In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. Here, we show that two homeobox genes at the Bar locus (BarH1 and BarH2) may belong to a different class of prepattern genes expressed latitudinally, and suggest that the developing notum consists of checker- square-like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar homeobox gene activity. Bar homeobox gene expression is restricted dorsally and posteriorly by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar homeobox genes is determined by wingless whose expression is under the control of Decapentaplegic signaling.

The Drosophila notum is a two-dimensional sheet of patterned sensory bristles, macrochaetae and microchaetae. Macrochaetae are distributed in a limited number of loci while many microchaetae are formed in evenly spaced rows in wide areas of the notum. These bristles are derivatives of sensory organ precursors (SOPs), singled out from epidermal cells that express the proneural genes, achaete and scute (collectively called ac-sc; Ghysen et al., 1993). Thus, understanding how ac-sc expression is spatially and temporally regulated is a fundamental question in bristle patterning in the notum. ac-sc encodes basic helix-loop-helix (bHLH) proteins, and is regulated by many enhancer elements that may serve as targets for site-specific combinations of products of prepattern genes (Gomez-Skarmeta et al., 1995).

Several transcription factors have been shown to be involved in the regulation of ac-sc expression in the notum. iroquois (iro) homeobox genes are expressed longitudinally in the lateral notum and positively regulate ac-sc (Gomez-Skarmeta et al., 1996; Leyns et al., 1996; Kehl et al., 1998). pannier (pnr) encodes a GATA1-related transcriptional factor and is expressed longitudinally in the medial notum (Ramain et al., 1993; Calleja et al., 1996). Positive regulation of ac-sc by pnr is blocked by binding of U-shaped, a zinc finger protein (Cubadda et al., 1997; Haenlin et al., 1997). It has been proposed that the developing notum is divided into several longitudinal subdomains by the expression of prepattern genes essential for proneural gene expression (Calleja et al., 1996). hairy (h) and extramacrochaetae (emc) encode HLH proteins that negatively regulate ac-sc. H directly represses ac-sc transcription (Ohsako et al., 1994; Van Doren et al., 1994) while Emc does so indirectly. Emc prevents Daughterless (Da) from forming a heterodimer with Ac or Sc. Ac-Da and Sc-Da may be essential for the auto- and cross-activation of ac-sc (Skeath and Carroll, 1991; Van Doren et al., 1991; Cubas and Modolell, 1992; Van Doren et al., 1992). The concerted actions of these negative factors and prepattern gene products may be essential for temporal and spatial regulation of ac-sc expression and bristle formation.

Morphogen gradients are essential for cell fate determination in many developmental contexts (Lecuit et al., 1996; Nellen et al., 1996; Zecca et al., 1996; Lecuit and Cohen, 1997; Neumann and Cohen, 1997). In developing notum, Wingless (Wg), a secreted protein of Wnt family (Cadigan and Nusse, 1997), is expressed in a narrow longitudinal stripe and is necessary for bristle formation in its expression domain. wg is required for the formation of presutural (PS) macrochaetae, situated several cell diameters from the wg domain (Phillips and Whittle, 1993). Decapentaplegic (Dpp), a BMP family member (Hogan, 1996), is expressed along the anterior- posterior compartment border (Basler and Struhl, 1994; Kojima et al., 1994; Tabata and Kornberg, 1994). The formation of dorso-central (DC) macrochaetae has recently been shown to require the concerted action of Dpp and Wg (Tomoyasu et al., 1998).

The present work shows BarH1 and BarH2, homeobox genes at the Bar locus (Kojima et al., 1991; Higashijima et al., 1992a), to belong to a new class of putative prepattern genes expressed latitudinally and suggests that the developing notum consists of checker-square-like subdomains, each governed by a different combination of longitudinal and latitudinal prepattern genes. Bar homeobox genes are expressed in the anterior-most region (prescutum) and positively regulates ac- sc expression. The area of prescutum Bar expression is delimited by Dpp and Wg signaling. In addition, our results indicates that the gradient of Dpp signaling activity determines the domain of wg expression.

Fly strains

Flies were raised at 25°C except for temperature sensitive mutants, which were raised at 16.5°C and 29°C (or 25°C), as the permissive and non-permissive temperatures respectively. BarP058 is an enhancer trap line at the Bar locus and was obtained from FlyView. Deletion mutants at the Bar locus, Df(1)B263-20 and Df(1)BH2, and fSH (a strong hypomorphic allele of f) have been described (Higashijima et al., 1992b). As Df(1)BH2, its transgenic derivative possessing a Fimbrin (Fim) gene fragment was used. This derivative shows improved viability and fertility without changing bristle phenotypes and Bar expression (unpublished data). B25B is a BarH2 deletion mutant obtained upon relocation of P058 (see Fig. 3). Fly lines with hs-BarH1 or hs-BarH2 have been described (Kojima et al., 1991, 1993). neurA101, an enhancer trap line at the neuralized locus, was used to mark SOPs and their progeny (Huang et al., 1991). iro1 (a viable hypomorphic allele of iro) and irorF209 (an enhancer trap line at the iro locus) have been described (Gomez-Skarmeta et al., 1996). Allelic combinations of pnrVX1, pnrV1, and pnrD1 were used as pnr mutants (Ramain et al., 1993). wgSp is a regulatory mutant of wg (Neumann and Cohen, 1996), while wgIL114 (wgts) is a temperature sensitive allele of wg, having defects in Wg secretion (Gonzalez et al., 1991). Sources for Hw49c, wg17en40,dppd12, dppd14, dppP10638 and hh9K (hhts) are described in FlyBase. Sources for Gal4 trap lines (pnrmd237 and ap-Gal4) and flies with UAS-dpp, UAS-CD2, and UAS-NZ (UAS-lacZ) are also described in FlyBase. Flies with hs-FLPase, tkva12FRT40A (Nellen et al., 1996), armH8.6FRT101 (Neumann and Cohen, 1997), 2πM FRT40A, NM FRT101, y FRT18A (Xu and Rubin, 1993), arm- lacZ FRT40A (Lecuit and Cohen, 1997), and Df(1)B263-20,y FRT18A were used for FLP/FRT-mediated mosaic analysis. Flies with UAS>CD2, y+>tkvQ253D, UAS>CD2, y+>flu-Δarm, C765-Gal4 (Nellen et al., 1996; Zecca et al., 1996), and arm-Gal4 (FlyBase) were used for generating transgene-expressing clones.

