-to-antenna or antenna-to-leg transformations caused by mutations in genes regulating development of the two appendages (Casares and Mann, 1998; Dong et al., 2000; Frischer et al., 1986; Pai et al., 1998; Postlethwait and Schneiderman, 1969; Struhl, 1981). Also, there are a number of genes that regulate development of both appendages including those involved in the PD patterning (Dong et al., 2001). In line with this idea, dAP-2 is expressed as multiple concentric rings in antennal discs as well as in leg discs (Fig. 7E), and dAP-2 null mutants display antennal phenotypes including loss of the basal cylinder (Monge et al., 2001). To investigate whether the two appendages share enhancers to regulate dAP-2, we examined reporter expression in L3 antennal discs. We found that the distal leg enhancer E7 drives lacZ expression in the distal domain of antennal discs (Fig. 1N, Suppl. Fig. S1A), and the EB enhancer, which is specific for the two proximal rings in leg discs, drives lacZ expression in one proximal ring in antennal discs (Fig. 1Q). These results demonstrate the sharing of some enhancers between the leg and antennal discs. However, we identified antennal segment 3 (AIII)-specific enhancer activity in BE, which does not show significant activity in leg discs (Fig. 1O, Suppl. Fig. S1B). In addition, we later found that a femur-specific enhancer is inactive in antennal discs (see below). Together, these findings reveal both shared and distinct mechanisms regulating dAP-2 expression in these two homologous appendages.

Fig. 7. Ectopically expressed Antp activates PrF in antennal discs.

Fig. 7

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(A–B) Ectopic activity of PrF in the antennal discs of gain-of-function Antp73b mutants. In wild-type animals, PrF-lacZ expression is detected only in the distal antennal discs (A). However, with ectopic expression of Antp, PrF-lacZ expression is induced in the proximal antennal disc of Antp73b mutants (A’). PrFC-lacZ also shows similar ectopic expression in the mutants (B–B’).

(C–D) The conserved Exd/Hox site is necessary for the ectopic activity of PrF. Mutations in either the Exd (C’) or Hox (D’) half site significantly reduce ectopic expression in the proximal antennal disc of Antp73b mutants.

(E–F) Immunostaining shows that dAP-2 is normally expressed as two concentric rings in the proximal antennal disc (E). Additional domains of dAP-2 coincide well with ectopic PrF-lacZ expression pattern in the proximal antennal disc of Antp73b mutants (F–F”). Double immunostaining against β-gal (green) and dAP-2 (red). Bars 30 µm

E6-lacZ co-localizes with endogenous dAP-2 and E6-driven dAP-2 can rescue leg mutant phenotypes

Since the E6 fragment displayed enhancer activity in multiple ring domains within leg discs, we performed double immunostaining to investigate whether the β-galactosidase (β-gal) domain of the E6-lacZ reporter represents the endogenous pattern of dAP-2. In the distal leg discs, the E6-lacZ pattern was broader showing only partial overlap with endogenous dAP-2 (Data not shown). In the proximal leg discs, four rings of β-gal in E6-lacZ transgenic leg discs precisely coincided with those of endogenous dAP-2 (Fig. 2A).

Fig. 2. E6 contains enhancer activity for the proximal coxa, trochanter, femur and tibia.

Fig. 2

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(A–A”) E6-driven β-gal coincides with the four proximal ring domains of dAP-2 in third instar leg discs.

(B) odd-lacZ expression marks the non-tarsal leg joints, pretarsus (pr) and the boundary between the coxa and thorax in pupal legs.

(C–D) Merged images show that dAP-2 domains are more distal than the odd-lacZ expression domains with partial overlap in each leg segmental boundary (arrows). The images were taken from the same disc at two different focal planes to show the proximal and distal region. th, thorax; co, coxa;tr, trochanter; fe, femur; ti, tibia; ta, tarsus. Double immunostaining against β-gal (green) and dAP-2 (red) (A–A”, C–D).

