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Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg
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Developmental Biology 312 (2007) 147 – 156 www.elsevier.com/developmentalbiology
Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg Tetsuya Shirai a,1 , Takeshi Yorimitsu a , Naruto Kiritooshi a , Fumio Matsuzaki b , Hideki Nakagoshi a,⁎ a
Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan b Laboratory for Cell Asymmetry, Center for Developmental Biology, RIKEN, Kobe, Japan Received for publication 6 April 2007; revised 21 August 2007; accepted 6 September 2007 Available online 18 September 2007
Abstract Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal–distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints. © 2007 Elsevier Inc. All rights reserved. Keywords: Drosophila; Leg; Segment; Notch; EGFR; Planar cell polarity; dve
Introduction Studies in the developing Drosophila leg have provided important insights into the intercellular signaling pathways regulating positional information, cell fate specification, and tissue growth (reviewed in Galindo and Couso, 2000; Kojima, 2004; Milán and Cohen, 2000). Adult Drosophila legs consist of several segments along the proximal–distal (P–D) axis: from proximal to distal, the coxa, trochanter, femur, tibia, tarsus and pretarsus bearing two claws. Neighboring leg segments are separated by flexible structures called joints, which allow them to move. In leg imaginal discs, decapentaplegic (dpp) and wingless (wg) are expressed in anterior dorsal and anterior ventral sectors, respectively, in response to the secreted protein Hedgehog, which is only expressed in posterior cells (Basler and Struhl, ⁎ Corresponding author. Fax: +81 86 251 7876. E-mail address: [email protected] (H. Nakagoshi). 1 Present address: Institute for Research in Humanities, Kyoto University, Japan. 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.09.003
1994). The combined activities of Dpp and Wg activate another set of P–D patterning genes such as homothorax (hth), dachshund (dac), and Distal-less (Dll), which are expressed in ringshaped territories in different domains along the P–D axis (AbuShaar and Mann, 1998; González-Crespo et al., 1998; Lecuit and Cohen, 1997; Wu and Cohen, 1999). At the most central portion of the leg discs, vein (vn), a neuregulin-like ligand of epidermal growth factor receptor (EGFR), is induced in response to Dpp and Wg. A graded activity of EGFR signaling from a distal tip (central portion of the disc) is required for patterning the distal leg segments (Campbell, 2002; Galindo et al., 2002). Distinct combinations of P–D patterning genes are independently required for each segmental expression of the Notch ligand Serrate (Ser) (Rauskolb, 2001). Notch signaling is activated in several rows of distal cells in each segment, which are distally adjacent to the Notch ligand-expressing domain, while in cells proximally adjacent to them, Notch signaling is not activated (Bishop et al., 1999; de Celis et al., 1998; Rauskolb and Irvine, 1999). The glycosyltransferase Fringe (Fng) modulates the ability of Notch ligands Delta (Dl) and Ser,
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and therefore is involved in the restricted activation of Notch signaling (Bishop et al., 1999; Brückner et al., 2000; Fleming et al., 1997; Irvine and Wieschaus, 1994; Moloney et al., 2000; Panin et al., 1997; Rauskolb and Irvine, 1999). Activation of Notch signaling in the distal region of each segment is required for leg growth and joint formation. Notch mutant mosaic legs result in severe segment truncation due to the loss of joint structures and reduced growth, while ectopic activation of Notch signaling induces extra joint formation and outgrowth of the leg tissue, indicating that Notch signaling is sufficient to establish the joint structure (Bishop et al., 1999; de Celis et al., 1998; Rauskolb and Irvine, 1999; Shellenbarger and Mohler, 1978). In contrast to embryonic segmentation, leg segmentation occurs in a growing tissue, thus the subdivision must be coordinated with tissue growth. Several Notchregulated genes have been identified, such as E(spl)mβ, oddskipped (odd), nubbin (nub), four-jointed (fj), big brain (bib), Drosophila AP-2 (dAP-2), and brother of odd with entrails limited (bowl) (Bishop et al., 1999; de Celis Ibeas and Bray, 2003; de Celis et al., 1998; Hao et al., 2003; Rauskolb and Irvine, 1999); however, the coordinated regulation between leg growth and segmentation remains unknown. The homeobox gene defective proventriculus (dve) is expressed in a pattern of concentric rings in leg discs, and is temporally regulated in the Notch-activated region. Our previous studies have shown that temporally regulated Dve repression is crucial for gut function and for wing patterning (Nakagoshi et al., 1998; Nakagoshi et al., 2002). In wing discs, Dve is expressed in the prospective wing region, and is subsequently repressed along the dorsal–ventral (D–V) compartment boundary through Notch-mediated signaling. In the midgut, the absorptive function of copper cells is established through Notch signaling in adjacent cells and through temporally regulated dve repression in copper cells. In Notch mutants, dve expression is derepressed in copper cells (Tanaka et al., 2007), suggesting that temporally regulated dve repression depends on the Notch activation in two distinct tissues. Here, we provide evidence that temporally regulated Dve repression is crucial for leg joint formation, during which Notch signaling relieves the joint-suppressive activity of Dve.
(Nakagoshi et al., 1998), dAP-2 (1:100) (Monge et al., 2001), and β-Gal (Cappel; 1:100) were used. Cy3- or FITC-conjugated secondary antibodies to mouse immunoglobulin G (Jackson; 1:100) and Cy3- (Jackson; 1:100) or FITCconjugated (Cappel; 1:100) secondary antibodies to rabbit immunoglobulin G were used. Confocal images were obtained with an OLYMPUS FluoView300. Nomarski images were obtained with a Zeiss AxioVision 3.0, and photo images of adult legs (Figs. 1 and 6A–D) were assembled into montages using Photoshop software (Adobe).
Mosaic analyses Mosaic clones were induced with the use of an FRT- and FLP-mediated recombination system (Xu and Rubin, 1993). To generate dve1 mosaic clones, FRT42D-dve1 flies were crossed with y w hs-flp; FRT42D y+M(2) or f hs-flp; FRT42D M(2) f+ ones, and the offspring were subjected to heat shock at 38 °C for 90 min in the late first to early second larval instars. For collecting the staged mutant prepupal leg discs, FRT42D locus was recombined with an ey-FLP5 transgene, which expresses FLPase also in the leg and antennal discs from early stages of development (Tsuji et al., 2000), leading to generate mosaic clones without heat shock. To generate Notch mutant mosaic clones, N55e11 FRT18A/ FM7 was crossed with w ubi-GFP.nls FRT18A; hs-flp38, and heat-shocked at 38 °C for 30 min in the late second to early third larval instars.
Results Extra joint formation in dve mutant legs The homeobox gene defective proventriculus (dve) is expressed in various tissues including the midgut and wing imaginal discs (Carr et al., 2005; Fuss and Hoch, 1998; Nakagoshi, 2005; Nakagoshi et al., 1998, 2002; Shirai et al., 2003). dveBG02382 is a hypomorphic allele, and homozygous escaper adults are rarely observed. In dveBG02382 homozygous wings, P–D patterning is affected due to reduced proliferation
Materials and methods Fly stocks The following mutant strains were used: dve1 (Nakagoshi et al., 1998), dve (Kölzer et al., 2003), pksple-1 (Held et al., 1986), and the Notch temperature-sensitive allele l(1)Nts1 (Shellenbarger and Mohler, 1975). To monitor Notch- and EGFR-activated cells, E(spl)mβ-CD2 and bib-lacZ, and argos-lacZ (argosstyP1) were used, respectively (de Celis et al., 1998; Okano et al., 1992). The following GAL4/UAS strains were used: bibNP4281 (Hayashi et al., 2002), UAS-GFP.nls (Bloomington Stock Center), UAS-dve-9B2 (Nakagoshi et al., 1998), UAS-d.n.N (Go et al., 1998), and UAS-DrafGOF (Brand and Perrimon, 1994). BG02382
Immunohistochemistry Mouse monoclonal antibodies to β-galactosidase (Gal) (Promega; 1:100 dilution) and CD2 (Serotec; 1:100), as well as rabbit antibodies to Dve (1:500)
Fig. 1. Extra joint formation in dve mutant legs. (A) A wild-type (WT) leg has joints between segments (arrows). (B) A dveBG02382 homozygous leg has extra joint-like structures (arrowheads). (C) Extra joints are clearly observed in a dve1 mosaic leg (f hs-flp/Y; FRT42D dve1/FRT42D M(2) f +), and they have reverse polarity (arrowheads). Distal is to the bottom, and proximal is to the top. The orientation is the same in following figures except Figs. 2A and 7.