Plasmid construction and germline transformation

pUAS-BarH1 and pUAS-BarH2 were made by inserting a 2.1 kb BarH1 cDNA fragment of pBH1B (Kojima et al., 1991) and a 2.2 kb BarH2 cDNA fragment of p1-12 (Higashijima et al., 1992a) into the NotI site of pUASV, respectively. pUASV is a derivative of pYC1.8 (Fridell and Searles, 1991) and includes the UAS sequence (Brand and Perrimon, 1993) along with a single NotI site (unpublished data). pBgaV is a Gal4 /vermilion transformation vector, in which Gal4 expression is regulated by B4.5 (see Fig. 3; unpublished data). pBN- Gal4 was constructed by inserting S8 (see Fig. 3) into the NotI site of pBgaV so that S8 was situated just upstream of B4.5. In pB4.5-lacZ, lacZ is under the control of B4.5, while, in pS8-BH2, BarH2 associated with its core promoter is under the control of S8 (unpublished data). UAS>CD2, y+>BarH1 was constructed by T. Chimura and K. S. (unpublished data). Germline transformation was performed by standard procedures.

Immunohistochemistry

Immunostaining was carried out as described by Hayashi et al. (1998). Mouse anti-Ac (1:1 dilution; Skeath and Carroll, 1991), mouse anti- Wg (1:100; Neumann and Cohen, 1997), and rabbit anti-Sc (1:1000; Vaessin et al., 1994) antibodies were obtained from S. B. Carroll, S. M. Cohen and Y. N. Jan, respectively. Rabbit anti-LacZ (anti-β-gal; cappel), mouse anti-LacZ (promega), rabbit anti-BarH1 and anti- BarH2 (Higashijima et al., 1992a), respectively, were used in 1:2000, 1:1000, 1:100 and 1:20 dilution in the case of DAB staining. Five times higher concentrations of antibodies were used for fluorescence staining. Mouse anti-HA antibody (BAbCO), which recognizes the Flu-epitope, mouse anti-CD2 antibody (OX-34; Cedarlane Laboratories Ltd.), and mouse anti-Myc antibody (Ab-1; Oncogene Science) were used in 1:100 dilution. Target-gene-specific expression of LacZ was examined using BarP058 (Bar-lacZ), neurA101 (neur- lacZ), irorF209 (iro-lacZ), wg17en40 (wg-lacZ) or dppP10638 (dpp-lacZ) backgrounds.

Clonal analysis

FLP/FRT-mediated mosaic analysis was carried out as described previously (Nellen et al., 1996; Neumann and Cohen, 1997). Late second to early third or first to early second instar larvae were heat shocked and dissected after 44-48 or 68-84 hours, respectively. To obtain larger clones of armH8.6, heat-shocked larvae (first to early second instar) were raised at 16.5°C for 3 days and then shifted to 25°C for 44-48 hours before fixation. hs-FLPase was induced by a heat shock at 37°C for 60-90 minutes to generate mutant clones, or at 34°C for 30-60 minutes to generate clones expressing transgenes.

Restricted expression of Bar homeobox genes along the anterior notal edge

The Drosophila notum is considered genetically divided into several longitudinal domains (Calleja et al., 1996). To further clarify relative locations of iro, wg and pnr expression areas, third-instar larval and pupal future notum were stained with various combinations of molecular markers (Fig. 1A-K) and the results are schematically shown in Fig. 1N-P. In larval and pupal future notum, pnr-Gal4 was expressed medially and iro- lacZ laterally (Fig. 1A,D,E,G). At variance with a previous prediction (Calleja et al., 1996), pnr-Gal4 and iro-lacZ domains were found to partially overlap each other (compare Fig. 1A,H (I) with D,K), and wg-lacZ (or Wg) expression was noted in the pnr-iro overlapping region and its immediate neighbors (Fig. 1C,E,F).

Fig. 1.

Expression of putative prepattern genes in the developing notum at 8 hours APF (after puparium formation; A-D,M) and in late third-instar (E-L). Dorsal is left and anterior is up. Vertical bars, midlines. Macrochaetae cells were marked with BarH1 (A,B), neur-LacZ (D,J,M) or Ac (H,I,K,L). Arrowheads, dorso-central macrochaetae, aDC or pDC. Arrows in A and B, indicate postnotum BarH1 expression. (A) BarH1 (black); UAS-LacZ driven by pnr-Gal4 (brown). Note that BarH1 is expressed in the anterior-most notum and postnotum. (B) BarH1 (black); wg-LacZ (brown). (C) wg-lacZ/+; pnr- Gal4 /UAS-CD2 flies were stained for CD2 (black) and LacZ (brown). The bracket shows that wg-LacZ expression partially overlaps pnr-Gal4 expression. (D) neur-LacZ and iro-LacZ staining which were carried out simultaneously. neur-LacZ signals are much stronger than iro-LacZ signals. (E) Wg (red); UAS-LacZ driven by pnr-Gal4 (green). (F) Wg (red); iro-LacZ (green). (G) BarH1(red); iro-LacZ (green). Note that iro-LacZ expression overlaps BarH1 expression in the anterolateral region. (H,I) Ac (red); UAS- LacZ driven by pnr-Gal4 (green). The arrow in H indicates the PS macrochaetae cell position. Note the absence of clear Ac signals in this preparation. (J) neur-LacZ (red); Wg (green). (K) Ac (red); iro-LacZ (green). The DC macrochaetae proneural region is enclosed by a white line with an arrowhead. The discs in I,K are slightly younger than those in other panels. (K′) An enlargement of a part of K. Note that the DC proneural region is positive for both iro-LacZ (K,K′) and pnr-Gal4 (I), indicating that pnr-Gal4 and iro-LacZ domains partially overlap each other not only in pupae (see A-D) but also in larvae. (L,M) PS macrochaetae are situated near the posteroventral corner of the Bar prescutum at both larval (L) and pupal (M) stages. (L) BarH1 (red); Ac (green). (M) Simultaneous staining for neur-LacZ and BarH1. Isolated strong signals labeled PS, pNP and aNP correspond to neur-LacZ signals. (N-P) Schematic drawings of putative prepattern gene expression in the developing notum at late third instar larval (N) and early pupal (O) stages, and the extrapolated adult pattern (P). Bar is expressed latitudinally in the anterior edge, while pnr, wg and iro expression occur longitudinally. Small open circles show the position of 11 macrochaetae. IS, intrascutal suture. Scale bar (K) 50 μm for (A,B,D-K), 40 μm (C), 20 μm (L), and 30 μm (M).

Fig. 1.