(E–G) E6-driven dAP-2 in the E6-GAL4;UAS-dAP-2 flies can rescue the leg outgrowth defect of dAP-2 mutants. Bars 30 µm

This prompted us to test whether dAP-2 expression driven by E6 can compensate for the loss of the endogenous gene during leg development. For this experiment, an E6-GAL transgene was used to drive dAP-2 cDNA expression from a UAS-dAP-2 transgene (Brand and Perrimon, 1993). E6-driven dAP-2 resulted in rescue of dAP-2 null mutant leg phenotypes to varying degrees. This variation in the extent of rescue is likely due to variegated transgene expression (data not shown). In some cases, almost completely rescue of the outgrowth defect in the proximal and intermediate leg segments (coxa, trochanter, femur and tibia) was observed (Fig. 2E–F). This suggests that E6 is sufficient for spatio-temporal regulation of dAP-2 in the proximal and intermediate leg segments. Since no other fragments from the 32 kb genomic region displayed enhancer activity for those segments, it is very likely that E6 is also necessary for dAP-2 expression in the proximal and intermediate segments. In the distal leg, E6-driven dAP-2 significantly rescued the outgrowth defect of the tarsus, but failed to restore tarsal joints (Fig. 2F). This is consistent with the observation that E6 is insufficient to restrict lacZ expression to segmental boundaries in the tarsus since joint formation requires precisely regulated expression of dAP-2 (Kerber et al., 2001). This suggests that proper regulation of dAP-2 in the tarsal joints requires the E7/B6d region, which can recapitulate dAP-2 expression patterns in the tarsus (Fig. 1D,E; Zou et al., unpublished data).

dAP-2 is expressed in the proximal ends of each leg segment except the tarsal segments

It was previously shown that dAP-2 is expressed in the distal ends of each tarsal segment, in other word, in the presumptive proximal joint cells (Kerber et al., 2001). To locate dAP-2 domains relative to leg segment boundaries in segments other than the tarsus, the expression pattern of dAP-2 was compared with that of a known joint marker, Odd-skipped (Odd). Odd is expressed in the presumptive mid-distal joint cells at each leg segment boundary except in the tarsal joints (Mirth and Akam, 2002). Consistent with previous reports, X-gal staining of an enhancer trap line for odd labeled the boundaries between thorax/coxa, coxa/trochanter, trochanter/femur, femur/tibia and tibia/tarsal1, and in the pretarsus (Fig. 2B). Immunostaining of leg discs indicates that odd-lacZ is expressed as five concentric rings as well as in the presumptive pretarsus in late third instar larvae. Cells expressing dAP-2 were located distal to odd-lacZ expressing cells with only partial overlap (Figs. 2 C–D). This suggests that dAP-2 is expressed in the proximal ends of each leg segment. Based on the above data, we conclude that the 5 proximal and intermediate rings of dAP-2 in the third instar leg disc correspond to the proximal ends of the presumptive coxa, trochanter, femur, tibia and tarsal1, respectively.

Dissection of the EB fragment identified two separate enhancers for the proximal coxa and proximal femur

EB contains enhancer activity in the proximal coxa and proximal femur as well as in a diffused pattern in the tarsus as shown by X-gal staining of both leg discs and pupal legs (Figs. 3B–C). Double immunostaining confirmed that EB-lacZ covers a subset of the E6-lacZ pattern coinciding with the first and third proximal rings of endogenous dAP-2 (Fig. 4A).

Fig. 3. Dissection of EB identified two separate enhancers for the proximal femur and coxa.

Fig. 3

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(A) Structure of reporter constructs with the conservation profile of EB in comparison with DPEB, a corresponding sequence from D. pseudoobscura. The 0.4 kb proximal femur enhancer (PrF) contains two highly conserved motifs (CM1 and CM2). EB1.7R includes both PrF and the 1.3 kb proximal coxa enhancer (PrC) and, hence, is named as PrFC.

