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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01315

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Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA

View larger version (51K): [in a new window]   Fig. 1. Proximodistal wing patterning. (A) Adult wing: distal (green), proximal wing and hinge (blue), and WG-expressing cells (red). The boundary between distal and proximal cells is an approximation. We adopt the term proximal wing for the blue region, but note that this entire region is sometimes referred to as the wing hinge. (B) Relative gene expression domains along the proximodistal axis, related to C by the dashed lines. Although for simplicity all genes are shown as having uniform expression levels, some are subject to modulation within their spatial domains (this work) (Williams et al., 1991; Clark et al., 1995; Villano and Katz, 1995; Brodsky and Steller, 1996; Ng et al., 1996; Rieckhof et al., 1997; Azpiazu and Morata, 2000; Casares and Mann, 2000; St Pierre et al., 2002; Wu and Cohen, 2002; Kolzer et al., 2003; Whitworth and Russell, 2003). (C) A wing imaginal disc, shaded as for the adult wing shown in A. Approximate location of DPP-expressing cells (yellow) is also indicated.

View larger version (81K): [in a new window]   Fig. 2. fat mutant clones upregulate targets of distal-to-proximal signalling. In this and subsequent figures, all panels show third instar wing discs, oriented with ventral down and anterior left, and panels marked prime show separate stains of the same disc. Clones of cells mutant for fat8 are marked by the absence of MYC (green). Arrows indicate clones with ectopic gene expression. Discs are stained for WG (red), rn-lacZ (blue/white in C, red in E), spd-fg-lacZ (blue/white in D, red in F), and NUB (blue). (A) Early third instar disc. (B) Late third instar disc. (C) Mid-third instar disc. Ectopic WG expression is associated with an expansion of the rn domain. (D) Mid-late third instar disc. The spd-fg enhancer and endogenous WG are both ectopically expressed, although differences in subcellular localization result in apparent differences within a focal plane. (E) Late third instar disc with a fat clone that extends beyond the NUB domain; arrows here point to the edges of the clone where it extends proximally; rn is induced only within NUB-expressing cells. (F) Mid-third instar disc with a fat clone in the NUB domain with spd-fg-lacZ expression (arrow), and a clone just proximal to this without spd-fg-lacZ expression (asterisk).

View larger version (134K): [in a new window]   Fig. 3. WG expression in mutant discs. Late third instar discs, stained for WG (red), with inner (arrow) and outer (arrowhead) rings marked. (A) vg83b27r. (B) fat8 vg83b27r. (C) fjd1 dsUA071. (D) fat8/fatG-rv. Because the disc is more folded (see Fig. 4), WG expression is only partially visible.

View larger version (71K): [in a new window]   Fig. 4. wg and dachs are required for overgrowth in fat mutant discs. Discs stained for VG (green) and NUB (magenta); discs shown in A-F are at 36-48 hours of third instar, those in G-H are at 48-60 hours. Cells that express only NUB correspond to the distal half of the proximal wing (Fig. 1). (A) Wild-type. (B) fat8/fatG-rv. (C) wgspd-fg. (D) wgspd-fg fat8/wgspd-fg fatG-rv. (E) dachs1. (F) fat8 dachs1. (G) fat8/fatG-rv disc, the overgrowth of the proximal wing is even more pronounced at this age, and the wing becomes highly folded. (H) wgspd-fg fat8/wgspd-fg fatG-rv. Wild type and fat8 dachs1 are not shown at this age, as they begin to pupate. Scale bar in B: 80 µm for A-H.

View larger version (129K): [in a new window]   Fig. 5. Expression of four-jointed and dachsous during wing development. Expression of VG (green), fj-lacZ (cyan), ds-lacZ (green), DS (green) and WG (red) are shown. (A) Early (0-12 hours of third instar) disc, stained for VG and fj-lacZ. (B) Early third instar disc, stained for ds-lacZ and WG. White bars identify WG expression in the proximal wing. Images to the right of the dashed line show different channels of vertical sections of the same disc. (C) Mid-late third instar disc (24-36 hours) stained for VG and fj-lacZ. Asterisks highlight proximal regions where VG expression remains elavated relative to fj. (D) Early third instar disc, stained for DS and WG. DS protein is predominantly apical (arrow). (E-G) Early (E), mid (F) and late (G) third instar discs, stained for WG and fj-lacZ.

View larger version (107K): [in a new window]   Fig. 6. Scalloped-Vestigial regulates four-jointed expression. (A) Mid-third instar disc with sd58 clones, marked by the absence of MYC (green), and stained for fj-lacZ (magenta). Arrows indicate examples of clones with reduced fj expression. (B) Mid-late third instar disc with VG-expressing clones, marked by GFP (green), and stained for fj-lacZ (blue/white) and WG (red). Arrow indicates a clone with ectopic fj.