Expression of putative prepattern genes in the developing notum at 8 hours APF (after puparium formation; A-D,M) and in late third-instar (E-L). Dorsal is left and anterior is up. Vertical bars, midlines. Macrochaetae cells were marked with BarH1 (A,B), neur-LacZ (D,J,M) or Ac (H,I,K,L). Arrowheads, dorso-central macrochaetae, aDC or pDC. Arrows in A and B, indicate postnotum BarH1 expression. (A) BarH1 (black); UAS-LacZ driven by pnr-Gal4 (brown). Note that BarH1 is expressed in the anterior-most notum and postnotum. (B) BarH1 (black); wg-LacZ (brown). (C) wg-lacZ/+; pnr- Gal4 /UAS-CD2 flies were stained for CD2 (black) and LacZ (brown). The bracket shows that wg-LacZ expression partially overlaps pnr-Gal4 expression. (D) neur-LacZ and iro-LacZ staining which were carried out simultaneously. neur-LacZ signals are much stronger than iro-LacZ signals. (E) Wg (red); UAS-LacZ driven by pnr-Gal4 (green). (F) Wg (red); iro-LacZ (green). (G) BarH1(red); iro-LacZ (green). Note that iro-LacZ expression overlaps BarH1 expression in the anterolateral region. (H,I) Ac (red); UAS- LacZ driven by pnr-Gal4 (green). The arrow in H indicates the PS macrochaetae cell position. Note the absence of clear Ac signals in this preparation. (J) neur-LacZ (red); Wg (green). (K) Ac (red); iro-LacZ (green). The DC macrochaetae proneural region is enclosed by a white line with an arrowhead. The discs in I,K are slightly younger than those in other panels. (K′) An enlargement of a part of K. Note that the DC proneural region is positive for both iro-LacZ (K,K′) and pnr-Gal4 (I), indicating that pnr-Gal4 and iro-LacZ domains partially overlap each other not only in pupae (see A-D) but also in larvae. (L,M) PS macrochaetae are situated near the posteroventral corner of the Bar prescutum at both larval (L) and pupal (M) stages. (L) BarH1 (red); Ac (green). (M) Simultaneous staining for neur-LacZ and BarH1. Isolated strong signals labeled PS, pNP and aNP correspond to neur-LacZ signals. (N-P) Schematic drawings of putative prepattern gene expression in the developing notum at late third instar larval (N) and early pupal (O) stages, and the extrapolated adult pattern (P). Bar is expressed latitudinally in the anterior edge, while pnr, wg and iro expression occur longitudinally. Small open circles show the position of 11 macrochaetae. IS, intrascutal suture. Scale bar (K) 50 μm for (A,B,D-K), 40 μm (C), 20 μm (L), and 30 μm (M).

Bar homeobox genes may belong to a class of notal subdivision genes different from iro, wg and pnr. Staining for BarH1 indicated that BarH1 is expressed latitudinally in the anterior-most region of future notum and postnotum (Figs 1A,B,G, 2A,B). BarH1 expression began at early to mid third instar. Anti-Ac antibody staining and neur-lacZ expression indicated PS macrochaetae to be situated in the vicinity of posterior-ventral corners of the anterior BarH1 expression domain (Fig. 1L,M). BarP058 is an enhancer trap line at the Bar locus. Double-staining BarP058 larval discs for LacZ and BarH1 or BarH2 showed that Bar-lacZ, BarH1 and BarH2 are coexpressed (Fig. 2E,F). In the following, BarH1 and BarH2 are referred to as Bar collectively and the anterior portion of the prescutum or its precursor expressing Bar as Bar prescutum. Bar expression similar to that in wing discs was observed in haltere discs (data not shown).

Fig. 2.

Regulation of notal Bar by two enhancers. Dorsal (medial) is left, and anterior is up. (A,C,E-K) Late third-instar larval wing discs. (B,D) A part of the notum at 8 hours APF is shown. Vertical lines, midlines. (A-D) BarH1 expression in wild type (A,B) and in Df(1)BH2 (C,D). Arrowheads in A and B indicate BarH1 expression in the prescutum, while those in C and D indicate the absence of BarH1 expression in the medial prescutum. Arrow, BarH1 expression in the future postnotum (a part of the peripordial membrane). (E) BarH1 (red); Bar-LacZ (green). That only yellow cells can be seen indicates the coexpression of BarH1 and Bar-LacZ in the anterior-most notal region. (F) BarH2 (red); Bar-LacZ (green). E and F together indicate that BarH1 and BarH2 are coexpressed in the notum. (G) BarH2 (red) driven by S8 in a Df(1)BH2 background. Wg (green). As shown by the arrowhead, BarH2 expression is restricted to the dorsal Bar prescutum (arrowhead), suggesting that S8 is a medial prescutum Bar enhancer. (H) A wild type disc. B4.5- LacZ(red); Wg (green). (I) A Df(1)BH2 disc. BarH1(red); wg-LacZ (green). (J) Coexpression of BarH1 (red) and B4.5-LacZ (green) in Df(1)BH2. H-J indicate that B4.5 is a lateral prescutum Bar enhancer. However, it should be noted that B4.5-LacZ expression in Df(1)BH2 is restricted more ventrally than that in a wild-type background (compare H with J). (K) X-gal staining of BN- Gal4/UAS-lacZ nota. LacZ expression mimics wild-type BarH1 expression (see A). Scale bar in D, 50 μm (A,C,E-K), 30 μm (B,D).

Fig. 2.

Regulation of notal Bar by two enhancers. Dorsal (medial) is left, and anterior is up. (A,C,E-K) Late third-instar larval wing discs. (B,D) A part of the notum at 8 hours APF is shown. Vertical lines, midlines. (A-D) BarH1 expression in wild type (A,B) and in Df(1)BH2 (C,D). Arrowheads in A and B indicate BarH1 expression in the prescutum, while those in C and D indicate the absence of BarH1 expression in the medial prescutum. Arrow, BarH1 expression in the future postnotum (a part of the peripordial membrane). (E) BarH1 (red); Bar-LacZ (green). That only yellow cells can be seen indicates the coexpression of BarH1 and Bar-LacZ in the anterior-most notal region. (F) BarH2 (red); Bar-LacZ (green). E and F together indicate that BarH1 and BarH2 are coexpressed in the notum. (G) BarH2 (red) driven by S8 in a Df(1)BH2 background. Wg (green). As shown by the arrowhead, BarH2 expression is restricted to the dorsal Bar prescutum (arrowhead), suggesting that S8 is a medial prescutum Bar enhancer. (H) A wild type disc. B4.5- LacZ(red); Wg (green). (I) A Df(1)BH2 disc. BarH1(red); wg-LacZ (green). (J) Coexpression of BarH1 (red) and B4.5-LacZ (green) in Df(1)BH2. H-J indicate that B4.5 is a lateral prescutum Bar enhancer. However, it should be noted that B4.5-LacZ expression in Df(1)BH2 is restricted more ventrally than that in a wild-type background (compare H with J). (K) X-gal staining of BN- Gal4/UAS-lacZ nota. LacZ expression mimics wild-type BarH1 expression (see A). Scale bar in D, 50 μm (A,C,E-K), 30 μm (B,D).

