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The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing

1998; Springer Nature; Volume: 17; Issue: 23 Linguagem: Inglês

10.1093/emboj/17.23.6846

1460-2075

Pedro Fernández-Fúnez, Cheng-Hsin Lu, Diego E. Rincón-Limas, Antonio Garcı́a-Bellido, Juan Botas,

Wnt/β-catenin signaling in development and cancer

Article1 December 1998free access The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing Pedro Fernández-Fúnez Pedro Fernández-Fúnez Centro de Biología Molecular/CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Cheng-Hsin Lu Cheng-Hsin Lu Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Diego E. Rincón-Limas Diego E. Rincón-Limas Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Antonio García-Bellido Antonio García-Bellido Centro de Biología Molecular/CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Juan Botas Corresponding Author Juan Botas Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Pedro Fernández-Fúnez Pedro Fernández-Fúnez Centro de Biología Molecular/CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Cheng-Hsin Lu Cheng-Hsin Lu Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Diego E. Rincón-Limas Diego E. Rincón-Limas Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Antonio García-Bellido Antonio García-Bellido Centro de Biología Molecular/CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049 Spain Search for more papers by this author Juan Botas Corresponding Author Juan Botas Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA Search for more papers by this author Author Information Pedro Fernández-Fúnez2, Cheng-Hsin Lu1, Diego E. Rincón-Limas1, Antonio García-Bellido2 and Juan Botas 1 1Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030 USA 2Centro de Biología Molecular/CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049 Spain ‡P.Fernández-Fúnez and C.-H.Lu contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6846-6853https://doi.org/10.1093/emboj/17.23.6846 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein (Ap) in dorsal cells. Here we report that dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the DLDB/CHIP co-factor, and suggest that the Ap and DLDB/CHIP proteins form a multimeric functional complex. In support of this model, we show that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression. Introduction Growth and patterning of the Drosophila appendages require the formation of compartments that are defined by the expression of selector genes (Diaz-Benjumea and Cohen, 1993; Blair, 1995; Tabata et al., 1995; reviewed by Lawrence and Struhl, 1996). The restricted expression of the selector gene apterous (ap) in the dorsal compartment of the wing is required for dorsal cell identity and for maintaining cells of the dorsal and ventral compartment in different lineages. The border between cells differing in ap expression defines the dorso-ventral compartment boundary that functions as an organizing center (Diaz-Benjumea and Cohen, 1993; Blair et al., 1994). At the dorso-ventral boundary, the interface between dorsal and ventral cells triggers a series of cell interactions mediated by the genes fringe, Serrate, Notch and Delta (reviewed in Irvine and Vogt, 1997). These interactions lead to the activation of wingless and vestigial whose activities mediate the patterning and growth of the wing along the dorso-ventral axis (Couso et al., 1995; Kim et al., 1995, 1996; Neumann and Cohen, 1997). ap encodes a putative transcription factor containing a homeodomain, and two LIM domains (Bourgouin et al., 1992; Cohen et al., 1992). Each LIM domain is composed of two cysteine-rich zinc fingers joined by a two amino acid spacer. Besides homeodomain transcription factors, LIM domains are found in many other proteins including kinases and a variety of LIM-only proteins. These can be nuclear or cytoplasmic, and the latter are often associated with the cytoskeleton. In contrast to other related zinc fingers, LIM domains do not appear to mediate protein–DNA interactions, instead they have been shown to mediate interactions among proteins. Thus, LIM domains are thought to function as adaptor modules bringing different proteins together in a complex (reviewed in Sanchez-Garcia and Rabbitts, 1994; Gill, 1995; Curtiss and Heilig, 1998; Dawid et al., 1998). In the context of homeodomain proteins, it was proposed that the LIM domains negatively regulate the activity of the homeodomain, and that a LIM domain-binding co-factor would liberate the homeodomain from the inhibitory action of the LIM domains (Taira et al., 1994). We have isolated a protein factor on the basis of its ability to bind Ap LIM domains. Sequence analysis reveals that it is related to a family of vertebrate proteins known as LDB, NLI or CLIM. These vertebrate homologs interact with a variety of proteins including certain (but not all) LIM domain proteins (Agulnick et al., 1996; Jurata et al., 1996; Bach et al., 1997; Wadman et al., 1997). We generated mutations in the Drosophila Ldb (dLdb) gene. These mutations are allelic to mutations in Chip, a previously described gene. Chip is known for its functions during segmentation (Morcillo et al., 1997), a process in which no LIM homeodomain protein is known to play a role. The DLDB/CHIP protein accumulates in the nuclei of all dorsal and ventral cells of the wing imaginal disc. Surprisingly, however, alterations in dLdb/Chip activity produce the specific phenotypes expected for a dorsal selector gene such as ap. Both lack and excess of dLdb/Chip function mimic ap lack-of-function phenotypes without affecting normal Ap protein accumulation. Thus the function of ap as a selector gene in cells of the dorsal compartment requires the correct amount of expression of a ubiquitously expressed co-factor. We show that the phenotype produced by excess dLdb/Chip can be rescued when ap is overexpressed. We conclude that the stoichiometry of Ap and DLDB/CHIP is critical for dorso-ventral compartmentalization. Results Identification of a protein binding to Apterous LIM domains To identify co-factors involved in ap function, we used the yeast two-hybrid interactor approach. A third instar Drosophila cDNA library was screened using the Ap LIM domains as bait. Five positive clones were obtained from ∼2×106 colonies screened, and two of these identified the fly homolog of Ldb/NLI/CLIM (Agulnick et al., 1996; Jurata et al., 1996; Bach et al., 1997). Recent studies on Ldb/NLI/CLIM have defined an N-terminal homodimerization domain, and a C-terminal domain that mediates interactions with LIM domains (Jurata and Gill, 1997; Breen et al., 1998; Jurata et al., 1998). The overall amino acid identity of the fly and vertebrate proteins is ∼64%, but they are up to 84% identical in the LIM-interacting domain (Figure 1A). Sequence comparison analysis also suggested that dLdb may be the same gene as Chip, a gene previously characterized for its functions during embryonic segmentation (Morcillo et al., 1997). We generated antibodies raised against the DLDB protein in rats. All the antisera revealed the same distribution of DLDB protein. Figure 1B–E shows that DLDB accumulates in the nuclei of cells in all tissues examined, and throughout development. The specificity of the antisera was demonstrated by staining dLdb mutants (see below, and Figure 3A–A″). The nuclear localization of DLDB was confirmed further by double staining with DLDB antibodies and propidium iodide (Figure 1F–H). Figure 1.The Drosophila LDB protein: comparison with vertebrate orthologs and pattern of accumulation. (A) Comparison of Drosophila and vertebrate LDB proteins. XLDB1 is a Xenopus protein, LDB1 (also known as CLIM2 or NLI) and CLIM1 are murine proteins (Agulnick et al., 1996; Jurata et al., 1996; Bach et al., 1997). Numbers refer to amino acid percentage identity in the homodimerization domain (HDD), LIM interaction domain (LID) or overall identity (numbers on the right). NLS: nuclear localization signal. (B–H) DLDB accumulates in the nuclei of most if not all cells. (B) Lateral view of a stage 5 embryo. (C) Ventral view of a stage 16 embryo. (D) Third instar wing–notum imaginal disc. (E) Third instar central nervous system and ring gland (arrow). (F) Nuclear immunodetection of DLDB (green) in salivary glands. (G) Merged image from green and red channels shown in (F) and (H), respectively. (H) Specimen from (F) showing nuclear staining with propidium iodide (red). Download figure Download PowerPoint Figure 2.dLdb/Chip regulates the expression of the wing margin markers Serrate and vestigial, but not of apterous. Green panels in the middle rows (′) show cells expressing the c-myc marker, and mutant dLdbM4 clones appear as unlabeled cells. Red panels on the right (″) show expression of the markers specified to the left of each row. Left panels show the merging of green and red channels. (A–A″) DLDB protein is not detectable in dLdb mutant clones. The clone (arrow) causes an outgrowth of surrounding tissue deforming the disc. (B–B″) Ser is expressed ectopically around dLdbM4 dorsal clones. Only non-mutant cells close to the clone, that maintain their dorsal identity, accumulate SER (arrows in B and B″). (C–C″) lacZ expression from the vestigial-B dorso-ventral boundary enhancer. Note the lacZ ectopic expression in a dLdbM4 dorsal clone (arrow). Cells in the border of the clone (mutant and non- mutant) express lacZ. (D–D″) A mutant dLdbM4 clone in a wing disc stained with anti-Ap antibody. Note the presence of Ap label inside the clone (top arrow in D″), and the abnormal growth of wild-type tissue surrounding the clone (bottom arrow). In all discs, anterior is left and dorsal is down. Download figure Download PowerPoint Generation of dLdb (Chip) mutations We isolated dLdb mutants in a screen for genes involved in wing margin development. One complementation group defined by four lethal ethyl methanesulfonate (EMS) alleles shows a characteristic dominant haploinsufficient phenotype consisting of nicks in the wing margin (Figure 2A). This complementation group was mapped to cytological position 60A, the same position as the dLdb gene as determined by in situ hybridization to polytene chromosomes (not shown). The four alleles of this complementation group were shown to be mutations in dLdb using the following approaches: (i) sequencing of the dLdb coding region in the mutant dLdbM3 revealed a deletion of seven nucleotides resulting in a stop codon that causes the deletion of 122 amino acids (not shown); (ii) rescue of the dLdb haploinsufficient phenotype by expressing the dLdb cDNA from C5-GAL4 (Yeh et al., 1995), a driver directing expression in the wing pouch at relatively low levels (not shown); (iii) lack of anti-DLDB immunoreactivity in somatic recombination clones (Figure 3A–A″); and (iv) rescue of the dLdb null phenotype in clones by expression of the dLdb cDNA using the C5-GAL4 driver (Figure 2H). Figure 3.dLdb/Chip lack-of-function phenotypes in the wing. (A) dLdbM4/+ wing showing nicks in the margin, a characteristic haploinsufficient phenotype of dLdb/Chip mutations. (B) ap78j/ap54 wing showing the margin and size reductions characteristic of weak ap mutations. (C) Notum and wing rudiments (arrows) of fly with the same genotype as in (B), but also heterozygous for dLdbM4. Note that reduction of the wings is much more severe than in (A) or (B), indicating a strong genetic interaction between ap and dLdb mutations. (D) Homozygous mutant dLdbM4 clone in the dorsal compartment of the wing inducing an ectopic wing margin (arrow). (E) Magnification of (D). The mutant cells are labeled with the cell markers forked and pawn. The border of the clone (marked by a dotted line) differentiates sensory elements of the ventral wing margin, thus the cells of the clone have changed their identity. Wild-type cells bordering the clone are induced to differentiate as sensory elements of the dorsal surface. (F) The small dLdbM4 mutant clone (arrow) far from the wing margin induces the outgrowth of the surrounding non-mutant cells. (G) The dLdbM4 mutant clone in the ventral compartment, indicated with the dotted line, has wild-type phenotype (the somewhat abnormal shape of the wing is caused by the slightly small size of pawn cells). (H) The phenotype of the dLdbM4 mutant clone in the dorsal compartment is rescued by expressing dLdb from a transgene at a relatively low level (C5 GAL4; UAS: dLdb/Chip). The border of the clone, marked with pawn, is outlined. Download figure Download PowerPoint After the work described here was completed, complementation tests between dLdb and Chip mutations revealed that they are alleles of the same gene. dLdb/Chip