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First published online 9 January 2008
doi: 10.1242/dev.014670

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Logic of Wg and Dpp induction of distal and medial fates in the Drosophila leg

Department of Biochemistry and Molecular Biophysics, Columbia University, 701 West 168th Street, HHSC 1104, New York, NY 10032, USA.

View larger version (102K): [in this window] [in a new window]   Fig. 1. brk is not required for DV axis specification. (A) Schematic representation of a third instar leg imaginal disc, summarizing the functions of Dpp and Wg in DV and PD axis development. The Wg (orange), Dpp (blue), Dll (green), Dll+Dac (yellow) and Dac-only (red) domains are indicated for a third instar leg disc. The Dac-only domain is larger in the dorsal disc. While Dpp promotes dorsal fates and Wg promotes ventral fates, Dpp and Wg act combinatorially to establish the PD axis. (A') Expression of Dll (green) and dac (red) in a third instar leg disc. All images of leg discs are oriented anterior leftwards and dorsal upwards. (B) A wild-type third instar leg disc stained for brk-lacZ (red), Wg (green) and P-Mad (blue). brk-lacZ expression is reciprocal to P-Mad, which is highest dorsally but is also weakly observed ventrally. The inset shows brk expression, which is weakly downregulated in ventral cells at this stage. (C-F'') brk- clones (absence of GFP, arrows) have no effect on dpp-lacZ (C-C''), wg-lacZ (D-D''), omb-lacZ (E-E'') or H15-lacZ (F-F''). Engrailed (En, blue) marks the posterior compartment. The ' and '' panels show enlargements of the clones as indicated.

View larger version (104K): [in this window] [in a new window]   Fig. 2. brk is required for PD axis specification. (A) A brk- clone originating in the ventral proximal femur marked by yellow and forked (arrow, outlined in white) creating a cell-autonomous outgrowth. (A') The entire outgrowth appears to have a single, tibia-like DV identity. co, coxa; tro, trochanter; fem, femur; tibia, tib; tib', tibia-like outgrowth; t, tarsal segments 1-5; cl, tarsal claw. (B) brk- clone proximal to the Dll and Dac domains (absence of GFP, arrow) showing cell-autonomous derepression of Dll and dac. No effects on Dll or dac are observed in brk- clones within their normal domains (asterisks). (C-D''') gro- (C) or CtBP- (D) clones (absence of GFP, green) derepress Dll and dac in proximoventral regions of the disc (arrows and clones labeled `2'; isolated clones are outlined in white and shown in C'', C''', D'' and D'''). No derepression is observed in dorsal gro- or CtBP- clones (arrowheads and clones labeled `1'; isolated clones are outlined in yellow and shown in C' and D'). Clones within the Dac or Dll domains (outlined, but not numbered) have no effect on Dac or Dll expression.

View larger version (78K): [in this window] [in a new window]   Fig. 3. A PD axis does not require Dpp signaling in the absence of Brk. (A) dppdiscs mutants have very small discs and the normal distal and medial Dll and dac domains are absent. The Dll and dac expression that remains corresponds to the Dpp- and Wg-independent trochanter region (arrow), which also expresses Homothorax (Hth, blue). (B) brkXA discs are overgrown, and the Dll and dac expression domains are expanded in the ventral and lateral parts of the disc. (C) brkXA; dppdiscs discs are overgrown and Dll, Dll+Dac and Dac expression domains are all present. Dll expression is expanded dorsally and the dorsal Dac-only domain is not present (arrow). Dac expression is expanded laterally compared with wild type (asterisks; compare with Fig. 1A'). (D) In brkXA; dppdiscs discs, wg (red) is expressed along the entire DV axis. Dll is also expanded dorsally, following the dorsal Wg expression (arrow). (E) In brkXA; dppdiscs discs, the distal domain of al is not present (arrow), whereas other, Dpp- and Wg-independent domains of al are still present (compare with wild type, F). (F) Expression of al in a wild-type disc. The distal al domain is indicated by the arrow.