Requirement of Bar for bristle formation in anterior notum

For clarification of possible roles of Bar in notal development, we first carried out FLP/FRT-mediated mosaic analysis (Xu and Rubin, 1993) using the Df(1)B263-20 chromosome which uncovers BarH1 and BarH2 along with forked (f), Fimbrin (Fim), a broken 297 retrotransposon and an unknown gene X2 (Fig. 3; Higashijima et al., 1992b; S. Ishimaru, T. K. and K. S., unpublished data). In this system, mutant cells are marked by f. Few microchaetae were generated in Df(1)B263-20 clones within the prescutum (Fig. 4B). PS macrochaetae formation took place only when PS macrochaetae formation sites were not included within Df(1)B263-20 clones. Df(1)BH2 is a deletion at the Bar locus, uncovering f, Fim and BarH2 (Fig. 3; Higashijima et al., 1992b). In flies hemizygous or homozygous for Df(1)BH2, the expression of BarH1 in the medial Bar prescutum was totally absent (compare Fig. 2C,D with Fig. 2A,B), implying that Df(1)BH2 uncovers a Bar enhancer specific to the medial Bar prescutum. In Df(1)BH2 flies, microchaetae were lost medially in the anterior three quarters of the prescutum (Fig. 4C). PS macrochaetae were lost60% of the time. Loss of BarH1 expression and the microchaetae-less phenotype in the medial Bar prescutum were also detected in Bar25B (Fig. 4D and data not shown), uncovering only BarH2 and its 3′ Bar enhancer sequences (Fig. 3; see below). In Bar25B, there was no PS macrochaetae formation (Fig. 4D). Taken together, these results suggest that Bar homeobox genes are essential for bristle formation in the Bar prescutum. Should Bar homeobox genes be redundant in function and involved in bristle formation in the Bar prescutum, bristle defects would certainly be rescued by their targeted expression. These considerations were confirmed through the use of the GAL4/UAS system (Brand and Perrimon, 1993) along with BsY in a Df(1)BH2 background. UAS-BarH1 or UAS-BarH2 was driven by BN-Gal4, a Gal4 driver capable of mimicking wild-type Bar expression (see below). Loss of microchaetae but not PS macrochaetae was virtually restored by the targeted expression of Bar homeobox genes (Fig. 4E,F). PS macrochaetae loss in Df(1)BH2 was rescued 100% of the time (n=30) by BsY containing BarH1 but lacking the S8 Bar enhancer (see below) without rescuing microchaetae defects (Figs 3 and 4I). BarH1 and BarH2 would thus appear functionally redundant to each other and essential for microchaetae formation in the medial Bar prescutum and PS macrochaetae formation.

Fig. 3.

Physical map of the Bar locus. Sizes and locations of BarH1, BarH2, f, Fim, and X2 transcription units are indicated by filled boxes or bars, while 297 is indicated by three vertical bars. Stippled boxes labeled S8 and B4.5, respectively, show Bar enhancer specific for medial and lateral Bar prescutum expression. Sizes and locations of Bar deletions are shown above the scale, while the region covered by the BSY chromosome is given under the scale. Open boxes, ambiguities. Triangle, P058 insertion site.

Fig. 3.

Physical map of the Bar locus. Sizes and locations of BarH1, BarH2, f, Fim, and X2 transcription units are indicated by filled boxes or bars, while 297 is indicated by three vertical bars. Stippled boxes labeled S8 and B4.5, respectively, show Bar enhancer specific for medial and lateral Bar prescutum expression. Sizes and locations of Bar deletions are shown above the scale, while the region covered by the BSY chromosome is given under the scale. Open boxes, ambiguities. Triangle, P058 insertion site.

Fig. 4.

Effects of Bar activity on notal bristle formation. Anterior is up. Black arrowheads show the presence or absence of microchaetae in the medial prescutum. Black horizontal lines drawn along IS indicate possible boundary between the prescutum and scutum. Thick filled and open arrows, respectively, show the presence and absence of PS macrochaetae. (A) Control (fSH mutants). Except for the f phenotype, microchaetae are normally formed in the prescutum. (B) The absence of microchaetae from Df(1)B263-20 mosaic clones in the prescutum. Note that there are only a few microchaetae in the mutant clone surrounded by a white line. Mutant clones were generated in first to early second instar larvae. (C,D) The absence of microchaetae from the medial prescutum of Df(1)BH2 (C) and B25B (D). The microchaetae-less phenotype of Df(1)BH2 was rescued by BN-Gal4-driven UAS- BarH1 (E) or UAS-BarH2 (F). (G,H) Ectopic microchaetae formation in the scutellum due to heat-induction of hs- BarH1. The scutellum is enclosed by a white circle. (G) No heat induction (control). (H) A 20 minute, 36°C heat induction at 6 hours APF. Thin arrows show ectopic microchaetae, most of which are out of focus. (I) Df(1)BH2 defects in PS macrochaetae formation but not microchaetae formation were eliminated by BsY. (J) Suppression of the bristle- less phenotype of Df(1)BH2 by Hw49c. (K) The absence of medial (black arrowhead) and lateral (white arrowhead) microchaetae from Df(1)BH2; wgts/wgSp flies raised at 25°C. Thin vertical arrows indicate the absence of microchaetae from the wg domain. This phenotype is not due to Bar activity. (L) Loss of scutum macrochaetae and notal size reduction by ubiquitous BarH1 expression. BarH1 was misexpressed throughout the notum by driving UAS-BarH1 by ap-GAL4. All macrochaetae characteristic of the scutum (aSA, pSA, aPA, pPA, aDC and pDC) were lost. In addition, scutellum and prescutum macrochaetae were variably lost; aSC (90%, n=28), pSC (20%, n=28), PS (46%, n=28), pNP (96%, n=28) and aNP (0%, n=28). As with hs-BarH1 induction, ectopic microchaetae formation occurred in the scutellum. Scale bar in (L) 100 μm.

Fig. 4.

Effects of Bar activity on notal bristle formation. Anterior is up. Black arrowheads show the presence or absence of microchaetae in the medial prescutum. Black horizontal lines drawn along IS indicate possible boundary between the prescutum and scutum. Thick filled and open arrows, respectively, show the presence and absence of PS macrochaetae. (A) Control (fSH mutants). Except for the f phenotype, microchaetae are normally formed in the prescutum. (B) The absence of microchaetae from Df(1)B263-20 mosaic clones in the prescutum. Note that there are only a few microchaetae in the mutant clone surrounded by a white line. Mutant clones were generated in first to early second instar larvae. (C,D) The absence of microchaetae from the medial prescutum of Df(1)BH2 (C) and B25B (D). The microchaetae-less phenotype of Df(1)BH2 was rescued by BN-Gal4-driven UAS- BarH1 (E) or UAS-BarH2 (F). (G,H) Ectopic microchaetae formation in the scutellum due to heat-induction of hs- BarH1. The scutellum is enclosed by a white circle. (G) No heat induction (control). (H) A 20 minute, 36°C heat induction at 6 hours APF. Thin arrows show ectopic microchaetae, most of which are out of focus. (I) Df(1)BH2 defects in PS macrochaetae formation but not microchaetae formation were eliminated by BsY. (J) Suppression of the bristle- less phenotype of Df(1)BH2 by Hw49c. (K) The absence of medial (black arrowhead) and lateral (white arrowhead) microchaetae from Df(1)BH2; wgts/wgSp flies raised at 25°C. Thin vertical arrows indicate the absence of microchaetae from the wg domain. This phenotype is not due to Bar activity. (L) Loss of scutum macrochaetae and notal size reduction by ubiquitous BarH1 expression. BarH1 was misexpressed throughout the notum by driving UAS-BarH1 by ap-GAL4. All macrochaetae characteristic of the scutum (aSA, pSA, aPA, pPA, aDC and pDC) were lost. In addition, scutellum and prescutum macrochaetae were variably lost; aSC (90%, n=28), pSC (20%, n=28), PS (46%, n=28), pNP (96%, n=28) and aNP (0%, n=28). As with hs-BarH1 induction, ectopic microchaetae formation occurred in the scutellum. Scale bar in (L) 100 μm.