and apterous mutations show genetic interactions and cause identical phenotypes in wing mosaics As shown in Figure 2A–C, dLdb/Chip mutations behave as strong enhancers of wing phenotypes produced by hypomorphic ap mutations. This synergistic interaction suggests that dLdb/Chip and ap have related functions. To investigate further the function of dLdb/Chip during wing development, we generated genetic mosaics by induced mitotic recombination using the Minute technique (Morata and Ripoll, 1975). Clones of dLdb/Chip mutant cells in the wing ventral compartment (n = 145) show a wild-type phenotype and appear with normal frequencies, indicating that dLdb/Chip is not required in this compartment (Figure 2G). In contrast, dLdb/Chip clones in the dorsal compartment (n = 35) are associated with wing outgrowths and ectopic wing margins (Figure 2D–F). Cells within these clones have a ventral identity revealed by the morphology of the wing margin bristles they differentiate. Normal cells abutting the mutant clone are induced to form the dorsal structures characteristic of the wing margin (Figure 2E). The ectopic margins can be visualized in undifferentiated imaginal discs with the use of molecular markers that label the wing margin (Figure 3B–B″ and C–C″). The largest wing outgrowths correspond to clones far from the normal wing margins. These clones cause the outgrowth of wild-type tissue, with the mutant clone located at the tip of the outgrowth (Figures 2F and 3D′). Thus, although dLdb/Chip is expressed in all wing cells, dLdb/Chip mutations produce specific phenotypes that are indistinguishable from ap phenotypes in clones (Diaz-Benjumea and Cohen, 1993; Blair et al., 1994). One possibility is that normal dLdb/Chip function is required for ap expression. To test this possibility, we monitored ap expression in dLdb/Chip mutant clones induced in wing imaginal discs. As shown in Figure 3D–D″, Ap protein accumulates normally in dLdb/Chip mutant cells. Thus dLdb/Chip does not regulate ap expression but it shows genetic interactions with ap, and it also produces the same mutant phenotypes in genetic mosaics. Taken together, these results are consistent with the hypothesis that dLdb/Chip encodes a co-factor required for ap function as a dorsal selector gene. Overexpression of dLdb/Chip causes the same phenotype as apterous lack of function If Ap and DLDB/CHIP physically interact forming a functional complex, their stoichiometry may be important for the formation of the complex and for dorso-ventral patterning. To test whether the levels of dLdb/Chip expression are important for dorso-ventral patterning, we overexpressed dLdb/Chip in various patterns using the GAL4/UAS system (Brand and Perrimon, 1993). Overexpression of dLdb/Chip using a decapentaplegic (dpp) GAL4 driver (Figure 4A) results in wing outgrowths, the creation of an ectopic wing margin on the dorsal compartment and a cut in the wing (Figure 4B). These phenotypes are also evident in imaginal discs using wingless-lacZ (wglacZ) expression as a marker of the wing margin (Figure 4C and D). Because lack of ap function in clones also causes wing outgrowths and ectopic wing margins (Diaz-Benjumea and Cohen, 1993; Blair et al., 1994), we investigated the distribution of Ap protein in these wing imaginal discs. Figure 4E and F shows that dLdb/Chip overexpression does not alter the distribution of Ap protein; these results indicate that the phenotypes produced by dLdb/Chip overexpression are not caused by dLdb/Chip repressing ap. Figure 4.dLdb/Chip overexpression causes apterous lack-of-function phenotypes. (A) lacZ reporter gene expression in dppGAL4; UAS: lacZ wing disc. The dorso-ventral compartment boundary is indicated by the dotted line. (B) Results of overexpressing dLdb/Chip in the dpp pattern shown in (A). Note the ectopic wing margin in the distal antero-posterior boundary (arrowhead), and the outgrowth of this region in the dorsal surface. Wings of this genotype also show a cut of the anterior wing margin (arrow), probably the result of the lack of a dorso-ventral compartment boundary where the dpp expression domain meets the normal dorso-ventral boundary. (C) Wild-type wglacZ expression pattern in third instar imaginal disc. Note