View larger version (99K): [in this window] [in a new window]   Fig. 4. Dll and dac expression in the absence of Dpp signaling and brk. (A-A'') Mitotic clones mutant for brk (arrow; marked by the absence of GFP, green, and outlined in red in A'') and tkv [arrow; marked by the absence of arm-lacZ (red) and outlined in yellow in A''] derepress Dll. These clones were induced separately and therefore result in independent clonal events. Cells mutant for both genes show no GFP (green) and no β-gal staining (red) (A'). (A'') Dll is derepressed cell autonomously throughout the brk- clone (marked by the red line), including cells that are also tkv- (marked by the yellow line). (B-B'') Cross-section of the same disc in A showing that the brk-; tkv- clone is proximal to the Dll expression domain (arrow, outlined in yellow). (B'') Schematic representation of the cross-section shown in B. Dll expression is shown in gray and the yellow arrow marks the region where Dll is derepressed (dark gray). tr, trocanter. (C-C'') A proximal clone doubly mutant for brk (absence of GFP, green) and tkv (absence of arm-lacZ, green) showing derepression of Dll (arrowhead, red) and dac (arrow, blue). These clones were induced at the same time, and are therefore congruent. (D-D') Cross-section of the same disc as in C showing that the brk-;tkv- clone (outlined in yellow) is proximal to the Dll and dac expression domains.

View larger version (104K): [in this window] [in a new window]   Fig. 5. Different levels of Wg signaling activate Dll and dac. (A) Expression of Dll (green), dac (blue) and brk (red) in a wild-type third instar leg disc. The individual channels are shown below. (A') Expression of Dll (green) and Dac (blue) in a wild-type third instar leg disc. (B-B''') In wgCX4/CX3 discs, the Dll (green) domain is reduced in size compared with wild type (compare with A) and the Dac (blue) domain is expanded ventrally (arrow). brk is repressed in the ventral domain because of dpp derepression (data not shown). (C-C''') In brkXA;wgCX4/CX3 discs, the Dll domain is reduced in size compared with wild type and the Dac domain is nearly circular. Compare discs in B and C with a wild-type disc in A and A'. The Dll-only domains in leg discs of this genotype and in wgCX4/CX3 discs vary from small (as in the example in B) to its complete absence (as in the example in C). (D-E'') brk-; tub>axin+ clones marked positively by lacZ (green) grown at different temperatures. (D) At 25°C, axin levels are sufficient to block both Dll and dac derepression (arrow). Compare with the brk- clone in Fig. 2B, showing both Dll and dac derepression. (D'') Cross-section of the same disc as in D showing the location of the clone (broken line, arrow). (E) Leg disc of the same genotype in D grown at 17°C. The Wg signaling that remains permits dac but not Dll derepression (arrow). Approximately one-third of these clones show dac only derepression like the clone shown here, a phenotype that is never observed in brk- clones. Approximately two thirds of these clones show derepression of both genes similar to the clone shown in Fig. 2B. (E'') Cross-section of the same disc in E showing the location of the clone (broken line, arrow).

View larger version (72K): [in this window] [in a new window]   Fig. 6. Different levels of Brk activate Dll and dac. (A-B'') brk-; tub>brk+ clones (lacZ, green) grown at different temperatures. (A) At 25°C, the levels of Brk are sufficient to prevent both Dll and dac derepression (arrow). Compare with the brk- clone in Fig. 2B, where both Dll and dac are derepressed. (B) At 17°C the levels of Brk are sufficient to prevent Dll derepression but not dac derepression (arrow). Approximately half of the clones examined with this genotype showed dac, but not Dll, derepression like the clone shown here, a phenotype that was never observed in brk- clones. The other (approximately) half of these clones failed to derepress either Dll or dac. The ' and '' panels show the single Dll or Dac expression patterns, respectively.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Temporal progression of Dll and dac expression during leg development. (A-C) Expression of brk, Dll and dac during leg development. The discs are not shown to scale. (A) At 60 hours AEL Dll (green) is present in the center of the disc where there is no detectable Brk (red). In the ventral and lateral regions of the disc (arrow), there is a sharp border that separates Dll and brk expression. Wg (blue) is highest in the ventral region of the disc but is also observed in Dll-expressing cells. (B) At about 72 hours AEL, dac (blue) is broadly expressed in the dorsal part of the leg disc where there is no brk (arrowhead) and in the lateral and ventral regions where there are low levels of Brk (arrow). In the cells where dac is activated, Dll is also expressed at low levels. The ' and '' panels show subsets of these expression patterns as indicated. (C) At about 108 hours AEL, the expression of Dll and dac overlaps with brk. At this time these genes are refractory to Brk repression. (D) In the early second instar (left), Dll is activated in the center of the disc where there is no Brk and high Wg levels. In a late second instar disc (middle), dac is activated: (1) in the dorsal region of the disc where there is no Brk and low levels of Wg signaling (region 1); and (2) in lateral and ventral cells, where there is high Wg signaling and low Brk (region 2). In late third instar (right), Dll and dac expression is maintained and become refractory to Brk repression. The bottom set of schematics illustrate the unique Brk:Wg ratios required to activate Dll and dac. For dac activation, two ratios are suggested.