Identification of medial and lateral Bar enhancers

While searching for Bar enhancers, two notum enhancers were identified (T. M., unpublished data). S8, absent from Df(1)BH2 and B25B (Fig. 3), was found responsible for Bar expression in the medial Bar prescutum (Fig. 2G). B4.5 was capable of driving reporter gene (lacZ) expression in the lateral Bar prescutum in wild-type and Df(1)BH2 backgrounds (Fig. 2H-J), although lacZ expression in the latter was restricted more ventrally than in the former. The Bar enhancer included in BN-Gal4 is a composite of S8 and B4.5 so that expression of UAS-lacZ driven by BN-Gal4 is capable of mimicking the endogeneous Bar expression (compare Fig. 2K and A).

Extra microchaetae formation by Bar misexpression at early pupal stages

To assess the capability of Bar for inducing microchaetae formation, hs-BarH1 or hs-BarH2 transgenes (Kojima et al., 1991, 1993) were heat-induced during larval or pupal development and the same results were obtained for each. Ectopic microchaetae were generated in the scutellum, wing blades and head capsule (Fig. 4H; data not shown). In the scutellum, normally possessing no microchaetae (Fig. 4G), 30-50 ectopic microchaetae were generated by a 36°C 10 minute heat-shock at 6 hours APF (after puparium formation), but few ectopic microchaetae were formed by heat shock before 2 hours APF or after 12 hours APF. In contrast to ectopic microchaetae formation in neurogenic mutations (Ghysen et al., 1993), extra microchaetae induced by hs-Bar were unclustered (Fig. 4H), suggesting that Bar acts as an activator of proneural genes.

Bar as an ac-sc activator

Staining for Ac showed that pupal notum-specific Ac expression begins in characteristic regions at 6 hours APF, peaking at 8 hours APF and eventually disappearing at 12 hours APF (Fig. 5D). These Ac-positive regions appeared to correspond to microchaetae proneural regions since microchaetae SOP formation starts at 8 hours APF (Usui and Kimura, 1993). Macrochaetae SOP are formed during third instar (Huang et al., 1991). In the Bar prescutum, the area of Ac expression was seen to overlap that of Bar (Fig. 5A). Sc expression was quite similar, if not identical, to Ac expression (data not shown).

Fig. 5.

Ac activation by Bar in the prescutum. Vertical lines, midline. (A) Ac expression overlaps Bar in the Bar prescutum. The notum at 8 hours APF was stained for Bar-LacZ (brown) and Ac (black). (B,C) Ac signals (nuclear signals black or brown) in Df(1)BH2 (C) were significantly reduced in the medial Bar prescutum in comparison to wild type (B). B4.5-LacZ (non-nuclear brown signals) was used to mark the lateral prescutum (see Fig. 2H,I). Horizontal line, the posterior limit of the Bar prescutum inferred from B4.5- LacZ expression. (D) Ac expression of a wild type notum. Pupal nota were prepared at 8 hours APF, and stained for Ac (brown) and neur- LacZ (black; a marker for macrochaetae cells). (E) A hs-BarH1 notum heat-shocked at 6 hours APF, and stained for Ac (brown) and neur-LacZ (black) at 8 hours APF. Half circles indicate scutella. Ac is strongly misexpressed in the scutellum. Scale bar in E 35 μm (A- C), 50 μm (D,E).

Fig. 5.

Ac activation by Bar in the prescutum. Vertical lines, midline. (A) Ac expression overlaps Bar in the Bar prescutum. The notum at 8 hours APF was stained for Bar-LacZ (brown) and Ac (black). (B,C) Ac signals (nuclear signals black or brown) in Df(1)BH2 (C) were significantly reduced in the medial Bar prescutum in comparison to wild type (B). B4.5-LacZ (non-nuclear brown signals) was used to mark the lateral prescutum (see Fig. 2H,I). Horizontal line, the posterior limit of the Bar prescutum inferred from B4.5- LacZ expression. (D) Ac expression of a wild type notum. Pupal nota were prepared at 8 hours APF, and stained for Ac (brown) and neur- LacZ (black; a marker for macrochaetae cells). (E) A hs-BarH1 notum heat-shocked at 6 hours APF, and stained for Ac (brown) and neur-LacZ (black) at 8 hours APF. Half circles indicate scutella. Ac is strongly misexpressed in the scutellum. Scale bar in E 35 μm (A- C), 50 μm (D,E).

Study was thus made to find whether Bar is capable of acting as an activator of ac-sc to control microchaetae formation. Ac expression was almost entirely absent from the Df(1)BH2 medial Bar prescutum (compare Fig. 5B and C), where no microchaetae formation took place (Fig. 4C). Following induction of hs-Bar, not only ectopic microchaetae formation (Fig. 4H) but strong Ac (and Sc) expression was apparent in the scutellum (compare Fig. 5D and E). hs-Bar dependent microchaetae formation was significantly suppressed in ac-sc hypomorphic mutant backgrounds (data not shown) while bristle defects including PS macrochaetae loss in Df(1)BH2 were eliminated by Hw49c, a gain-of-function allele of ac-sc (Fig. 4J). Bar may thus be considered to be an activator of ac- sc essential for producing proneural clusters for microchaetae and possibly PS macrochaetae as well.

Owing to weak transient Ac expression in the putative proneural region for PS macrochaetae, loss of the PS proneural region of Ac expression in a Bar mutant background could not be clearly demonstrated by antibody staining.