the wglacZ expression along the dorso-ventral boundary. (D) wglacZ expression on the dorso-ventral boundary is altered when dLdb is overexpressed as in (B). Ectopic wglacZ expression is observed in the dorsal side of the wing antero-posterior compartment boundary (arrowhead). The arrow points to a gap in the expression of wglacZ that corresponds to the cut in the wing margin shown in (B). (E) Wild-type distribution of Ap protein. (F) Ap protein distribution is unaffected after dLdb overexpression as in (B) and (D). Note the normal Ap label where dLdb is overexpressed (arrow). Outgrowths in the wing disc can be seen as new disc foldings associated with the formation of ectopic margins shown in (B) and (D). Download figure Download PowerPoint Interestingly, overexpression of ap using the drivers dppGAL4 or apGAL4 described above does not result in wing abnormalities in the dorsal compartment (not shown). The results described above show that overexpression of dLdb/Chip results in the same phenotype as its lack of function, i.e. transformation of dorsal into ventral cells, and as a consequence wing margin formation and outgrowth. These observations suggest that the relative amounts of Ap and DLDB are important for dorso-ventral patterning. Overexpression of apterous rescues the phenotypes produced by dLdb/Chip overexpression If the relative amounts of Ap and DLDB are critical for dorso-ventral patterning, then it should be possible to rescue the excess of dLdb/Chip phenotype by overexpressing ap. The proposed domain structure of the LDB/NLI family of proteins provides a conceptual framework to understand the phenotypes produced by altering the dosage of dLdb/Chip and ap. The presence of homodimerization and LIM-interacting domains in LDB/NLI proteins suggests that LDB and LIM domain proteins may form tetrameric complexes. These complexes would be formed by two LDB molecules interacting through the N-terminal homodimerization domain; in addition, each LDB molecule would interact with a LIM domain through the C-terminal LIM-interacting domain. The occurrence of these complexes has been demonstrated between murine LDB and the hamster LIM/homeodomain protein LMX1 (Jurata and Gill, 1997). In the case of DLDB–Ap, this tetrameric complex (Figure 6A) may be the functional complex carrying out the dorso-ventral patterning functions. This model predicts that dLdb/Chip overexpression would lead to the formation of non-functional complexes, and it also predicts that functional DLDB–Ap complexes would be reconstituted by overexpressing ap in addition to dLdb/Chip (see Figure 6 and Discussion). Figure 5.A model for Ap/DLDB function. (A) An DLDB–Ap tetramer may be the functional complex carrying out dorso-ventral patterning functions. For simplicity, the Ap and DLDB molecules are depicted in the absence of DNA. However, one possibility is that DLDB may be instrumental in bringing together otherwise distant Ap-binding sites; DLDB may also facilitate contacts with the target promoter required for activation or repression of transcription. (B) Overexpression of DLDB would lead to the titration of Ap, and the formation of non-functional trimers. (C) Ap overexpression would not lead to the formation of non-functional complexes because overabundance of Ap should not be able to prevent interactions between DLDB molecules bound to Ap. (D) Ap overexpression would reconstitute functional complexes after DLDB overexpression. Download figure Download PowerPoint To test this model, we used a GAL4 insertion in ap (Calleja et al., 1996). Expression from this GAL4 driver faithfully reproduces ap expression in the wing imaginal disc (not shown). When dLdb/Chip is expressed from the ap promoter, the wing is reduced or eliminated depending on the UAS:dLdb/Chip transgenic line used. Most lines reduce the wings (Figure 5A), whereas the strongest line completely eliminates them (not shown). As shown in Figure 5B, the reduced wing phenotype can be completely rescued by ap overexpression. These results provide further evidence for the idea that the stoichiometry of Ap and DLDB is critical for dorso-ventral patterning. Figure 6.ap overexpression rescues the phenotypes caused by excess dLdb/Chip. (A) Notum and