(B–C) EB drives lacZ expression in the proximal ends of the femur and coxa in addition to a diffused distal pattern in L3 leg discs (B) and late pupal legs (C).

(D) DPEB produces an EB-like expression pattern in D. melanogaster leg discs.

(E–I) Deletion analysis reveals that EB contains two separate enhancers: PrF (H) and PrC (I).

(J–M) Deletion of either CM1 or CM2 results in loss of PrF activity. Note that PrC activity is not affected by these deletions.

Fig. 4. dAP-2 co-localizes with Hth and Dll in the proximal femur.

Fig. 4

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(A–A”, B–B”) EB-lacZ and DPEB-lacZ expression coincides with the dAP-2 domains in the femur and coxa in L3 leg discs.

(C–C”) The proximal femur dAP-2 ring domain lies in the distal end of the Hth domain.

(D–D”) The proximal femur dAP-2 ring domain corresponds to the distal portion of the proximal Dll domain. hth-lacZ and Dll-lacZ lines were used to mark Hth and Dll domains, respectively. Double immunostaining against β-gal (green) and dAP-2 (red). Bars 30 µm

A pairwise sequence alignment of EB and a corresponding D. pseudoobscura sequence (DPEB) indicates that this region contains multiple conserved motifs (Fig. 3A). When DPEB was tested in D. melanogaster, it faithfully reproduced the enhancer activity of EB in the proximal and intermediate leg discs (Fig. 3D; Fig. 4B). This suggests that the conserved motifs in EB are required for the leg enhancer activity. To narrow enhancer activity to a smaller fragment, a series of deletion constructs was generated based on the sequence comparison so that each construct contains a subset of the conserved motifs (Fig. 3A). In vivo reporter analysis with the deletion constructs revealed two separate enhancers in EB. A 368 bp fragment (PrF) directed expression to the proximal femur (Fig. 3H), and a longer 1.3 kb fragment (PrC) directed expression to the proximal coxa (Fig. 3I).

PrC contains two highly conserved motifs, one in the middle of the enhancer and the other at the 3’ end as well as several less conserved motifs at the 5’ end (Fig. 3A). Deletion of ~200 bp from either the 5’ or 3’ end of the fragment resulted in loss of coxa-specific enhancer activity (Fig. 3A; data not shown). While the strong effect of the 3’ deletion was consistent with the high level of sequence conservation at the 3’ end of PrC (95% identity in a 60 bp motif), it was unexpected that deletion of the weakly conserved 5’ end also abolished enhancer activity. This suggests that the small patches of conserved sequences within the 5’ 200 bp region of PrC might also be necessary for enhancer activity (data not shown).

While E6 drove expression as a complete ring in the proximal coxa, PrC-lacZ expression in the presumptive coxa appeared to be weaker in the lateral and ventral leg disc (Figs. 3I, L). In addition, as mentioned above, another sub-fragment of E6, B3, contains a coxa-specific enhancer activity which was stronger in the lateral leg disc (Fig. 1J; data not shown). Taken together, our data suggest that dAP-2 expression in the proximal coxa is controlled by at least two separate enhancers, EB and B3, which have differential activity along the DV axis of leg discs.

A 0.3 kb region at the 5’ end of EB is highly conserved between the two Drosophila species and is responsible for the diffused distal lacZ pattern (Figs. 3B, J). Although every fragment containing EB0.3 frequently drove expression in the proximal tarsal1 as well as in the rest of the tarsus, it is not clear whether EB0.3 directs dAP-2 expression the tarsal1 in vivo due to the relatively large variation among different reporter constructs (data not shown).