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rate in wing imaginal discs, and wing vein formation is impaired in longitudinal veins 2 (L2) and 5 (L5) (Kölzer et al., 2003). Interestingly, we found an additional phenotype in dveBG02382 homozygous legs. In wild type, leg segments are separated by a flexible structure called joint which consists of a ball-and-socket structure and invaginates into the proximal side (arrows in Fig. 1A); however, dveBG02382 homozygous legs have extra jointlike structures at the center of each tarsal segment 2–5 (ta2–ta5) (arrowheads in Fig. 1B). To further confirm this observation, we used a strong hypomorphic allele, dve1, which is homozygously lethal in the first instar larval stage. In dve1 mutant mosaic legs, an extra joint structure was clearly detected at the center of each ta2–ta5 segment, whereas no extra joints were found in more proximal segments. Furthermore, these extra joints had reverse polarity and invaginated into the distal side (arrowheads in Fig. 1C, see below). These results suggest that Dve activity is required to suppress inappropriate joint formation. Dve expression in leg imaginal discs Notch signaling is crucial for leg joint formation and the ectopic activation of Notch signaling induces extra joint structures (Bishop et al., 1999; de Celis et al., 1998; Rauskolb and Irvine, 1999). Thus, the expression pattern of Dve was compared with those of Notch target genes during leg development. The activity of Notch signaling was monitored as the expression of the reporter gene E(spl)mβ-CD2 (de Celis et al., 1998). In the third larval instar, Dve is expressed in a reiterated pattern of concentric rings in leg imaginal discs (Figs. 2A–A″). Some Dve-expressing regions are distally located to the Notch-activated region with partial overlapping, which is similar to those at later stages (Fig. 2A, arrows). In other regions, some Dve-expressing regions are proximally located to the Notch-activated region with partial overlapping (Fig. 2A, arrowheads), although we do not know yet how these patterns arise. In everting prepupal leg discs (4 h after puparium formation, APF), the segmental structure appears and Notch signaling is activated in several rows of distal cells in each segment (de Celis et al., 1998; Fig. 2B″). At this stage, the Dveexpressing region straddles the fold of the segment boundary and partly overlaps with the Notch-activated region (Figs. 2B– B″). Thereafter, Dve expression exhibits a more restricted pattern. At 6 h APF, Dve is repressed on the proximal side to the intersegmental fold, where Notch signaling is activated (Figs. 2C–C″). These expression patterns suggest that Notch signaling is required for temporally regulated Dve repression along the P–D axis.
Fig. 2. Temporally regulated Dve expression in leg discs. Expression patterns of Dve (magenta, A′–C′) and E(spl)mβ-CD2 (green, A″–C″) are shown. Merged images are shown in panels A–C. The Notch-activated region was monitored as the expression of E(spl)mβ-CD2. (A) In the late third larval instar, Dve is expressed in concentric rings. Distal is to the right, and proximal is to the left in a tangential optical section. Dve-expressing regions are located distally (arrows) or proximally (arrowheads) to those of E(spl)mβ-CD2. (B) At 4 h APF, Dve is expressed in regions both proximal and distal to intersegmental folds (dotted lines). (C) At 6 h APF, Dve is repressed in the Notch-activated region proximal to the folds. (D–D″) A Notch mutant mosaic leg (N55e11 FRT18A/w ubi-GFP.nls FRT18A; hs-flp38/+) at 6 h APF. Dve expression (magenta, D, D′) is strongly derepressed in Notch mutant clones (arrows), which was marked by the absence of GFP expression (green, D, D″).
Notch-mediated Dve repression is required for joint formation Notch signaling is critically required for temporally regulated dve repression in the midgut and wing discs. As described above, the relationship between Notch signaling and the Dve expression pattern in leg discs is very similar to those in the midgut and wing discs. Thus, we examined the effect of Notch signaling on Dve expression by generating Notch mutant mosaic clones. Within Notch mutant clones, Dve was strongly
derepressed outside the normal Dve-expressing domain (Figs. 2D–D″, arrows). These results support the notion that temporally regulated Dve repression is controlled by Notch signaling in leg discs as well as the midgut and wing discs. To determine the physiological function of Dve repression in prepupal leg discs, a specific GAL4 driver line should be required. We searched the GAL4 Enhancer-Trap Data Base
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(GETDB) (Hayashi et al., 2002), and identified the candidate line bibNP4281 which has a P-element insertion in the big brain (bib) locus. bib is a neurogenic gene that encodes a protein with sequence similarity to channel proteins, and is capable of augmenting the Dl–Notch pathway during neurogenesis (Doherty et al., 1997). It has been reported that bib is a target gene of the Notch signaling pathway, and is expressed in 1–2 cell rows in the distal region of each leg segment (de Celis et al., 1998). At 4 h APF, the bibNP4281-expressing region partly overlaps with that of Dve expression (Figs. 3A–A″); however, Dve expression is no longer observed in the bib NP4281expressing region at 6 h APF (Figs. 3B–B″). This temporal expression pattern of bibNP4281 coincides well with that of Notch-target gene E(spl)mβ. Furthermore, it is worth noting that the region of bibNP4281 expression seems to be narrower than that of E(spl)mβ-CD2, although a previous report showed the co-expression of bib-lacZ and E(spl)mβ-CD2 (de Celis et al., 1998). To trace the bibNP4281-expressing region at later stages, Green Fluorescent Protein (GFP) expression was induced in this region. In wild type, the segment structure disappears at 8–10 h APF, and thereafter new intersegmental folds are generated, and cells become small and columnar by 24 h APF. These columnar
cells invaginate into the proximal side and joint morphogenesis is completed by 40 h APF (Mirth and Akam, 2002). At 24 h APF, bibNP4281 expression straddles the newly formed intersegmental fold (Figs. 3C, D), and labels the joint structure at 40 h APF (Fig. 3E). Thus, the bibNP4281-expressing region, where Dve is repressed, corresponds to the joint structure itself, and dve mutant phenotypes suggest that the Dve activity is required to suppress joint formation (Fig. 1). Therefore, we hypothesized that normal joint formation is achieved through reduction of the joint-suppressive activity of Dve. To test this hypothesis, we induced forced dve expression in the presumptive joint region by using bibNP4281. The progeny was pharate adult lethal, and the resultant legs showed a characteristic ‘jointless phenotype’ that specifically lacks the joint structure without segment truncation (Figs. 4A, B). These results indicate that Dve repression in the bibNP4281-expressing region is specifically required for joint formation. If this is the case, inhibition of Notch signaling in the bibNP4281-expressing region should also induce a jointless phenotype without segment truncation. As expected, similar jointless phenotypes were observed on the expression of a dominant-negative form of Notch (d.n.N) in the bibNP4281-expressing region (Fig. 4C). Mutually antagonistic interaction between Notch and EGFR signaling is required for normal joint formation
Fig. 3. Expression of bibNP4281 labels the Notch-activated region and the joint structure. (A, B) Expression of the GAL4 enhancer-trap line bibNP4281 is observed in the Notch-activated region of everting leg discs (bibNP4281 N UAS-GFP.nls, green in panels A″, B″). Dve expression (magenta in panels A′, B′) and the merged images (A, B) are shown. The expression of bibNP4281 overlaps with that of Dve in regions proximal to intersegmental folds (dotted lines) at 4 h APF (A), but not at 6 h APF (B). (C–E) Expression of bibNP4281 in legs at 24 h APF (C, D) and 40 h APF (E) is detected in the presumptive joint structure.