Regulation of Bar expression by Dpp and Wg signaling

During late third instar, the expression domain of Bar in the prescutum is immediately adjacent to dpp and wg expression domains (Figs 6A, 7A). dpp likely regulates Bar expression negatively. When dpp was expressed throughout the notum using UAS-dpp driven by ap-Gal4, Bar expression was totally abolished (Fig. 6E). Conversely, reduction in dpp activity ectopically induced Bar expression in the medial region of future notum (Fig. 6B). Similar medial expansion of Bar expression was observed subsequent to reduction in the activity of hedgehog (hh), an inducer of dpp (Fig. 6C; Basler and Struhl, 1994; Kojima et al., 1994; Tabata and Kornberg, 1994). To more fully confirm the negative regulation of Bar expression by Dpp signaling, FLP/FRT-mediated mosaic analysis (Xu and Rubin, 1993; Nellen et al., 1996) was carried out. Clones in which Dpp signaling is constitutively active were generated using tkvQ253D, which encodes a constitutively active form of a type I receptor of Dpp (Nellen et al., 1996). Bar expression was completely abolished irrespective of clone position (Fig. 6F,G). Cells in the Bar prescutum would thus appear to receive Dpp signals at the intensity less than the threshold level for Bar repression. Clones homozygous for tkva12 (a strong hypomorphic allele of tkv) generated on the dorsal (medial) side of the notum were always associated with ectopic Bar expression (Fig. 6H,I). However, it should be noted that, in a large dorsal clone, Bar misexpression was restricted to its dorsal side (Fig. 6I). In clones generated near the posterior-dorsal edge of the endogenous Bar expression domain, Bar misexpression was restricted to a narrow region immediately adjacent to the endogeneous Bar domain (Fig. 6H). Bar misexpression was observed much less frequently in tkva12 clones generated in or near the wg domain (Fig. 6K,L). In contrast, no appreciable change in Bar expression was detected in clones generated in the lateral notum (Fig. 6J).

Fig. 6.

Regulation of notal Bar and wg expression by Dpp signaling. The future nota of late third instar wing imaginal discs were examined. Anterior is up and dorsal is left. (A) Wild-type disc. BarH1 (red); dpp-LacZ (green). (B-E) BarH1 (red); Wg (green). (B) In a dpp strong disc mutant (dppd12/dppd14) background, the anterior Bar domain was expanded and shifted to the dorsal-most region of the notum (see arrowhead). Wg expression, which normally occurs in the central notum, was scarcely detectable in the vicinity of the dorsal Bar misexpression region (thick arrow). The thin arrow indicates the absence of Bar expression in the lateral prescutum. (C) In hhts discs treated at 29°C for 48 hours before dissection, Bar and wg expression domains, respectively, shifted to the dorsal edge of the notum (arrowhead) and dorsally (thick arrow). (D) Prolonged hh inactivation (29°C for 54 hours) resulted in further dorsal shift of Bar and wg expression domains; Wg signals were extensively reduced. (E) Both BarH1 (red) and wg (green) were repressed in the future notum by UAS-dpp driven by ap-Gal4. (F,G) BarH1 expression was abolished in all tkvQ253D clones (arrowheads) generated in the Bar prescutum in late second to early third instar larvae. BarH1 (red); CD2 (green). The absence of CD2 shows the location of tkvQ253D clones. tkvQ253D was driven by arm- Gal4. (H-J) BarH1 (red); Myc (green). BarH1 misexpression occurred in tkva12 clones generated near the posterior border of the Bar prescutum (H; arrowhead) and in the dorsal side of the notum (I; arrowhead). No BarH1 misexpression was detected in lateral notum clones (J; arrow). Mutant clones were generated in late second to early third instar (H), or in first to early second instar larvae (I,J). The absence of Myc shows the location of tkva12 clones. Note that BarH1-positive clones in H and I, respectively, contain BarH1-negative cells in the posterior and ventral sides of the clones. (K,L) Less-frequent appearance of BarH1 misexpression in tkva12 clones generated in or near the central wg expression domain (see arrowheads in K,L) in first to second instar larvae. (K) BarH1 (red); Myc (green). (L) BarH1 (red); Wg (green). Note that the tkva12 clone (outlined in L) lacks Wg expression. (M,N) Wg (red); arm-LacZ (green). The absence of arm-LacZ shows the location of tkva12 clones. In tkva12 clones within the wg domain, Wg expression was often abolished (see arrows). In mutant clones dorsal to the wg domain, Wg misexpression occurred (see arrowheads),while no Wg misexpression could be detected in clones lateral to the wg domain. (O,P) wg-LacZ (red); CD2 (green). In tkvQ253D clones anterolateral to the wg domain, wg-LacZ was misexpressed (arrowheads), while wg-LacZ expression was abolished in clones within the wg domain (arrows). No Wg expression occurred in dorsal clones. Clones in M-P were generated in late second to early third instar larvae. Scale bar (P), 50 μm.

Fig. 6.

Regulation of notal Bar and wg expression by Dpp signaling. The future nota of late third instar wing imaginal discs were examined. Anterior is up and dorsal is left. (A) Wild-type disc. BarH1 (red); dpp-LacZ (green). (B-E) BarH1 (red); Wg (green). (B) In a dpp strong disc mutant (dppd12/dppd14) background, the anterior Bar domain was expanded and shifted to the dorsal-most region of the notum (see arrowhead). Wg expression, which normally occurs in the central notum, was scarcely detectable in the vicinity of the dorsal Bar misexpression region (thick arrow). The thin arrow indicates the absence of Bar expression in the lateral prescutum. (C) In hhts discs treated at 29°C for 48 hours before dissection, Bar and wg expression domains, respectively, shifted to the dorsal edge of the notum (arrowhead) and dorsally (thick arrow). (D) Prolonged hh inactivation (29°C for 54 hours) resulted in further dorsal shift of Bar and wg expression domains; Wg signals were extensively reduced. (E) Both BarH1 (red) and wg (green) were repressed in the future notum by UAS-dpp driven by ap-Gal4. (F,G) BarH1 expression was abolished in all tkvQ253D clones (arrowheads) generated in the Bar prescutum in late second to early third instar larvae. BarH1 (red); CD2 (green). The absence of CD2 shows the location of tkvQ253D clones. tkvQ253D was driven by arm- Gal4. (H-J) BarH1 (red); Myc (green). BarH1 misexpression occurred in tkva12 clones generated near the posterior border of the Bar prescutum (H; arrowhead) and in the dorsal side of the notum (I; arrowhead). No BarH1 misexpression was detected in lateral notum clones (J; arrow). Mutant clones were generated in late second to early third instar (H), or in first to early second instar larvae (I,J). The absence of Myc shows the location of tkva12 clones. Note that BarH1-positive clones in H and I, respectively, contain BarH1-negative cells in the posterior and ventral sides of the clones. (K,L) Less-frequent appearance of BarH1 misexpression in tkva12 clones generated in or near the central wg expression domain (see arrowheads in K,L) in first to second instar larvae. (K) BarH1 (red); Myc (green). (L) BarH1 (red); Wg (green). Note that the tkva12 clone (outlined in L) lacks Wg expression. (M,N) Wg (red); arm-LacZ (green). The absence of arm-LacZ shows the location of tkva12 clones. In tkva12 clones within the wg domain, Wg expression was often abolished (see arrows). In mutant clones dorsal to the wg domain, Wg misexpression occurred (see arrowheads),while no Wg misexpression could be detected in clones lateral to the wg domain. (O,P) wg-LacZ (red); CD2 (green). In tkvQ253D clones anterolateral to the wg domain, wg-LacZ was misexpressed (arrowheads), while wg-LacZ expression was abolished in clones within the wg domain (arrows). No Wg expression occurred in dorsal clones. Clones in M-P were generated in late second to early third instar larvae. Scale bar (P), 50 μm.