A conserved Exd/Hox binding site is required for the femur enhancer activity

The proximal femur enhancer, PrF, contains two highly conserved motifs (CM1 and CM2) of 45 and 38 bp in length, respectively (Fig. 3A). To test whether the two conserved motifs are necessary for enhancer activity, a series of 5’ or internal deletions were introduced into EB-LacZ (Fig. 3A). In this context, the PrC pattern was used as an internal control. Removal of either CM1 or CM2 led to loss of the PrF enhancer activity, while the PrC pattern remained the same (Figs. 3K–M).

The nucleotide sequences of CM1 and CM2 were examined for known transcription factor binding sites using the program, ConSite (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite). No strong candidate sites were found. We then manually searched the sequences for binding sites for transcription factors involved in leg development. We identified a consensus binding site for Exd/Hox heterodimers at the 5’ end of CM2. Exd is a good candidate to be an upstream regulator of dAP-2, especially in the proximal femur, because it is required for development of the proximal leg segments (Gonzalez-Crespo and Morata, 1995, 1996; Rauskolb et al., 1995). Double immunostaining indicated that the proximal femur dAP-2 domain resides within the Hth domain where Exd proteins are known to be nuclear. Furthermore, the distal end of the proximal femur dAP-2 domain precisely coincides with that of the Hth domain (Fig. 4C). Additional immunostaining revealed that the proximal femur dAP-2 domain overlaps also with Dll and Dac domains (Fig. 4D; data not shown) consistent with the previous observation that the three PD patterning genes are co-expressed at the boundary between trochanter and femur (Duncan et al., 2010; Wu and Cohen, 1999).

To test whether the conserved Exd/Hox binding site is necessary for enhancer activity, point mutations previously shown to eliminate Exd/Hox binding were introduced into either the Exd or Hox half-site (Fig. 5B). Both of the mutations resulted in loss of the PrF pattern without affecting the PrC pattern indicating that the conserved binding site is essential for the femur-specific enhancer activity (Figs. 5C–E).

Fig. 5. A conserved Exd/Hox binding site is required for activity of the proximal femur enhancer.

Fig. 5

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(A) Alignment of the conserved motif 2 (CM2) from D. melanogaster (EB) and D. pseudoobscura (DPEB). The sequence deleted in Fig. 3M is underlined. A consensus Exd/Hox binding site is marked in a box.

(B) The sequences of the known Exd/Hox site, fkh[250con], the wild-type Exd/Hox site in CM2 (PrFWT), and mutated Exd/Hox sites (PrFEXD and PrFHOX) are shown. These mutations in each half site are known to abolish heterodimer binding.

(C–E) In vivo reporter analysis with the mutated PrFC fragments indicates that the Exd/Hox binding site is necessary for the PrF enhancer activity. lacZ expression in the proximal femur is lost by mutations in either the Exd (D) or the Hox (E) half site while lacZ expression in the coxa remains the same.

(F–H) EMSA using the fkh[250con], wild-type and mutant oligos with Exd and three Hox proteins, Antp, Ubx and Scr. All three Exd/Hox heterodimers bind strongly to the wild-type site, and this binding is abolished by mutations in the half sites (arrows). Antp and Scr show weak binding as a monomer (arrowheads).

Exd/Hox proteins bind to a conserved site within the PrF enhancer in vitro

The above mutational analysis suggested that Exd/Hox heterodimers bind to the consensus site in PrF and directly regulate dAP-2 expression. Therefore, we performed electrophoretic mobility shift assays (EMSA) on an oligonucleotide covering the sequence around the Exd/Hox binding site in PrF with Exd and three Hox proteins, Scr, Antp and Ubx, which are expressed in leg discs (Casanova et al., 1985; Casares and Mann, 1998; Emerald and Cohen, 2004; Struhl, 1982). Another oligonucleotide, fkh[250con], was used as a positive control because it was previously shown to bind to all three Exd/Hox heterodimers (Ryoo and Mann, 1999). Compared to the control, our wild-type oligo showed stronger binding of all three Exd/Hox heterodimers (Figs. 5F–H). As expected, Exd alone did not bind to the oligos and the three Hox proteins showed no or poor binding as monomers. To confirm that the Exd/Hox binding site, not the surrounding sequences, is responsible for the strong binding, EMSA was performed with oligos containing point mutations in either the Exd or Hox half-site (Figs. 5B, F–H). The mutation in the Exd half-site prevented heterodimer binding with minor effect on Hox monomer binding, and the mutation in the Hox half-site prevented both heterodimer binding and Hox monomer binding. In summary, PrF contains a conserved Exd/Hox binding site which is essential for enhancer activity and is bound strongly by heterodimers of Exd and the three Hox proteins expressed in leg discs.