wing (arrow) of a apGAL4/+; UAS: dLdb/+ fly. Note that dLdb/Chip overexpression in the ap domain causes severe reductions in the margin and size of the wing. (B) Wing of a apGAL4/+; UAS: dLdb +/+ UAS: ap fly. Note the complete rescue of the phenotype shown in (A) by ap overexpression. Download figure Download PowerPoint Discussion Using the yeast two-hybrid system, we cloned dLdb/Chip, a gene whose protein product interacts with Ap LIM domains. DLDB/CHIP is the Drosophila homolog of a family of vertebrate proteins known for binding certain LIM domain proteins, and at least one non-LIM protein (Agulnick et al., 1996; Jurata et al., 1996; Bach et al., 1997; Wadman et al., 1997). As reported recently, DLDB/CHIP is required for normal embryonic segmentation (Morcillo et al., 1997). However, no known LIM protein (including Ap) is known to play a role during Drosophila segmentation. Thus, this early function of DLDB/CHIP is likely to be independent of LIM proteins. The analysis of DLDB/CHIP function in genetic mosaics described here reveals that it has very specific functions during wing development. We found no requirement for DLDB in cells of the ventral compartment, and an exclusive role for DLDB in dorsal cells specifying dorsal identity in cooperation with Ap. Mutations in dLdb/Chip are haploinsufficient (Figure 2A; see also Figure 3A in Morcillo et al., 1997); in addition, dLdb/Chip and ap show strong genetic interactions (Figure 2A–C). These results suggested that dorso-ventral patterning is very sensitive to dLdb/Chip dosage. This sensitivity was confirmed in overexpression experiments; we found that excess dLdb/Chip expression mimics ap lack-of-function phenotypes. How can these results be explained in terms of a DLDB–Ap functional complex? In principle, Ap and DLDB/CHIP may form a dimer. However proteins of the LDB family contain a homodimerization domain, suggesting that they may form tetramers with LIM proteins. The formation of these tetrameric complexes has been demonstrated between the LIM/homeodomain protein LMX1 and LDB (Jurata and Gill, 1997). The LDB homodimerization domain maps to its N-terminus, and this region is ∼64% identical between the fly and the vertebrate proteins. Thus, Ap and DLDB may form a tetramer as shown in Figure 6A, and this may be the functional complex carrying out the activity as a dorsal selector protein. We made three observations that support the hypothesis of the DLDB–Ap tetramer as a functional complex. (i) Excess DLDB causes ap lack-of-function phenotypes. According to the tetramer model, excess DLDB would lead to the titration of Ap and the formation on non-functional trimers (Figure 6B). This result is more difficult to explain in terms of a DLDB–Ap dimer because Ap would not be titrated. (ii) ap overexpression does not produce a mutant phenotype. The tetramer model predicts that excess Ap should not prevent two DLDB molecules interacting with one another; thus it should not interfere with formation of the tetramer (Figure 6C). (iii) The excess DLDB is rescued by overexpressing ap. The addition of Ap to an excess of DLDB should result in the transformation of non-functional trimers into functional tetramers (Figure 6D). It will be interesting to learn if LDB/NLI/CLIM proteins in vertebrates show dosage-sensitive interactions with their protein partners, and how many and what classes of proteins they interact with. DLDB/CHIP was proposed to be a general facilitator of enhancer–promoter interactions (Morcillo et al., 1997); accordingly, DLDB/CHIP may help Ap make contacts with the basal transcription machinery at the promoters of genes under Ap control. The hypothesis of DLDB/CHIP as a general enhancer–promoter facilitator implies that it has many partners. However, our mosaic analysis in the wing suggests that the functions of DLDB/CHIP are very specific and that the number of its protein partners may be very limited. Materials and methods Library screening The DNA sequence encoding the ap LIM domains was amplified with specific primers and cloned into the yeast bait vector pAS1. The resulting construct was sequenced and transformed into the yeast strain HF7C (Clontech). A λACT library from Drosophila third instar larvae (a gift from