View larger version (117K): [in a new window]   Fig. 7. Four-jointed can influence gene expression in the proximal wing. FJ-expressing clones are marked by GFP (green). (A) Late third instar disc, stained for fj-lacZ (blue/white) and WG (red). Arrow points to a clone that induces WG expression in flanking cells in the proximal wing. No induction of fj occurs. (B) Mid-late third instar disc, stained for VG (blue/white) and WG (red). Arrows point to clones that induce WG. No induction of VG occurs. (C) Mid-third instar disc, stained for expression of rn-lacZ (blue/white) and WG (red). Arrow indicates induction of WG and rn flanking a clone. Their expression is also decreased within the FJ-expressing cells. (D) Late third instar disc, stained for rn-lacZ (blue) and WG (red). Arrows point to clones that alter WG expression. (E) Mid-late third instar disc, stained for NUB (blue) and spd-fg-lacZ (red). spd-fg-lacZ is induced only up to the edge of the NUB domain, even though the clone (arrow) extends proximally. Although spd-fg-lacZ expression is broader and more diffuse than endogenous WG (Neumann and Cohen, 1996), it does not extend to the edge of NUB expression in the absence of ectopic FJ. (F) Mid-third instar disc, stained for NUB (blue) and rn-lacZ (red). rn-lacZ is induced only up to the edge of the NUB domain, even though the clone (arrow) extends proximally. (G) Early third instar disc, stained for Fat (magenta). Fat is tightly localized apically; because the disc is not flat this figure is a composite projection of different focal planes to allow visualization of Fat over a broad region. Fat appears to be concentrated along the edge of the clone (arrow).

View larger version (118K): [in a new window]   Fig. 8. four-jointed influences the initiation of WG expression in the proximal wing. fjd1 mutant clones, made using the Minute technique and marked by absence of ß-galactosidase (green). (A) Early third instar disc. WG expression is reduced within large fj mutant clones (arrows). The requirement for fj is non-autonomous, as fj mutant cells in the proximal wing express WG normally (asterisk). Arrowhead identifies elevated WG expression in cells immediately adjacent to wild-type cells. (B) Mid-third instar disc, arrows point to reduced expression. (C) Mid-late third instar disc, with most tissue mutant. WG expression is no longer noticeably reduced by absence of fj (asterisk).

View larger version (63K): [in a new window]   Fig. 9. Dachsous influences WG expression. Discs stained for WG (red), Fat (magenta), MYC (green) or GFP (green). (A) Early third instar disc with dsUA071 mutant clones, marked by absence of GFP. In some cases, WG is relatively decreased within clones (asterisk), and relatively increased in flanking wild-type cells (arrows). (B) Late third instar disc with dsUA071 mutant clones, marked by the absence of MYC. WG expression is increased within a clone (arrow). (C) Early to mid-third instar disc with clone overexpressing DS, marked by GFP. WG appears elevated in cells at the edge of the clone (arrows), and slightly decreased in more internal cells. (D) Mid-third instar disc with clone overexpressing DS, marked by GFP. Ectopic expression of WG is detectable outside the clone (arrows). (E) Clone overexpressing DS and FJ, marked by GFP. Arrows point to examples of ectopic WG. Asterisk indicates a region where WG expression is out of the plane of focus. (F) Early-mid third instar with clones overexpressing DS, stained for Fat. Image is a composite of projections through different focal planes. Fat appears to accumulate at the clone border (arrow), and to be depleted from neighboring cells.

View larger version (88K): [in a new window]   Fig. 10. Dachs is required for distal-to-proximal signalling. Discs stained for WG (red), with mutant clones marked by the absence of MYC or GFP (green). Arrows point to clones with reduced WG, asterisks mark clones with essentially normal WG. (A) gug35 mutant clones. (B) Early third instar disc with dachs1 Minute clones. (C) Mid-late third instar disc with dachs1 clones. (D) Early third instar disc with fat8 dachs1 clones. (E) Mid-third instar disc with fat8 dachs1 clones. WG expression is still reduced in these clones, but is starting to recover.

View larger version (24K): [in a new window]   Fig. 11. Models for Fat and distal-to-proximal signalling. (A) Analysis of WG regulation, together with studies of tissue polarity, imply that Fat activity is modulated by the juxtaposition of cells with different levels of FJ or DS activity. Both normal expression patterns and analysis of genetic mosaics imply that at FJ expression borders, Fat is inhibited in cells with less FJ, and activated in cells with more FJ. The effects of DS are more variable, but in some cases Fat is inhibited in cells with more DS, and activated in cells with less DS. Fat functions normally to inhibit WG expression. As Dachs is required for WG, and is epistatic to Fat, the simplest genetic pathway would have Fat antagonizing Dachs activity. Fat regulates some processes via Grunge; however, it is not currently known whether these also require Dachs. (B) SD-VG specifies distal wing fate, and is regulated by Notch, WG and DPP signaling. We hypothesize that FJ acts redundantly with some other gene (X), which would also be regulated by SD-VG, and which would also act through Fat to regulate WG in the proximal wing. We further suggest that DS might be repressed by WG and DPP independently of SD-VG regulation, providing an additional input into Fat signaling. Induction of WG also appears to require NUB, and to be repressed distally.