Exd and Hth are required for dAP-2 expression in the proximal femur

The data from our deletion/mutational analyses and from EMSA suggest that dAP-2 is a direct target of Exd/Hox heterodimers in the proximal femur. We tested this idea by examining whether endogenous dAP-2 expression in the region is changed in exd mutant clones generated by FLP/FRT mediated recombination (Xu and Rubin, 1993). It appeared that loss of exd has region-specific effects on dAP-2 expression; dAP-2 expression was lost in the exd mutant clones in the proximal femur, but not altered in the mutant clones elsewhere (Figs. 6A–A”).

Fig. 6. exd, hth and Antp are required for dAP-2 expression in the proximal femur.

Fig. 6

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(A–B) dAP-2 expression is lost in exd−/− (A–A”) and hth−/− (B–B”) clones generated in the proximal femur by FRT/FLP recombination (arrow). Ectopic dAP-2 expression is occasionally observed in exd−/− and hth−/− clones positioned between dAP-2 domains in the trochanter and femur (arrowheads).

(C–E) Antp is required for dAP-2 expression in the proximal femur of T2 leg discs. dAP-2 expression is not affected in Antp−/− clones in T1 and T3 leg discs (C–C”, E–E”). However, dAP-2 expression in the proximal femur is lost in Antp−/− clones generated in T2 leg discs (D–D”, arrow). dAP-2 expression is often retained in large clones which disrupt the overall shape of the leg disc (D–D”, arrowhead). Double immunostaining against β-gal (green) and dAP-2 (red). Mutant clones are marked by lack of lacZ expression. Bars 30 µm

Since Hth is necessary for the nuclear localization and function of Exd, we also examined dAP-2 expression in hth mutant clones and found that hth is required for dAP-2 expression in the femur (Figs. 6B–B”). Interestingly, ectopic expression of dAP-2 was frequently observed in exd and hth mutant clones in the presumptive inter-joint cells located mostly in the femur and trochanter (Fig. 6B; data not shown). There is no clear explanation for the ectopic dAP-2 expression in the mutant clones outside of the Hth domain, but developmental functions of exd and hth in the intermediate region of the leg segments have been previously reported (Casares and Mann, 2001; Rauskolb et al., 1995).

We also examined changes in dAP-2 expression in Dll and dac mutant leg discs since both genes are expressed in the proximal femur. While the femur dAP-2 domain was retained in dac mutant, PrF activity was lost in the hypomorphic Dll mutant leg discs implying that Dll is required for dAP-2 expression in the femur (Suppl. Fig. S2).

Antp is required for dAP-2 expression in the proximal femur in mesothoracic leg discs