S.J.Elledge) was used for the yeast two-hybrid screening as described (Durfee et al., 1993). Two 1.9 kb clones were obtained from the screening (pACT-23-22 and pACT-24-1). Both clones contain the full open reading frame (ORF) but differ by 47 nucleotides in length at the 3′ end. A longer 2.2 kb dLdb cDNA was later isolated from a Drosophila early pupae cDNA library (Poole et al., 1985) using the pACT-23-22 cDNA as probe. Sequence analysis All DNA sequence analyses were performed on both strands using the ABI PRISM dye terminator kit (Perkin Elmer). To identify the mutations in dLdb, four sets of primers were used to amplify sequences covering all the dLdb coding region using genomic DNA from dLdb heterozygous flies as templates. The resulting PCR products were cloned into the pGEM T-Vector (Promega) and six independent clones were analyzed for each specific cloning. Antibody production and immunostaining The full ORFs of dLdb and ap cDNAs were cloned into pRSET C (Invitrogen) and pET15b (Novagen), respectively. The resulting constructs were used for protein production in Escherichia coli strain BL21DE3 (Novagen). The His-tagged proteins were purified through His-Bind Resin according to the manufacturer's instructions (Novagen). The purified fusion proteins were isolated by SDS–PAGE and used to immunize rats and guinea pigs by Cocolico Co. (Reamstown, PA). Immunostaining was performed as described (Vachon et al., 1992). The following primary antisera were used: mouse anti-β-GAL (1:10 000, Promega), guinea pig anti-Ap (1:2000, see above), rat anti-DLDB (1:3000, see above), mouse anti-C-MYC (1:10, American Type Culture Collection) and rabbit anti-SER (Speicher et al., 1994). For horseradish peroxidase staining, subsequent steps were conducted according with the manufacturer of the ABC kit (Vector Laboratories). For confocal image, the secondary antisera conjugated with fluorescein isothiocyanate or Cy5 were used at 1:200 according to the manufacturer's instructions (Jackson ImmunoResearch laboratories). Propidium iodide staining was conducted following standard techniques. Drosophila stocks and crosses To generate the UAS: dLdb transgenic lines, the dLdb cDNA was swapped from pACT-23-22 to the XhoI site of pUAST (Brand and Perrimon, 1993). UAS: ap was made with a 1740 bp XbaI–SmaI fragment from the ap cDNA that contains the ORF, 5′ and 3′ sequences cloned into the KpnI site of pUAST. The resulting constructs were used to inject yw embryos by standard procedures (Rubin and Spradling, 1982). Three independent lines for both dLdb and ap were tested. All fly strains were obtained from the Bloomington Drosophila Stock Center and crosses were grown at 25°C unless otherwise indicated in the text. The genetic interaction between ap and dLdb was tested by crossing ap54/SM5 flies to ap78j/CyO and to ap78j dLdbM4/CyO flies at 18°C. Generation of mosaic clones pwn dLdbM4 flies were crossed to f36a; f+44 f+52 M(2)58F and the progeny were irradiated (X-rays, 1000 rad) at 48–72 h after egg-laying. To generate clones on wing imaginal discs, dLdbM4/T(2;3)SM6a-TM6b and dLdbM4; vg(B)/T(2;3)SM6a-TM6b flies were mated with heat-shock-inducible 42πM myc flies (Xu and Rubin, 1993). Mitotic recombination was induced in 48–60 h AEL staged larvae using 1000 rad X-rays. After 60 h, larvae were heat-shocked for 2 h at 37°C to induce myc expression. Wing imaginal discs were immunostained after a 90 min recovery as described (Kim et al., 1995). Mutagenesis dLdb mutations were induced by EMS (0.2 M) in adult males using standard protocols (Lewis and Bacher, 1968) on a Vallecas wild-type strain. Acknowledgements We are very grateful to Jaeseob Kim and the Carroll laboratory for advice during the initial stages of this work, to the Bloomington stock center for Drosophila strains, to Stephen Elledge for the Drosophila cDNA library used for screening with Ap LIM domains, to Gerard Karsenty and Hugo Bellen for critically reading the manuscript, and to Wei-Chi She for helping with preparation of figures. C.-H.L., D.E.R.-L. and P.F.-F. were supported by a NSF training grant, a PEW postdoctoral fellowship and a fellowship from Comunidad de Madrid, respectively. 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