To further clarify the mechanism of joint formation, we examined the effects of other signaling molecules with forced expression in the bib NP4281 -expressing region. One such candidate is the EGFR signaling pathway, because the subdivision of distal segments depends on a graded activity of EGFR signaling from the distal tip of a leg disc (Campbell, 2002; Galindo et al., 2002). Furthermore, EGFR signaling molecules such as vn, rhomboid, and pntP2 are expressed in a segmental pattern at later stages, and that the EGFR–Ras– MAPK signal transduction pathway antagonizes Notch activity (Galindo et al., 2005). Thus, the forced activation of EGFR signaling was performed by expressing an activated form of Draf or ras. The resultant legs also showed a similar jointless phenotype (Fig. 4D and data not shown). The complementary pattern between Notch and EGFR signaling raises the possibility that Notch signaling also represses EGFR signaling to create its differential activity within each segment of a leg disc. To test this possibility, we used a temperature-sensitive allele of Notch (Nts1) with an argos-lacZ (argos styP1 ) marker, which reflects high levels of EGFR signaling in various tissues (Golembo et al., 1996; MartinBlanco et al., 1999; Spencer et al., 1998; Fig. 5A). Hemizygous males (Nts1 /Y; argos-lacZ/+) were reared at a permissive temperature (18 °C) until third larval instar, and then shifted to a restrictive temperature (29 °C) for 12 h. At 4–6 h APF, argoslacZ expression had broadly expanded to the distal side of each segment, where Notch signaling is normally activated in prepupal leg discs (compare Fig. 5B with Fig. 5A′). Thus, Notch activity antagonizes EGFR signaling to generate differential activation of EGFR signaling along the P–D axis, which is proximally higher and distally lower in a segment. It is
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Fig. 4. Jointless phenotype caused by Notch signal inhibition or EGFR signal activation in the bibNP4281 region. (A–D) Tarsal segments are shown at the same magnification. (A′–D′) High magnification views of the boxed regions in panels A–D are shown. (A) A wild-type leg has ball-and-socket structures between segments. (B) Forced expression of dve in the bibNP4281 region, where Dve is normally repressed, caused jointless phenotype (bibNP4281 N UAS-dve at 21 °C). (C) Forced expression of a dominant negative form of Notch (d.n.N) in the bibNP4281 region caused jointless phenotype (bibNP4281 N UAS-d.n.N at 25 °C). (D) Forced expression of an activated form of Draf, an effector of the EGFR signaling pathway, in the bibNP4281 region caused jointless phenotype (bibNP4281 N UAS-Draf at 25 °C).
worth noting that intersegmental folds were normally formed despite the failure of subdivision within a segment when Notch activity was reduced at this stage (Fig. 5B). Relationship between Notch signaling and Dve activity The above results prompted us to examine whether there is also an antagonistic interaction between Notch signaling and Dve activity at the interface of segment boundary. Expression of the Notch-target gene bib-lacZ is detected in 1–2 cell rows within the E(spl)mβ-expressing domain, which is observed in 3–4 cell rows (Figs. 5C–C″). Although a previous report showed the co-expression of these two reporter genes bib-lacZ and E(spl)mβ-CD2 (de Celis et al., 1998), our results indicate that their co-expression is restricted in a subset of E(spl)mβexpressing cells. As described above, bib NP4281 is also expressed in a subset of E(spl)mβ-expressing domain, therefore it is assumed that forced dve expression or Notch signal inhibition by using bibNP4281 specifically antagonized the Notch signaling pathway required for joint formation. Furthermore, we found that dAP-2 expression in wild type straddles the fold of segment boundary just like Dve expression pattern at 4 h APF (Figs. 5D–D″). Thereafter, dAP-2 expression is refined to the distal region of a segment, which is a nearly identical pattern to that of E(spl)mβ-CD2 expression, and this
refined pattern coincides well with a previous report (Kerber et al., 2001). To examine the relationship between Notch signaling and Dve activity, we generated dve mutant mosaic clones. Expression of E(spl)mβ-CD2 was not affected in dve mutant clones (Figs. 5E–E″). Then, we examined the effect of dve mutation on refinement of dAP-2 expression, because dAP-2 activity is essential for joint formation (Kerber et al., 2001). In dve mutant clones, dAP-2 expression was strongly retained compared to the neighboring normal cells in which dAP-2 expression is refined (Figs. 5F–F″). Therefore, these results suggest that Dve activity does not affect the primary Notch target gene expression, whereas it is required to suppress the expression of further downstream target genes including dAP-2. Extra joint formation in planar cell polarity mutants In mutant legs for the planar cell polarity (PCP) signaling pathway such as prickle (pk), dishevelled (dsh), frizzled (fz), and inturned (in), an extra joint is formed in each tarsal segment except ta5, and pk mutant legs exhibit the most severe phenotype (Held et al., 1986). Expressivity of the extra joint formation varies between segments, and it is relatively higher in ta2–ta4 than in ta1 (Held et al., 1986; Figs. 6A, C).
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Although the mechanism underlying the extra joint formation has been unclear, these extra joints have reverse polarity as observed for dve1 mutant mosaic legs (Figs. 6B, D). These results suggest a possible link between Dve activity and the PCP signaling pathway, although dve mutant mosaic wings do not exhibit any abnormality in wing hair polarity. In adult legs, bristle polarity can be checked by the position of a bract, which is normally located on the proximal side of a bristle socket (arrows in Fig. 6E). In pk mutant legs, bristle polarity is disturbed, whereas dve1 mutant bristles marked by the cuticular marker yellow exhibit normal polarity (Figs. 6F, G). These results indicate that dve mutations did not affect the PCP signaling pathway, whereas they induced the extra joint phenotype as do PCP mutations. Thus, we hypothesized that Dve activity is regulated downstream of the PCP signaling pathway and independent of its activity to form PCP. Therefore, we examined the Dve expression pattern in pk mutants, and found that Dve expression was substantially reduced in ta2–ta4, but not in ta1 and ta5 (Figs. 6H, H′). This expression pattern well accounts for the PCP mutant phenotype in which extra joints are frequently formed in ta2–ta4, but never in ta5 (Figs. 6A, C). Furthermore, this reduced Dve expression appears to depend on the expanded Notch signal activation in these regions, because E(spl)mβ expression has broadly expanded (compare Figs. 6H, I with Figs. 5D, E). In pk mutants, dAP-2 expression is normally refined at the ta4/ta5 segment boundary (arrowheads in Fig. 6I) but expanded at other boundaries, suggesting that ectopic Notch signal activation occurs at least in ta2–ta4. Taken together, these results strongly suggest that Dve expression is maintained downstream of the PCP signaling pathway, which is independent of the bristle polarity. Discussion Antagonistic interaction between Notch and EGFR signaling
Fig. 5. Relationship between EGFR, Notch, and Dve activities. (A) Expression patterns of argos-lacZ (magenta) and E(spl)mβ-CD2 (green) at 4 h APF in an everting leg disc are shown. The region of active EGFR signaling is monitored as the expression of argos-lacZ, and is observed in a segmental pattern complementary to the Notch-activated region. A single channel image is shown in panel A′. (B) Expression of argos-lacZ in a Nts1 everting leg disc at 29 °C. argos-lacZ expression has expanded to the distal region of each segment where Notch signaling is normally activated. (C, D) Expression patterns of Notch target genes: bib-lacZ (magenta in panels C, C′), E(spl)mβ-CD2 (green in panels C, C″, D, D″), and dAP-2 (magenta in panels D, D′). (E, F) Notch target gene expression in dve mutant mosaic clones: E(spl)mβ-CD2 (green in panels E, E″) and dAP-2 (magenta in panels F, F′). Everting leg discs of eyFLP5 FRT42D y+M(2)/FRT42D dve1; E(spl)mβ-CD2/+ (E) and eyFLP5 FRT42D ubiGFP M(2)/FRT42D dve1 (F). Mosaic clones of dve mutant cells were marked by anti-Dve staining (magenta in panels E, E′) or the absence of GFP signal (green in panels F, F″).