We explored which Hox protein dimerizes with Exd and binds to PrF to regulate dAP-2 expression in the proximal femur. Among the three Hox genes expressed in leg discs, only Antp is expressed in all of the leg discs, and is required for specifying leg identity (Casares and Mann, 1998; Emerald and Cohen, 2004; Struhl, 1981). In addition to its function as a homeotic selector gene, Antp has been proposed to contribute to the growth and segmentation of the proximal leg (Casares and Mann, 2001). Importantly, it has been shown that Antp is upregulated in the proximal femur region (Duncan et al., 2010). Therefore, we examined dAP-2 expression in Antp mutant clones. Interestingly, Antp was required for dAP-2 expression in the proximal femur of the mesothoracic (T2) leg discs, but was dispensable for dAP-2 expression in the prothoracic (T1) and metathoracic (T3) leg discs (Figs. 6C–E). This result is consistent with the previous observation that Antp mutant clones cause defects mainly in T2 legs (Emerald and Cohen, 2004; Struhl, 1982). It is possible that Scr and Antp can also bind to PrF and compensate for the loss of Antp in T1 and T2 legs, respectively (See Discussion).

Ectopic expression of Antp in the antenna activates the PrF enhancer

Given the importance of the Exd/Hox binding site for the PrF enhancer activity, it is not surprising that PrF is not active in the antennal discs where no Hox gene is expressed in antennal discs (Brower, 1987; Diederich et al., 1991; Levine et al., 1983; Martinez-Arias et al., 1987; Randazzo et al., 1991). In a dominant gain-of-function mutant, Antp73b, ectopic expression of Antp in the antennal discs led to transformation of an antenna into a leg-like structure (Jorgensen and Garber, 1987; Schneuwly et al., 1987). We hypothesized that ectopically expressed Antp can activate PrF in the antennal discs considering the similar developmental potential of the two appendages including the presence of nuclear-localized Exd (Casares and Mann, 1998; Pai et al., 1998). Indeed, we observed ectopic PrF-lacZ expression in the intermediate antennal discs of Antp73b mutants (Fig. 7B). Double-immunostaining indicated that ectopic dAP-2 expression was also induced in the mutants, and coincided with PrF-lacZ pattern (Figs. 7E–F). Importantly, the ectopic PrF-lacZ expression was strongly repressed by the mutations in the Exd/Hox binding site (Figs. 7B–D).

PrF-lacZ expression was also examined in antennal discs with dpp-GAL4-driven expression of Scr and Ubx, which was shown to induce an antenna-to-leg transformation (Mann and Hogness, 1990; Zeng et al., 1993; Zhao et al., 1993). As predicted, ectopic expression of Scr and Ubx also activated PrF-lacZ expression in the Dpp domain of antennal discs (Suppl. Fig. S3). These data demonstrate that the appendage-specific activity of PrF is well correlated with the presence of Hox proteins further supporting that dAP-2 is directly regulated by Exd/Hox heterodimers in the proximal femur.

Discussion

The segmentally repeated expression of dAP-2 in the developing leg and antennal discs may suggest that its expression in each segment is regulated in a similar manner by upstream segmentation genes. Alternatively, each domain (ring) of dAP-2 expression could result from the combinatorial activities of multiple transcription factors, which themselves are not expressed in a repeated pattern, but instead occupy distinct and broader domains along the PD axis of the appendages. Current data provide strong evidence that the latter strategy is utilized to establish dAP-2 expression in all but the tarsal segments. It seems that the tarsus has adopted a strategy different from that of other leg segments to regulate dAP-2 expression (Kerber et al., 2001)(Zou et al., unpublished data).

dAP-2 regulation by multiple segment-specific enhancers

In an effort to understand molecular mechanisms controlling dAP-2 expression during leg development, the regulatory potential of dAP-2 genomic fragments was tested using transgenic reporter analyses. We have successfully isolated multiple enhancers which can independently direct reporter expression in specific leg segments and together recapitulate, almost completely, the endogenous expression pattern (Fig. 8A). It is intriguing that the relative positions of these enhancers on the chromosome are well correlated with the position of their activity along the PD axis of the leg. Importantly, the presence of segment-specific enhancers suggests that dAP-2 expression is differentially regulated in each leg segment. It is likely that each domain of dAP-2 expression in the true joints is regulated by a combination of upstream regulators involved in PD patterning using segment-specific enhancers similar to the distinct enhancers used to regulate expression of the pair-rule gene, even-skipped, in every other parasegment during embryonic segmentation (Harding et al., 1989). Interestingly, dAP-2 expression in the coxa is differentially regulated along the DV axis and depends on two region-specific enhancers. In addition, the EB fragment displayed relatively weaker activity in the ventral region compared to the larger E6 fragment. These data raise the possibility that DV patterning genes are also involved in dAP-2 regulation in the proximal segments. It is possible that the use of multiple region-specific enhancers is a general mechanism establishing expression of segmentation genes during leg development.