Possible effects of wg on Bar expression was sought using wgts, in which Wg secretion but not production is temperature- sensitive (Gonzalez et al., 1991). Bar expression in the lateral Bar prescutum was abolished after 48 hours, but not 24 hours, incubation at 29°C (Fig. 7C,D). Consistent with this, not only medial but also lateral microchaetae and PS macrochaetae were absent from Df(1)BH2 flies transheterozygous for wgts and wgSp, raised at the non-permissive temperature (25°C) (Fig. 4K). armadillo (arm), a β-catenin homologue, is a signal transducer of Wg signaling (Cadigan and Nusse, 1997). Bar expression was lost in clones mutant for arm when generated in the lateral Bar prescutum (Fig. 7E), while Bar misexpression was present in lateral prescutum clones expressing a constitutively active form of armarm; Fig. 7F). Bar expression in lateral prescutum is thus concluded to require Wg signals, whose levels determine the ventral border of the Bar prescutum.

Fig. 7.

Interactions between Bar and wg in the notum. Late third instar wing imaginal discs were examined. In all panels, BarH1 expression is shown by red. (A,B) wg-LacZ (green). (A) Wild type. The anterior BarH1 expression domain abuts on the central Wg expression domain. (B) wg-LacZ expression expanded anteriorly in Df(1)BH2 (arrowhead) 50% of the time. (C,D) Wg (green). Bar expression in the lateral prescutum (see arrowheads) disappeared when wgts flies were raised at 29°C for 48 hours (D), but not 24 hours (C), just before dissection. (E) Myc (green). armH8.6 clones are visualized by the absence of Myc. The arrowhead indicates the absence of Bar expression. (F) BarH1 misexpression in Δarm clones. Green, Flu-positive clones expressing the flu-tagged Δarm driven by C765-Gal4. BarH1 was misexpressed in a part of lateral prescutum clones (arrowhead). (G,H) notal Wg expression (green) was repressed by misexpression of BarH1 throughout the notum (arrowheads, G; ap-Gal4/UAS-BarH1), or in clones ectopically expressing BarH1 by UAS- BarH1/ap-Gal4 (H). Clones were generated in first to early second (E,F), or in late second (H) instar larvae. (I) The regulation of Bar expression by dpp and wg is schematically shown. Bar expression is restricted dorsally and posteriorly by dpp, and activated by wg in the lateral prescutum. wg expression may be repressed by Bar in the anterior-most notum. ac-sc is positively regulated by Bar. (J) The regulation of wg expression by Dpp signaling is schematically shown. Notal wg expression requires optimal levels of Dpp signaling activity. Scale bar in H, 50 μm.

Fig. 7.

Interactions between Bar and wg in the notum. Late third instar wing imaginal discs were examined. In all panels, BarH1 expression is shown by red. (A,B) wg-LacZ (green). (A) Wild type. The anterior BarH1 expression domain abuts on the central Wg expression domain. (B) wg-LacZ expression expanded anteriorly in Df(1)BH2 (arrowhead) 50% of the time. (C,D) Wg (green). Bar expression in the lateral prescutum (see arrowheads) disappeared when wgts flies were raised at 29°C for 48 hours (D), but not 24 hours (C), just before dissection. (E) Myc (green). armH8.6 clones are visualized by the absence of Myc. The arrowhead indicates the absence of Bar expression. (F) BarH1 misexpression in Δarm clones. Green, Flu-positive clones expressing the flu-tagged Δarm driven by C765-Gal4. BarH1 was misexpressed in a part of lateral prescutum clones (arrowhead). (G,H) notal Wg expression (green) was repressed by misexpression of BarH1 throughout the notum (arrowheads, G; ap-Gal4/UAS-BarH1), or in clones ectopically expressing BarH1 by UAS- BarH1/ap-Gal4 (H). Clones were generated in first to early second (E,F), or in late second (H) instar larvae. (I) The regulation of Bar expression by dpp and wg is schematically shown. Bar expression is restricted dorsally and posteriorly by dpp, and activated by wg in the lateral prescutum. wg expression may be repressed by Bar in the anterior-most notum. ac-sc is positively regulated by Bar. (J) The regulation of wg expression by Dpp signaling is schematically shown. Notal wg expression requires optimal levels of Dpp signaling activity. Scale bar in H, 50 μm.

During late third instar, the expression domain of Bar in the prescutum overlaps with those of pnr and iro (see Fig. 1E,G). Since no appreciable change in Bar expression was detected in flies mutant for iro or pnr (iro1, pnrVX1/pnrV1, and pnrD1/pnrV1), Bar expression may be regulated independently of pnr and iro.

Negative regulation of wg expression by Bar

In wg17en40 flies mutant for Df(1)BH2, anterior expansion of wg-lacZ expression was found 50% of the time (Fig. 7B). Wg expression was repressed by BarH1 misexpression (Fig. 7G,H). Thus, Bar may at least have partial involvement in the negative regulation of wg.

When Bar was expressed throughout the notum using ap- Gal4 and UAS-Bar, nearly all notal structures including pNP, aSA, pSA, aPA, pPA, aDC, pDC, and aSC macrochaetae were lost (Fig. 4L). Occasional loss of PS and pSC macrochaetae was also noted. Some of these phenotypes may be attributed to wg repression by Bar, since (1) the absence of wg results in loss of PS, aDC, pDC, pPA, aSC, pSC macrochaetae (Phillips and Whittle, 1993), and (2) notal wg expression was lost in ap- Gal4/UAS-Bar flies (Fig. 7G). Restriction of Bar expression to the anterior-most notum seems essential for normal subdivision of the notum.

Negative and positive regulation of notal wg expression by Dpp signaling

Fig. 6B,E indicates notal Wg expression to have been virtually completely abolished in strong disc mutants of dpp (dppd12/dppd14) and UAS-dpp/ap-Gal4 flies expressing dpp throughout the notum. The wg expression domain shifted dorsally with reduction in the activity of Hh, a dpp inducer (Fig. 6C). The Hh activity of hhts flies was removed by a shift from 16.5 to 29°C in mid-late second instar and a subsequent 48-hour incubation at 29°C. Prolonged incubation at 29°C not only enhanced dorsal shift of the wg domain but also caused considerable loss of Wg expression area (Fig. 6D). It thus follows that proper levels of Dpp signals may be essential for notal wg expression.