To achieve specific developmental programs, antagonism between Notch and EGFR signaling has been widely observed (Culi et al., 2001; Kumar and Moses, 2001; Miller and Cagan, 1998; Tsuda et al., 2002; zur Lage and Jarman, 1999). A graded activity of EGFR signaling from the distal tip of a leg disc is crucial for patterning the distal structure, and it should be converted into the segmental activation, which is critical for suppression of inappropriate joint formation (Galindo et al., 2005). One possible explanation is that P–D patterning genes define the segment boundary, and thereby refine the Notch signaling pathway in the distal region of each segment, where EGFR signaling should be repressed. The expanded expression of argos-lacZ in Nts mutants strongly suggests that the Notch signaling pathway antagonizes EGFR signaling (Fig. 5B). Interestingly, a similar type of regulation has been reported for Caenorhabditis elegans vulval development (Shaye and Greenwald, 2002; Wang and Sternberg, 1999; Yoo et al., 2004). Thus, the antagonistic interaction between EGFR and Notch signaling establishes the complementary activation of these pathways in
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Notch signaling in the posterior PSM is translated into the segmental units in the wavefront, which is generated in the anterior PSM in response to the decreased activities of graded Wnt and FGF signaling from the tail end. As an embryo grows caudally, the wavefront moves backwards at a constant rate. Thus, the segment boundary is set at the interface between the Notch-activated and -repressed domains in the anterior PSM (reviewed in Aulehla and Herrmann, 2004; Giudicelli and Lewis, 2004; Pourquié, 2003; Saga and Takeda, 2001). During vertebrate somitogenesis, it has been shown that the interface between the Notch-activated and -repressed domains is generated on suppression of Notch activity through induction of the lunatic-fringe (Lfng) gene in the segment boundary (Morimoto et al., 2005). This refinement is under the control of the basic helix–loop–helix type transcription factor Mesp2, which is expressed in the rostral half of the anterior PSM, indicating that rostral–caudal polarity within a somite is important for restricted Notch activation. Our results indicate that the restricted Notch activation during Drosophila leg segmentation also occurs at the segment boundary rather than the center of each segment (see below), suggesting that a conserved mechanism in both Drosophila legs and vertebrate somites underlies the activation of Notch signaling adjacent to the segment boundary. The mechanism of joint formation
Fig. 6. Dve activity suppresses extra joint formation downstream of the PCP signaling pathway. (A–D) The ta1–ta2 (A, B) and ta3–ta5 (C, D) segments of pk (A, C) and dve1 (B, D) mutant legs are shown, respectively. Arrows indicate normal joints, and red arrowheads indicate ectopic joints, which have a complete ball-and-socket structure with reverse polarity. White arrowheads in ta1 indicate incomplete joint-like structures, which have only a socket (A) or a rarely observed tiny dot (B). Note that there is no ectopic joint in ta5 of a pk mutant leg (C). (E–G) The bract–socket–vector polarity of tibia is shown. (E) In wild type, bracts are localized proximally to bristles (arrows). (F) In pk mutants, the bract positions are abnormal (arrowheads). (G) In dve1 mutant mosaic legs, dve1 mutant bristles marked by yellow exhibit normal polarity (arrows). (H, I) In pk mutants, E(spl)mβ expression (green) has broadly expanded on the distal side to the intersegmental folds. The folds between ta4 and ta5 are indicated by arrowheads. Dve expression (magenta in panels H, H′) is substantially reduced in the proximal region of ta2–ta4, and dAP-2 expression (magenta in panel I) is refined in ta5, but not in ta2–ta4.
neighboring cells, and is crucial for both vulval cell fate determination and leg joint formation. Conserved mechanism of segment boundary specification In vertebrates, the early process of body segmentation, i.e. somitogenesis, takes place sequentially from head to tail. Somites are generated from the presomitic mesoderm (PSM), the unsegmented paraxial mesoderm at the tail end of the embryo. A ‘clock and wavefront’ model has been proposed to explain the mechanism of sequential somite formation. Oscillated gene expression, i.e., the clock, driven by Wnt and
Dve-expressing region straddles the fold of the segment boundary (Fig. 7A), and the following observations indicate that Dve has joint-suppressive activity: (1) dve mutant legs resulted in extra joint formation and (2) forced expression of dve in the presumptive joint region suppressed joint formation. Thus, the mechanism of joint development can be explained as follows; Notch-mediated Dve repression on the proximal side to the intersegmental fold relieves the above joint-suppressive activity, leading to normal leg joint formation. This is reminiscent of the abdomen-suppressive activity of Hunchback, which is relieved by Nanos to induce the abdominal structure (Hülskamp et al., 1989; Irish et al., 1989). In contrast, Dve expression on the distal side to the fold should be maintained to suppress inappropriate joint formation, because dve mutation leads to extra joint formation with reverse polarity. It appears that Dve activity is only induced to suppress joint formation and that temporally regulated Dve repression is crucial for normal leg joint formation, because dve mutations did not affect normal joint formation. Extra joints with reverse polarity (reverse joints) are derived from mutants deficient in the PCP or EGFR signaling pathway (Bishop et al., 1999; Galindo et al., 2005; Held et al., 1986). Previous reports have suggested a model in which the Notch signal activation proximal to the Notch ligand-expressing domains is blocked by these signals, only allowing the Notch signal activation in a distally adjacent region, i.e., the distal region of each segment (Bishop et al., 1999; Galindo et al., 2005). Based on the expression pattern of the Notch ligand Ser, it is assumed that the center of a segment is highly potent for receiving Notch signaling. This idea can explain the reverse
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The segment boundary generates bidirectional joint-forming activity Based on our results, we propose a model in which jointforming activity is generated from the intersegmental fold in a bidirectional manner, and that an inappropriate signal having reverse polarity is blocked by Dve activity, and the PCP and EGFR signaling pathways (Fig. 7B). In our model, Dve activity is required to suppress Notch target genes, such as dAP-2, involved in joint formation. This is very similar to the situation observed in wing discs, where the Notch target gene wg is repressed by Dve in regions adjacent to the Notch-activated D– V boundary (Nakagoshi et al., 2002). It is an intriguing possibility that the vertebrate somite boundary generates similar bidirectional signals, and that the inhibition of either one is closely linked to the rostral–caudal polarity within a somite. Further characterization of Drosophila leg segmentation is needed to determine whether this model is applicable to vertebrate somitogenesis or other segmentation processes. Acknowledgments
Fig. 7. Schematic representation of gene expression in everting leg discs. (A) Notch signaling is activated in the distal region of each segment (green), and EGFR signaling is activated in a segmental pattern complementary to the Notchactivated region (red). At 4 h APF, Dve is expressed in regions both proximal and distal to the intersegmental folds (magenta). Subsequently, Dve is repressed in the Notch-activated region proximal to the fold at 6 h APF (magenta). The expression of bibNP4281 is observed where Dve is normally repressed (blue), and this region corresponds to the presumptive joint structure. The intersegmental folds are shown by arrowheads. The proximal (P)–distal (D) axis is indicated by double arrows. (B) The segment boundary is refined by a mutually antagonistic interaction between Notch and EGFR signaling, and generates bidirectional joint-forming activity (blue arrows). An inappropriate activity having reverse polarity should be blocked by Dve activity (T-shaped bar). Furthermore, the planar cell polarity (PCP) and EGFR signaling pathways are also required to suppress an inappropriate one (Bishop et al., 1999; Galindo et al., 2005). The PCP signaling pathway independent of bristle polarity is required for the maintenance of Dve expression (broken arrow), presumably due to the inactivation of Notch signaling together with Fringe (Fng) activity (Bishop et al., 1999; Fig. 6). Notch signaling in the bibNP4281 region relives the jointsuppressive activity of Dve at 6 h APF and induces joint structures.
polarity of extra joints, because Ser activates the Notch signaling pathway in two different directions: from proximal to distal for normal joints, and distal to proximal for extra joints. However, it seems unlikely that ectopic activation of Notch signaling is restricted at the center of a segment. A Notch-target gene, dAP-2, is autonomously activated in response to ectopic Notch signaling (Kerber et al., 2001), and, in pk mutants, ectopic dAP-2 expression has expanded on the distal side to the intersegmental fold, the most proximal but not the central region in a segment (Fig. 6I). Furthermore, the joint-suppressive activity of Dve is also required to repress dAP-2 expression on the distal side to the intersegmental fold (Fig. 5F). These results led us to favor the possibility that reverse joints are derived from the distally adjacent region to the intersegmental fold.