Fig. 8. Summary: dAP-2 regulation in the Drosophila legs.

Fig. 8

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(A) Multiple segment-specific enhancers isolated by in vivo reporter analyses are shown. BXE is shared by E7 and B6d and is likely to direct expression in the presumptive tarsal joints. E6 contains enhancers for the intermediate and proximal leg segments. PrF is specific for the proximal femur. PrC and B3 together drive expression in the coxa.

(B) A proposed model for dAP-2 regulation in the proximal femur. Antp and probably other Hox proteins are required to repress hth in the intermediate leg. In contrast, concerted action of Hth and Hox proteins is necessary for growth and segmentation of the proximal leg (up to the level of the proximal femur). Hth mediates nuclear localization of Exd, and Exd/Hox heterodimers activate dAP-2 expression in the proximal femur through the binding site in PrF. Restricted activation of PrF could be accomplished either by an unknown positive regulator ‘X’ in the proximal femur or by an unknown repressor ‘Y’ in the more proximal region.

Antennal versus leg enhancers of dAP-2

Current data indicate that dAP-2 expression in antennal discs also requires multiple region-specific enhancers. Some of the leg enhancers showed an antennal expression pattern similar to their leg patterns with respect to the PD axis. However, there are also enhancers specific for either antennal or leg discs implying that the genes required for normal identity of the two homologous appendages might be involved in regulation of dAP-2 expression in some segments. One of the features that distinguish antennae from legs is that in antennal discs, hth expression is expanded to the intermediate region where dac expression is missing. In contrast, the expression patterns of Dll, dac and hth are very similar in the proximal and distal regions of the two appendages (Dong et al., 2000). It is interesting to note that the dAP-2 enhancers for the most proximal and distal regions are shared between the two appendages while the intermediate region utilizes distinct enhancers. This implies that dAP-2 expression in the intermediate region is more likely to be regulated by antennal- or leg-specific regulatory pathways. For example, although the femur and the AIII are homologous structures, the Hox-dependent proximal femur enhancer is active in the leg, but not in the antenna. Likewise, the BE enhancer is active in the proximal AIII of the Hox-free antenna, but not in the leg.

dAP-2 regulation by Hth, Exd and Hox proteins

The Hox gene Antp has been considered to be a key factor in determining leg identity since Antp mutant clones in the T2 leg cause a leg-to-antenna transformation, mainly outside of the Hth domain (Emerald and Cohen, 2004; Struhl, 1981). Previous studies suggested that Antp performs its selector function by acting as a repressor of hth and other antennal genes in the intermediate leg (Casares and Mann, 1998; Duncan et al., 2010). In contrast, both Antp and hth are expressed in the proximal leg, and are required for growth and segmentation of this region (Abu-Shaar and Mann, 1998; Casares and Mann, 2001; Pai et al., 1998; Wu and Cohen, 1999). Therefore, it has been proposed that the role of Antp as a repressor of hth is limited to the intermediate leg, and that both Antp and Hth contribute to proper development of the proximal leg (Casares and Mann, 2001).