To determine whether there is an optimal range in Dpp signals for notal wg expression, examination was made of wg expression in loss-of-function (tkva12) and gain-of-function (tkvQ253D) tkv clones. tkvQ253D was driven by arm-Gal4. In all cases, clones were generated during late second to early third instar and possible changes in wg expression were examined using anti- Wg or anti-LacZ antibodies in late third instar. wg misexpression was detected in all tkva12 clones (n=17) dorsal to the authentic domain, while wg expression was absent from 30% of clones (n=24) generated within the authentic wg expression domain (Fig. 6M,N). No wg misexpression was present in ventral tkva12 clones. The effects of tkvQ253D differed from those of tkva12. Two thirds of tkvQ253D clones (n=66) anterolateral to the authentic wg domain were found to be associated with wg misexpression, while wg expression was abolished in 90% of clones (n=29) generated within the authentic wg expression domain (Fig. 6O,P). No wg expression was found in dorsal clones. An optimal range in Dpp signals may thus be concluded for notal wg expression (Fig. 7J) and it follows then that the notal wg domain can shift both dorsally and laterally depending on Dpp signaling intensity. In extreme cases, the wg domain may be totally eliminated from the notal region.

Bar homeobox genes as putative prepattern genes

We have shown that BarH1 and BarH2, a pair of homeobox genes at the Bar locus, serve as putative prepattern genes for the anterior three quarters of the prescutum (Bar prescutum; see Fig. 1N-P). BarH1 and BarH2 are coexpressed in the Bar prescutum and required for transcriptional activation of proneural genes, ac-sc, and hence, bristle formation in the Bar prescutum. BarH1 and BarH2 appear functionally redundant to each other, since targeted expression of either BarH1 or BarH2 rescued microchaetae-less phenotypes of a Bar-deletion mutant (see Fig. 4E,F).

Bar genes may act as prepattern genes in tissues other than the notum. As with future notum, Bar is expressed in regions surrounding the compound eyes and regulates bristle formation (T. K., unpublished data). Bar misexpression brought about ectopic microchaetae formation in wing blades and the head capsule (unpublished data).

In embryonic external sensory organs, Bar is expressed in thecogen (glial cells) and neurons and is essential for sensillum subtype determination (Higashijima et al., 1992) Bar is also expressed in similar cells in nearly all adult sensory organs (T. K., unpublished data). However, in the progeny of SOPs, its expression was noted only in later stages of development and hence unrelated to region-specific Bar expression studied here.

Checker-board-like subdivisions of future notum by putative prepattern gene expression

Future notum may be divided into square subdomains in a checker-board-like manner, each with its own unique combinations of prepattern gene expression (see Fig. 1N-P). Putative prepattern genes, iro and pnr, form longitudinal domains. Here, we have shown Bar homeobox genes to form the anterior-most latitudinal domain. To our knowledge, this is the first demonstration of the presence of latitudinal prepattern genes in the notum. Bristle formation in each subdomain may be positively regulated by a region-specific combination of prepattern genes. Consistent with this, our unpublished data disclose that microchaetae formation in the anterolateral prescutum (the lateral Bar prescutum), where Bar and iro are coexpressed (see Fig. 1G), requires the concerted action of Bar and iro.

Ac-Sc expression patterns differ in some respects with those of putative prepattern genes (compare Fig. 1O and Fig. 5D) and this would suggest the presence of a factor that restricts Ac-Sc expression spatially and/or temporally. Emc may possibly be such a factor, since notal Emc expression patterns at early pupal stages are partially complementary to those of Ac-Sc (unpublished data; see also Cubas and Modolell, 1992; Van Doren et al., 1992).

In various developmental contexts, determination of fate of a group of cells apparently depends on checker-board-like field subdivision along the anteroposterior and dorsoventral axes (Lawrence and Struhl, 1996). For example, the destiny of any given portion of the ectoderm, mesoderm and neuroectoderm in Drosophila may be determined by a combination of various genes expressed under the control of two-dimensional positional information provided by secretory and/or transcriptional factors (Azpiazu et al., 1996; Lawrence and Struhl, 1996; Skeath, 1998). Our study demonstrated that the two-dimensional sheet of Drosophila notum may be subdivided by a combination of longitudinal and latitudinal prepattern genes, suggesting that the checker-board-like subdivision of a developmental field may thus be a general developmental mechanism.

Regulation of Bar expression by Dpp and Wg signaling

Dpp and Wg have been shown to provide two-dimensional positional information in the wing pouch (Lecuit et al., 1996; Nellen et al., 1996; Zecca et al., 1996; Neumann and Cohen, 1997), but their roles in gene regulation in the notum should extend far beyond this. Our results (see Fig. 6A-E, M-P) show that the size and location of the notal wg expression domain vary depending on Dpp signaling activity. As schematically shown in Fig. 7J, strong Dpp signaling activity in the dorsal or medial region may repress wg expression while weak Dpp signals may determine the ventral limit of the authentic wg expression domain.

Clonal analysis (see Fig. 6F-L) indicated Bar expression in the prescutum to be negatively regulated by Dpp signaling (see Fig. 7I). Posterior and dorsal (medial) borders of the Bar prescutum domain is likely to be restricted directly by Dpp signals. Bar expression was always abolished in tkv-active (tkvQ253D) clones no matter where situated in the Bar prescutum (Fig. 6F,G) while Bar misexpression was restricted to tkv-negative (tkva12) clones in the dorsal notum (Fig. 6H-L). Dpp signal activity in the ventral notum may thus perhaps be less than that required for Bar repression.

Lateral Bar expression in the prescutum is positively regulated by wg (see Fig. 7C-F). Ectopic Bar expression in Δarm clones generated in the lateral prescutum suggests that a narrow anterior region situated more ventral to the Bar prescutum is wg-susceptible and thus the ventral border of Bar expression domain in the prescutum may be determined by Wg signals (Fig. 7I). Loss of Bar expression from the lateral prescutum in dpp mutants (see thin arrow in Fig. 6B) may be a secondary effect arising from the loss of Wg expression in dpp mutants, since Bar expression was not abolished in tkv- negative clones in the lateral prescutum (see Fig. 6J).

Fig. 2H suggests that the lateral Bar enhancer for microchaetae formation may be included in fragment B4.5. Thus, a transcription factor situated downstream of Wg signaling might recognize and bind to sites in B4.5 so as to activate Bar and hence ac-sc. PS macrochaetae formation requires not only Bar but also wg (Phillips and Whittle, 1993) and accordingly a similar cascade including wg, Bar and ac-sc may be involved in PS macrochaetae formation. However, that PS macrochaetae defects in Df(1)BH2 are rescued by BS Y but not UAS-Bar driven by BN-Gal4 may be indication that cis- regulatory elements essential for PS macrochaetae formation are included in BS Y but neither in S8 nor B4.5.

We are grateful to K. Basler, A. Bejsovec, S. M. Cohen, J. L. Gomez-Skarmeta, S. Hayashi, J. Modolell, P. Simpson, G. Struhl, T. Tabata, R. Ueda, T. Uemura and the stock centers of Bloomington, and FlyView for fly strains, and to S. B. Carroll, S. M. Cohen and Y. N. Jan for antibodies. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan to K. S.

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