We are grateful to Drs. J. F. de Celis, P. J. Mitchell, T. Kojima, H. Okano, the Bloomington Stock Center, and the Drosophila Genetic Resource Center (National Institute of Genetics, Mishima) for the fly strains and antibodies; Drs. T. Kojima and T. Awasaki for helpful discussions. We also thank the Advanced Science Research Center (Okayama University) for the use of confocal microscope. This work was supported by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (to H. N.). References Abu-Shaar, M., Mann, R.S., 1998. Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development. Development 125, 3821–3830. Aulehla, A., Herrmann, B.G., 2004. Segmentation in vertebrates: clock and gradient finally joined. Genes Dev. 18, 2060–2067. Basler, K., Struhl, G., 1994. Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368, 208–214. Bishop, S.A., Klein, T., Arias, A.M., Couso, J.P., 1999. Composite signalling from Serrate and Delta establishes leg segments in Drosophila through Notch. Development 126, 2993–3003. Brand, A.H., Perrimon, N., 1994. Raf acts downstream of the EGF receptor to determine dorsoventral polarity during Drosophila oogenesis. Genes Dev. 8, 629–639. Brückner, K., Perez, L., Clausen, H., Cohen, S., 2000. Glycosyltransferase activity of Fringe modulates Notch–Delta interactions. Nature 406, 411–415. Campbell, G., 2002. Distalization of the Drosophila leg by graded EGF-receptor activity. Nature 418, 781–785. Carr, M., Hurley, I., Fowler, K., Pomiankowski, A., Smith, H.K., 2005. Expression of defective proventriculus during head capsule development is conserved in Drosophila and stalk-eyed flies (Diopsidae). Dev. Genes Evol. 215, 402–409. Culi, J., Martin-Blanco, E., Modolell, J., 2001. The EGF receptor and N signalling pathways act antagonistically in Drosophila mesothorax bristle patterning. Development 128, 299–308. de Celis Ibeas, J.M., Bray, S.J., 2003. Bowl is required downstream of Notch for elaboration of distal limb patterning. Development 130, 5943–5952.
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Developmental Biology 312 (2007) 147 – 156 www.elsevier.com/developmentalbiology
Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg Tetsuya Shirai a,1 , Takeshi Yorimitsu a , Naruto Kiritooshi a , Fumio Matsuzaki b , Hideki Nakagoshi a,⁎ a
Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530, Japan b Laboratory for Cell Asymmetry, Center for Developmental Biology, RIKEN, Kobe, Japan Received for publication 6 April 2007; revised 21 August 2007; accepted 6 September 2007 Available online 18 September 2007
Abstract Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal–distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints. © 2007 Elsevier Inc. All rights reserved. Keywords: Drosophila; Leg; Segment; Notch; EGFR; Planar cell polarity; dve
Introduction Studies in the developing Drosophila leg have provided important insights into the intercellular signaling pathways regulating positional information, cell fate specification, and tissue growth (reviewed in Galindo and Couso, 2000; Kojima, 2004; Milán and Cohen, 2000). Adult Drosophila legs consist of several segments along the proximal–distal (P–D) axis: from proximal to distal, the coxa, trochanter, femur, tibia, tarsus and pretarsus bearing two claws. Neighboring leg segments are separated by flexible structures called joints, which allow them to move. In leg imaginal discs, decapentaplegic (dpp) and wingless (wg) are expressed in anterior dorsal and anterior ventral sectors, respectively, in response to the secreted protein Hedgehog, which is only expressed in posterior cells (Basler and Struhl, ⁎ Corresponding author. Fax: +81 86 251 7876. E-mail address: [email protected] (H. Nakagoshi). 1 Present address: Institute for Research in Humanities, Kyoto University, Japan. 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.09.003
1994). The combined activities of Dpp and Wg activate another set of P–D patterning genes such as homothorax (hth), dachshund (dac), and Distal-less (Dll), which are expressed in ringshaped territories in different domains along the P–D axis (AbuShaar and Mann, 1998; González-Crespo et al., 1998; Lecuit and Cohen, 1997; Wu and Cohen, 1999). At the most central portion of the leg discs, vein (vn), a neuregulin-like ligand of epidermal growth factor receptor (EGFR), is induced in response to Dpp and Wg. A graded activity of EGFR signaling from a distal tip (central portion of the disc) is required for patterning the distal leg segments (Campbell, 2002; Galindo et al., 2002). Distinct combinations of P–D patterning genes are independently required for each segmental expression of the Notch ligand Serrate (Ser) (Rauskolb, 2001). Notch signaling is activated in several rows of distal cells in each segment, which are distally adjacent to the Notch ligand-expressing domain, while in cells proximally adjacent to them, Notch signaling is not activated (Bishop et al., 1999; de Celis et al., 1998; Rauskolb and Irvine, 1999). The glycosyltransferase Fringe (Fng) modulates the ability of Notch ligands Delta (Dl) and Ser,
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and therefore is involved in the restricted activation of Notch signaling (Bishop et al., 1999; Brückner et al., 2000; Fleming et al., 1997; Irvine and Wieschaus, 1994; Moloney et al., 2000; Panin et al., 1997; Rauskolb and Irvine, 1999). Activation of Notch signaling in the distal region of each segment is required for leg growth and joint formation. Notch mutant mosaic legs result in severe segment truncation due to the loss of joint structures and reduced growth, while ectopic activation of Notch signaling induces extra joint formation and outgrowth of the leg tissue, indicating that Notch signaling is sufficient to establish the joint structure (Bishop et al., 1999; de Celis et al., 1998; Rauskolb and Irvine, 1999; Shellenbarger and Mohler, 1978). In contrast to embryonic segmentation, leg segmentation occurs in a growing tissue, thus the subdivision must be coordinated with tissue growth. Several Notchregulated genes have been identified, such as E(spl)mβ, oddskipped (odd), nubbin (nub), four-jointed (fj), big brain (bib), Drosophila AP-2 (dAP-2), and brother of odd with entrails limited (bowl) (Bishop et al., 1999; de Celis Ibeas and Bray, 2003; de Celis et al., 1998; Hao et al., 2003; Rauskolb and Irvine, 1999); however, the coordinated regulation between leg growth and segmentation remains unknown. The homeobox gene defective proventriculus (dve) is expressed in a pattern of concentric rings in leg discs, and is temporally regulated in the Notch-activated region. Our previous studies have shown that temporally regulated Dve repression is crucial for gut function and for wing patterning (Nakagoshi et al., 1998; Nakagoshi et al., 2002). In wing discs, Dve is expressed in the prospective wing region, and is subsequently repressed along the dorsal–ventral (D–V) compartment boundary through Notch-mediated signaling. In the midgut, the absorptive function of copper cells is established through Notch signaling in adjacent cells and through temporally regulated dve repression in copper cells. In Notch mutants, dve expression is derepressed in copper cells (Tanaka et al., 2007), suggesting that temporally regulated dve repression depends on the Notch activation in two distinct tissues. Here, we provide evidence that temporally regulated Dve repression is crucial for leg joint formation, during which Notch signaling relieves the joint-suppressive activity of Dve.
(Nakagoshi et al., 1998), dAP-2 (1:100) (Monge et al., 2001), and β-Gal (Cappel; 1:100) were used. Cy3- or FITC-conjugated secondary antibodies to mouse immunoglobulin G (Jackson; 1:100) and Cy3- (Jackson; 1:100) or FITCconjugated (Cappel; 1:100) secondary antibodies to rabbit immunoglobulin G were used. Confocal images were obtained with an OLYMPUS FluoView300. Nomarski images were obtained with a Zeiss AxioVision 3.0, and photo images of adult legs (Figs. 1 and 6A–D) were assembled into montages using Photoshop software (Adobe).
Mosaic analyses Mosaic clones were induced with the use of an FRT- and FLP-mediated recombination system (Xu and Rubin, 1993). To generate dve1 mosaic clones, FRT42D-dve1 flies were crossed with y w hs-flp; FRT42D y+M(2) or f hs-flp; FRT42D M(2) f+ ones, and the offspring were subjected to heat shock at 38 °C for 90 min in the late first to early second larval instars. For collecting the staged mutant prepupal leg discs, FRT42D locus was recombined with an ey-FLP5 transgene, which expresses FLPase also in the leg and antennal discs from early stages of development (Tsuji et al., 2000), leading to generate mosaic clones without heat shock. To generate Notch mutant mosaic clones, N55e11 FRT18A/ FM7 was crossed with w ubi-GFP.nls FRT18A; hs-flp38, and heat-shocked at 38 °C for 30 min in the late second to early third larval instars.