The similar loss-of-function phenotypes of hth and exd suggest that Hth and Exd act on common target genes during development of the proximal leg (Gonzalez-Crespo and Morata, 1995; Pai et al., 1998; Rauskolb et al., 1995; Rieckhof et al., 1997). In certain developmental contexts, Hth can directly bind to DNA through its homeodomain in a ternary complex including Exd and Hox proteins to regulate expression of target genes (Ferretti et al., 2005; Ryoo et al., 1999). However, it has been shown that a Hth isoform lacking the homeodomain can execute the function of Hth in PD patterning of Drosophila leg discs indicating that direct DNA binding is not necessary for its function in proximal leg discs (Noro et al., 2006). Since we were unable to find a conserved, consensus Hth binding site in the proximal femur enhancer of dAP-2, Hth is likely functioning in dAP-2 expression through a mechanism independent of its direct binding to DNA through its homeodomain. Instead, Hth may regulate dAP-2 expression in the proximal femur by facilitating the nuclear localization of Exd or by interacting with other transcription factors which bind DNA.

As a cofactor of Hox proteins, Exd, and its mammalian homolog Pbx, cooperatively bind DNA with Hox proteins and regulate expression of their target genes which are involved in a variety of developmental processes in both vertebrates and Drosophila (Chan et al., 1997; Gebelein et al., 2002; Maconochie et al., 1997; Popperl et al., 1995; Popperl et al., 2000; Ryoo and Mann, 1999). Although previous genetic analyses have revealed essential functions of Exd and Hox proteins in leg development, it has been unclear whether these factors act together on common target genes during this process. In this study, we identified a conserved Exd/Hox binding site which is required for activity of the proximal femur enhancer of dAP-2. Through clonal analyses, we demonstrated that hth, exd and Antp are necessary for dAP-2 expression in the presumptive proximal femur of leg imaginal discs (Fig. 8B). To the best of our knowledge, this is the first example of a direct target gene of an Exd/Hox complex in Drosophila limb development. Our study also provides insight into the molecular mechanism integrating the combinatorial actions of PD patterning genes in the regulation of region-specific expression of leg segmentation genes.

Redundancy of Hox proteins in dAP-2 regulation

Although Antp is expressed in all three pairs of legs, most of the prothoracic (T1) and metathoracic (T3) legs with Antp mutant clones appeared to be normal, except for a rare fusion between the femur and tibia (Casares and Mann, 2001; Struhl, 1982). However, Scr/Antp double mutant clones in T1 legs and Antp/Ubx double mutant clones in T3 legs generated leg defects indistinguishable from those generated by Antp mutant clones in T2 legs (Struhl, 1982). It was proposed that the low penetrance of the Antp mutant phenotypes in T1 and T3 legs is due to redundancy with Scr and Ubx, which are expressed in T1 and T3 leg discs, respectively (Brower, 1987; Casanova et al., 1985; Casares and Mann, 2001; Glicksman and Brower, 1988; Percival-Smith et al., 1997; Rozowski and Akam, 2002; Stern, 2003). This idea is consistent with the previous observations that Scr and Ubx both can induce antenna-to-leg transformations when ectopically expressed in antennal discs (Mann and Hogness, 1990; Zeng et al., 1993; Zhao et al., 1993). We propose that Antp, Scr and Ubx can redundantly activate dAP-2 expression in the proximal femur as Exd/Hox heterodimers based on the following observations (Fig. 8B). First, Antp is required for expression of dAP-2 in T2 leg discs, but not in T1 and T3 leg discs. Secondly, our EMSA results demonstrate that all three Hox proteins bind strongly to the binding site in PrF as Exd/Hox heterodimers. Thirdly, all three Hox proteins can activate PrF enhancer function when ectopically expressed in antennal discs.

Supplementary Material

01

Acknowledgements

We thank Bloomington Stock Center, G. Campbell, S.M. Cohen, T. Kojima, G. Mardon, G. Morata, G. Panganiban, S.W. Schaeffer and Y.H. Sun for providing fly stocks, and R.S. Mann for plasmids and fly stocks. We thank members of the Mitchell laboratory for discussion, and Tara Alexander for critical reading of the manuscript.

Footnotes

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