Results Extra joint formation in dve mutant legs The homeobox gene defective proventriculus (dve) is expressed in various tissues including the midgut and wing imaginal discs (Carr et al., 2005; Fuss and Hoch, 1998; Nakagoshi, 2005; Nakagoshi et al., 1998, 2002; Shirai et al., 2003). dveBG02382 is a hypomorphic allele, and homozygous escaper adults are rarely observed. In dveBG02382 homozygous wings, P–D patterning is affected due to reduced proliferation
Materials and methods Fly stocks The following mutant strains were used: dve1 (Nakagoshi et al., 1998), dve (Kölzer et al., 2003), pksple-1 (Held et al., 1986), and the Notch temperature-sensitive allele l(1)Nts1 (Shellenbarger and Mohler, 1975). To monitor Notch- and EGFR-activated cells, E(spl)mβ-CD2 and bib-lacZ, and argos-lacZ (argosstyP1) were used, respectively (de Celis et al., 1998; Okano et al., 1992). The following GAL4/UAS strains were used: bibNP4281 (Hayashi et al., 2002), UAS-GFP.nls (Bloomington Stock Center), UAS-dve-9B2 (Nakagoshi et al., 1998), UAS-d.n.N (Go et al., 1998), and UAS-DrafGOF (Brand and Perrimon, 1994). BG02382
Immunohistochemistry Mouse monoclonal antibodies to β-galactosidase (Gal) (Promega; 1:100 dilution) and CD2 (Serotec; 1:100), as well as rabbit antibodies to Dve (1:500)
Fig. 1. Extra joint formation in dve mutant legs. (A) A wild-type (WT) leg has joints between segments (arrows). (B) A dveBG02382 homozygous leg has extra joint-like structures (arrowheads). (C) Extra joints are clearly observed in a dve1 mosaic leg (f hs-flp/Y; FRT42D dve1/FRT42D M(2) f +), and they have reverse polarity (arrowheads). Distal is to the bottom, and proximal is to the top. The orientation is the same in following figures except Figs. 2A and 7.
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rate in wing imaginal discs, and wing vein formation is impaired in longitudinal veins 2 (L2) and 5 (L5) (Kölzer et al., 2003). Interestingly, we found an additional phenotype in dveBG02382 homozygous legs. In wild type, leg segments are separated by a flexible structure called joint which consists of a ball-and-socket structure and invaginates into the proximal side (arrows in Fig. 1A); however, dveBG02382 homozygous legs have extra jointlike structures at the center of each tarsal segment 2–5 (ta2–ta5) (arrowheads in Fig. 1B). To further confirm this observation, we used a strong hypomorphic allele, dve1, which is homozygously lethal in the first instar larval stage. In dve1 mutant mosaic legs, an extra joint structure was clearly detected at the center of each ta2–ta5 segment, whereas no extra joints were found in more proximal segments. Furthermore, these extra joints had reverse polarity and invaginated into the distal side (arrowheads in Fig. 1C, see below). These results suggest that Dve activity is required to suppress inappropriate joint formation. Dve expression in leg imaginal discs Notch signaling is crucial for leg joint formation and the ectopic activation of Notch signaling induces extra joint structures (Bishop et al., 1999; de Celis et al., 1998; Rauskolb and Irvine, 1999). Thus, the expression pattern of Dve was compared with those of Notch target genes during leg development. The activity of Notch signaling was monitored as the expression of the reporter gene E(spl)mβ-CD2 (de Celis et al., 1998). In the third larval instar, Dve is expressed in a reiterated pattern of concentric rings in leg imaginal discs (Figs. 2A–A″). Some Dve-expressing regions are distally located to the Notch-activated region with partial overlapping, which is similar to those at later stages (Fig. 2A, arrows). In other regions, some Dve-expressing regions are proximally located to the Notch-activated region with partial overlapping (Fig. 2A, arrowheads), although we do not know yet how these patterns arise. In everting prepupal leg discs (4 h after puparium formation, APF), the segmental structure appears and Notch signaling is activated in several rows of distal cells in each segment (de Celis et al., 1998; Fig. 2B″). At this stage, the Dveexpressing region straddles the fold of the segment boundary and partly overlaps with the Notch-activated region (Figs. 2B– B″). Thereafter, Dve expression exhibits a more restricted pattern. At 6 h APF, Dve is repressed on the proximal side to the intersegmental fold, where Notch signaling is activated (Figs. 2C–C″). These expression patterns suggest that Notch signaling is required for temporally regulated Dve repression along the P–D axis.
Fig. 2. Temporally regulated Dve expression in leg discs. Expression patterns of Dve (magenta, A′–C′) and E(spl)mβ-CD2 (green, A″–C″) are shown. Merged images are shown in panels A–C. The Notch-activated region was monitored as the expression of E(spl)mβ-CD2. (A) In the late third larval instar, Dve is expressed in concentric rings. Distal is to the right, and proximal is to the left in a tangential optical section. Dve-expressing regions are located distally (arrows) or proximally (arrowheads) to those of E(spl)mβ-CD2. (B) At 4 h APF, Dve is expressed in regions both proximal and distal to intersegmental folds (dotted lines). (C) At 6 h APF, Dve is repressed in the Notch-activated region proximal to the folds. (D–D″) A Notch mutant mosaic leg (N55e11 FRT18A/w ubi-GFP.nls FRT18A; hs-flp38/+) at 6 h APF. Dve expression (magenta, D, D′) is strongly derepressed in Notch mutant clones (arrows), which was marked by the absence of GFP expression (green, D, D″).
Notch-mediated Dve repression is required for joint formation Notch signaling is critically required for temporally regulated dve repression in the midgut and wing discs. As described above, the relationship between Notch signaling and the Dve expression pattern in leg discs is very similar to those in the midgut and wing discs. Thus, we examined the effect of Notch signaling on Dve expression by generating Notch mutant mosaic clones. Within Notch mutant clones, Dve was strongly
derepressed outside the normal Dve-expressing domain (Figs. 2D–D″, arrows). These results support the notion that temporally regulated Dve repression is controlled by Notch signaling in leg discs as well as the midgut and wing discs. To determine the physiological function of Dve repression in prepupal leg discs, a specific GAL4 driver line should be required. We searched the GAL4 Enhancer-Trap Data Base
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(GETDB) (Hayashi et al., 2002), and identified the candidate line bibNP4281 which has a P-element insertion in the big brain (bib) locus. bib is a neurogenic gene that encodes a protein with sequence similarity to channel proteins, and is capable of augmenting the Dl–Notch pathway during neurogenesis (Doherty et al., 1997). It has been reported that bib is a target gene of the Notch signaling pathway, and is expressed in 1–2 cell rows in the distal region of each leg segment (de Celis et al., 1998). At 4 h APF, the bibNP4281-expressing region partly overlaps with that of Dve expression (Figs. 3A–A″); however, Dve expression is no longer observed in the bib NP4281expressing region at 6 h APF (Figs. 3B–B″). This temporal expression pattern of bibNP4281 coincides well with that of Notch-target gene E(spl)mβ. Furthermore, it is worth noting that the region of bibNP4281 expression seems to be narrower than that of E(spl)mβ-CD2, although a previous report showed the co-expression of bib-lacZ and E(spl)mβ-CD2 (de Celis et al., 1998). To trace the bibNP4281-expressing region at later stages, Green Fluorescent Protein (GFP) expression was induced in this region. In wild type, the segment structure disappears at 8–10 h APF, and thereafter new intersegmental folds are generated, and cells become small and columnar by 24 h APF. These columnar
cells invaginate into the proximal side and joint morphogenesis is completed by 40 h APF (Mirth and Akam, 2002). At 24 h APF, bibNP4281 expression straddles the newly formed intersegmental fold (Figs. 3C, D), and labels the joint structure at 40 h APF (Fig. 3E). Thus, the bibNP4281-expressing region, where Dve is repressed, corresponds to the joint structure itself, and dve mutant phenotypes suggest that the Dve activity is required to suppress joint formation (Fig. 1). Therefore, we hypothesized that normal joint formation is achieved through reduction of the joint-suppressive activity of Dve. To test this hypothesis, we induced forced dve expression in the presumptive joint region by using bibNP4281. The progeny was pharate adult lethal, and the resultant legs showed a characteristic ‘jointless phenotype’ that specifically lacks the joint structure without segment truncation (Figs. 4A, B). These results indicate that Dve repression in the bibNP4281-expressing region is specifically required for joint formation. If this is the case, inhibition of Notch signaling in the bibNP4281-expressing region should also induce a jointless phenotype without segment truncation. As expected, similar jointless phenotypes were observed on the expression of a dominant-negative form of Notch (d.n.N) in the bibNP4281-expressing region (Fig. 4C). Mutually antagonistic interaction between Notch and EGFR signaling is required for normal joint formation
Fig. 3. Expression of bibNP4281 labels the Notch-activated region and the joint structure. (A, B) Expression of the GAL4 enhancer-trap line bibNP4281 is observed in the Notch-activated region of everting leg discs (bibNP4281 N UAS-GFP.nls, green in panels A″, B″). Dve expression (magenta in panels A′, B′) and the merged images (A, B) are shown. The expression of bibNP4281 overlaps with that of Dve in regions proximal to intersegmental folds (dotted lines) at 4 h APF (A), but not at 6 h APF (B). (C–E) Expression of bibNP4281 in legs at 24 h APF (C, D) and 40 h APF (E) is detected in the presumptive joint structure.
To further clarify the mechanism of joint formation, we examined the effects of other signaling molecules with forced expression in the bib NP4281 -expressing region. One such candidate is the EGFR signaling pathway, because the subdivision of distal segments depends on a graded activity of EGFR signaling from the distal tip of a leg disc (Campbell, 2002; Galindo et al., 2002). Furthermore, EGFR signaling molecules such as vn, rhomboid, and pntP2 are expressed in a segmental pattern at later stages, and that the EGFR–Ras– MAPK signal transduction pathway antagonizes Notch activity (Galindo et al., 2005). Thus, the forced activation of EGFR signaling was performed by expressing an activated form of Draf or ras. The resultant legs also showed a similar jointless phenotype (Fig. 4D and data not shown). The complementary pattern between Notch and EGFR signaling raises the possibility that Notch signaling also represses EGFR signaling to create its differential activity within each segment of a leg disc. To test this possibility, we used a temperature-sensitive allele of Notch (Nts1) with an argos-lacZ (argos styP1 ) marker, which reflects high levels of EGFR signaling in various tissues (Golembo et al., 1996; MartinBlanco et al., 1999; Spencer et al., 1998; Fig. 5A). Hemizygous males (Nts1 /Y; argos-lacZ/+) were reared at a permissive temperature (18 °C) until third larval instar, and then shifted to a restrictive temperature (29 °C) for 12 h. At 4–6 h APF, argoslacZ expression had broadly expanded to the distal side of each segment, where Notch signaling is normally activated in prepupal leg discs (compare Fig. 5B with Fig. 5A′). Thus, Notch activity antagonizes EGFR signaling to generate differential activation of EGFR signaling along the P–D axis, which is proximally higher and distally lower in a segment. It is
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Fig. 4. Jointless phenotype caused by Notch signal inhibition or EGFR signal activation in the bibNP4281 region. (A–D) Tarsal segments are shown at the same magnification. (A′–D′) High magnification views of the boxed regions in panels A–D are shown. (A) A wild-type leg has ball-and-socket structures between segments. (B) Forced expression of dve in the bibNP4281 region, where Dve is normally repressed, caused jointless phenotype (bibNP4281 N UAS-dve at 21 °C). (C) Forced expression of a dominant negative form of Notch (d.n.N) in the bibNP4281 region caused jointless phenotype (bibNP4281 N UAS-d.n.N at 25 °C). (D) Forced expression of an activated form of Draf, an effector of the EGFR signaling pathway, in the bibNP4281 region caused jointless phenotype (bibNP4281 N UAS-Draf at 25 °C).
worth noting that intersegmental folds were normally formed despite the failure of subdivision within a segment when Notch activity was reduced at this stage (Fig. 5B). Relationship between Notch signaling and Dve activity The above results prompted us to examine whether there is also an antagonistic interaction between Notch signaling and Dve activity at the interface of segment boundary. Expression of the Notch-target gene bib-lacZ is detected in 1–2 cell rows within the E(spl)mβ-expressing domain, which is observed in 3–4 cell rows (Figs. 5C–C″). Although a previous report showed the co-expression of these two reporter genes bib-lacZ and E(spl)mβ-CD2 (de Celis et al., 1998), our results indicate that their co-expression is restricted in a subset of E(spl)mβexpressing cells. As described above, bib NP4281 is also expressed in a subset of E(spl)mβ-expressing domain, therefore it is assumed that forced dve expression or Notch signal inhibition by using bibNP4281 specifically antagonized the Notch signaling pathway required for joint formation. Furthermore, we found that dAP-2 expression in wild type straddles the fold of segment boundary just like Dve expression pattern at 4 h APF (Figs. 5D–D″). Thereafter, dAP-2 expression is refined to the distal region of a segment, which is a nearly identical pattern to that of E(spl)mβ-CD2 expression, and this
refined pattern coincides well with a previous report (Kerber et al., 2001). To examine the relationship between Notch signaling and Dve activity, we generated dve mutant mosaic clones. Expression of E(spl)mβ-CD2 was not affected in dve mutant clones (Figs. 5E–E″). Then, we examined the effect of dve mutation on refinement of dAP-2 expression, because dAP-2 activity is essential for joint formation (Kerber et al., 2001). In dve mutant clones, dAP-2 expression was strongly retained compared to the neighboring normal cells in which dAP-2 expression is refined (Figs. 5F–F″). Therefore, these results suggest that Dve activity does not affect the primary Notch target gene expression, whereas it is required to suppress the expression of further downstream target genes including dAP-2. Extra joint formation in planar cell polarity mutants In mutant legs for the planar cell polarity (PCP) signaling pathway such as prickle (pk), dishevelled (dsh), frizzled (fz), and inturned (in), an extra joint is formed in each tarsal segment except ta5, and pk mutant legs exhibit the most severe phenotype (Held et al., 1986). Expressivity of the extra joint formation varies between segments, and it is relatively higher in ta2–ta4 than in ta1 (Held et al., 1986; Figs. 6A, C).
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Although the mechanism underlying the extra joint formation has been unclear, these extra joints have reverse polarity as observed for dve1 mutant mosaic legs (Figs. 6B, D). These results suggest a possible link between Dve activity and the PCP signaling pathway, although dve mutant mosaic wings do not exhibit any abnormality in wing hair polarity. In adult legs, bristle polarity can be checked by the position of a bract, which is normally located on the proximal side of a bristle socket (arrows in Fig. 6E). In pk mutant legs, bristle polarity is disturbed, whereas dve1 mutant bristles marked by the cuticular marker yellow exhibit normal polarity (Figs. 6F, G). These results indicate that dve mutations did not affect the PCP signaling pathway, whereas they induced the extra joint phenotype as do PCP mutations. Thus, we hypothesized that Dve activity is regulated downstream of the PCP signaling pathway and independent of its activity to form PCP. Therefore, we examined the Dve expression pattern in pk mutants, and found that Dve expression was substantially reduced in ta2–ta4, but not in ta1 and ta5 (Figs. 6H, H′). This expression pattern well accounts for the PCP mutant phenotype in which extra joints are frequently formed in ta2–ta4, but never in ta5 (Figs. 6A, C). Furthermore, this reduced Dve expression appears to depend on the expanded Notch signal activation in these regions, because E(spl)mβ expression has broadly expanded (compare Figs. 6H, I with Figs. 5D, E). In pk mutants, dAP-2 expression is normally refined at the ta4/ta5 segment boundary (arrowheads in Fig. 6I) but expanded at other boundaries, suggesting that ectopic Notch signal activation occurs at least in ta2–ta4. Taken together, these results strongly suggest that Dve expression is maintained downstream of the PCP signaling pathway, which is independent of the bristle polarity. Discussion Antagonistic interaction between Notch and EGFR signaling
Fig. 5. Relationship between EGFR, Notch, and Dve activities. (A) Expression patterns of argos-lacZ (magenta) and E(spl)mβ-CD2 (green) at 4 h APF in an everting leg disc are shown. The region of active EGFR signaling is monitored as the expression of argos-lacZ, and is observed in a segmental pattern complementary to the Notch-activated region. A single channel image is shown in panel A′. (B) Expression of argos-lacZ in a Nts1 everting leg disc at 29 °C. argos-lacZ expression has expanded to the distal region of each segment where Notch signaling is normally activated. (C, D) Expression patterns of Notch target genes: bib-lacZ (magenta in panels C, C′), E(spl)mβ-CD2 (green in panels C, C″, D, D″), and dAP-2 (magenta in panels D, D′). (E, F) Notch target gene expression in dve mutant mosaic clones: E(spl)mβ-CD2 (green in panels E, E″) and dAP-2 (magenta in panels F, F′). Everting leg discs of eyFLP5 FRT42D y+M(2)/FRT42D dve1; E(spl)mβ-CD2/+ (E) and eyFLP5 FRT42D ubiGFP M(2)/FRT42D dve1 (F). Mosaic clones of dve mutant cells were marked by anti-Dve staining (magenta in panels E, E′) or the absence of GFP signal (green in panels F, F″).
To achieve specific developmental programs, antagonism between Notch and EGFR signaling has been widely observed (Culi et al., 2001; Kumar and Moses, 2001; Miller and Cagan, 1998; Tsuda et al., 2002; zur Lage and Jarman, 1999). A graded activity of EGFR signaling from the distal tip of a leg disc is crucial for patterning the distal structure, and it should be converted into the segmental activation, which is critical for suppression of inappropriate joint formation (Galindo et al., 2005). One possible explanation is that P–D patterning genes define the segment boundary, and thereby refine the Notch signaling pathway in the distal region of each segment, where EGFR signaling should be repressed. The expanded expression of argos-lacZ in Nts mutants strongly suggests that the Notch signaling pathway antagonizes EGFR signaling (Fig. 5B). Interestingly, a similar type of regulation has been reported for Caenorhabditis elegans vulval development (Shaye and Greenwald, 2002; Wang and Sternberg, 1999; Yoo et al., 2004). Thus, the antagonistic interaction between EGFR and Notch signaling establishes the complementary activation of these pathways in
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Notch signaling in the posterior PSM is translated into the segmental units in the wavefront, which is generated in the anterior PSM in response to the decreased activities of graded Wnt and FGF signaling from the tail end. As an embryo grows caudally, the wavefront moves backwards at a constant rate. Thus, the segment boundary is set at the interface between the Notch-activated and -repressed domains in the anterior PSM (reviewed in Aulehla and Herrmann, 2004; Giudicelli and Lewis, 2004; Pourquié, 2003; Saga and Takeda, 2001). During vertebrate somitogenesis, it has been shown that the interface between the Notch-activated and -repressed domains is generated on suppression of Notch activity through induction of the lunatic-fringe (Lfng) gene in the segment boundary (Morimoto et al., 2005). This refinement is under the control of the basic helix–loop–helix type transcription factor Mesp2, which is expressed in the rostral half of the anterior PSM, indicating that rostral–caudal polarity within a somite is important for restricted Notch activation. Our results indicate that the restricted Notch activation during Drosophila leg segmentation also occurs at the segment boundary rather than the center of each segment (see below), suggesting that a conserved mechanism in both Drosophila legs and vertebrate somites underlies the activation of Notch signaling adjacent to the segment boundary. The mechanism of joint formation
Fig. 6. Dve activity suppresses extra joint formation downstream of the PCP signaling pathway. (A–D) The ta1–ta2 (A, B) and ta3–ta5 (C, D) segments of pk (A, C) and dve1 (B, D) mutant legs are shown, respectively. Arrows indicate normal joints, and red arrowheads indicate ectopic joints, which have a complete ball-and-socket structure with reverse polarity. White arrowheads in ta1 indicate incomplete joint-like structures, which have only a socket (A) or a rarely observed tiny dot (B). Note that there is no ectopic joint in ta5 of a pk mutant leg (C). (E–G) The bract–socket–vector polarity of tibia is shown. (E) In wild type, bracts are localized proximally to bristles (arrows). (F) In pk mutants, the bract positions are abnormal (arrowheads). (G) In dve1 mutant mosaic legs, dve1 mutant bristles marked by yellow exhibit normal polarity (arrows). (H, I) In pk mutants, E(spl)mβ expression (green) has broadly expanded on the distal side to the intersegmental folds. The folds between ta4 and ta5 are indicated by arrowheads. Dve expression (magenta in panels H, H′) is substantially reduced in the proximal region of ta2–ta4, and dAP-2 expression (magenta in panel I) is refined in ta5, but not in ta2–ta4.
neighboring cells, and is crucial for both vulval cell fate determination and leg joint formation. Conserved mechanism of segment boundary specification In vertebrates, the early process of body segmentation, i.e. somitogenesis, takes place sequentially from head to tail. Somites are generated from the presomitic mesoderm (PSM), the unsegmented paraxial mesoderm at the tail end of the embryo. A ‘clock and wavefront’ model has been proposed to explain the mechanism of sequential somite formation. Oscillated gene expression, i.e., the clock, driven by Wnt and
Dve-expressing region straddles the fold of the segment boundary (Fig. 7A), and the following observations indicate that Dve has joint-suppressive activity: (1) dve mutant legs resulted in extra joint formation and (2) forced expression of dve in the presumptive joint region suppressed joint formation. Thus, the mechanism of joint development can be explained as follows; Notch-mediated Dve repression on the proximal side to the intersegmental fold relieves the above joint-suppressive activity, leading to normal leg joint formation. This is reminiscent of the abdomen-suppressive activity of Hunchback, which is relieved by Nanos to induce the abdominal structure (Hülskamp et al., 1989; Irish et al., 1989). In contrast, Dve expression on the distal side to the fold should be maintained to suppress inappropriate joint formation, because dve mutation leads to extra joint formation with reverse polarity. It appears that Dve activity is only induced to suppress joint formation and that temporally regulated Dve repression is crucial for normal leg joint formation, because dve mutations did not affect normal joint formation. Extra joints with reverse polarity (reverse joints) are derived from mutants deficient in the PCP or EGFR signaling pathway (Bishop et al., 1999; Galindo et al., 2005; Held et al., 1986). Previous reports have suggested a model in which the Notch signal activation proximal to the Notch ligand-expressing domains is blocked by these signals, only allowing the Notch signal activation in a distally adjacent region, i.e., the distal region of each segment (Bishop et al., 1999; Galindo et al., 2005). Based on the expression pattern of the Notch ligand Ser, it is assumed that the center of a segment is highly potent for receiving Notch signaling. This idea can explain the reverse
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The segment boundary generates bidirectional joint-forming activity Based on our results, we propose a model in which jointforming activity is generated from the intersegmental fold in a bidirectional manner, and that an inappropriate signal having reverse polarity is blocked by Dve activity, and the PCP and EGFR signaling pathways (Fig. 7B). In our model, Dve activity is required to suppress Notch target genes, such as dAP-2, involved in joint formation. This is very similar to the situation observed in wing discs, where the Notch target gene wg is repressed by Dve in regions adjacent to the Notch-activated D– V boundary (Nakagoshi et al., 2002). It is an intriguing possibility that the vertebrate somite boundary generates similar bidirectional signals, and that the inhibition of either one is closely linked to the rostral–caudal polarity within a somite. Further characterization of Drosophila leg segmentation is needed to determine whether this model is applicable to vertebrate somitogenesis or other segmentation processes. Acknowledgments
Fig. 7. Schematic representation of gene expression in everting leg discs. (A) Notch signaling is activated in the distal region of each segment (green), and EGFR signaling is activated in a segmental pattern complementary to the Notchactivated region (red). At 4 h APF, Dve is expressed in regions both proximal and distal to the intersegmental folds (magenta). Subsequently, Dve is repressed in the Notch-activated region proximal to the fold at 6 h APF (magenta). The expression of bibNP4281 is observed where Dve is normally repressed (blue), and this region corresponds to the presumptive joint structure. The intersegmental folds are shown by arrowheads. The proximal (P)–distal (D) axis is indicated by double arrows. (B) The segment boundary is refined by a mutually antagonistic interaction between Notch and EGFR signaling, and generates bidirectional joint-forming activity (blue arrows). An inappropriate activity having reverse polarity should be blocked by Dve activity (T-shaped bar). Furthermore, the planar cell polarity (PCP) and EGFR signaling pathways are also required to suppress an inappropriate one (Bishop et al., 1999; Galindo et al., 2005). The PCP signaling pathway independent of bristle polarity is required for the maintenance of Dve expression (broken arrow), presumably due to the inactivation of Notch signaling together with Fringe (Fng) activity (Bishop et al., 1999; Fig. 6). Notch signaling in the bibNP4281 region relives the jointsuppressive activity of Dve at 6 h APF and induces joint structures.
polarity of extra joints, because Ser activates the Notch signaling pathway in two different directions: from proximal to distal for normal joints, and distal to proximal for extra joints. However, it seems unlikely that ectopic activation of Notch signaling is restricted at the center of a segment. A Notch-target gene, dAP-2, is autonomously activated in response to ectopic Notch signaling (Kerber et al., 2001), and, in pk mutants, ectopic dAP-2 expression has expanded on the distal side to the intersegmental fold, the most proximal but not the central region in a segment (Fig. 6I). Furthermore, the joint-suppressive activity of Dve is also required to repress dAP-2 expression on the distal side to the intersegmental fold (Fig. 5F). These results led us to favor the possibility that reverse joints are derived from the distally adjacent region to the intersegmental fold.
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