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First published online 5 May 2004
doi: 10.1242/dev.01127

Development 131, 2681-2692 (2004)
Published by The Company of Biologists 2004
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Opposing inputs by Hedgehog and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc

Chulan Kwon1, Rebecca Hays2,*, Jennifer Fetting1,{dagger} and Teresa V. Orenic1,{ddagger}

1 University of Illinois at Chicago, Department of Biological Sciences, Chicago, IL 60607, USA
2 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL, USA



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  		Fig. 1. D-h expression is activated by a Hh-response element. (A) A third instar leg imaginal disc depicting expression of the DV-h stripes (blue) relative to ac expression (green) and the compartment boundary. The D-h and V-h stripes occupy distinct registers relative to the AP boundary. (B) (Top) map of the h locus showing the position of the D-h enhancer. Below is shown a more detailed map of the D-h enhancer. Fragments tested in reporter constructs are shown beneath the detailed map: full-length D-h enhancer (light blue) and the Hh response element (HHRE, dark blue), which has two Ci binding sites and the repression element (REPE, red; see text and Fig. 3). The CMB site in the REPE is described in the text and in Fig. 8. (C) Sequence of two Ci-binding sites present in the HHRE. Underlined bases were altered in the Ci1, Ci2 or Ci1&2 double mutant (see Materials and methods). (D,E) lacZ expression in leg imaginal discs carrying: D-h-lacZ (D) and HHRE-lacZ (E). (F-G) lacZ expression directed by HHRE-lacZ site mutants: Ci1 mutant (F), Ci2 (G) and Ci1&2 (H).

 


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  		Fig. 2. Hh signaling is required for function of the D-h activation element. Clones lacking smo function were examined in a prepupal leg, 3 hours APF. (A) HHRE-GFP expression (green). (B) myc expression (red). Homozygous smoIIG26 clones are identified by the lack of Myc expression. (C) Merge of images in A and B (several smo-clones that overlap the HHRE-GFP stripe are outlined in white). Note the absence HHRE-GFP expression in smo-clones

 


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  		Fig. 3. Ci binds the D-h activation element, while Brk and Mad bind the repression element. Binding of the Ci DNA-binding domain to the Ci1 site in HHRE (lanes 1-7) and binding of full-length Brk or GST-N-Mad protein to the CMB element (lanes 8-27) were tested by EMSA. Sequences of Ci1 site wild-type (Ci1 wt), CMB site wild-type (CMB wt) and the following mutant probes are shown below: Ci1 site mutant (Ci1 mut), Brk site mutant (Brk mut), Mad site double-mutant (Mad dmut), Mad site single-mutant (Mad smut), CRE mutant (C mut), Mad2 site mutant (M2 mut), Brk/Mad2 site mutant (BM2 mut) and Mad1/Mad2 mutant (MM mut). Putative binding sites are underlined on the wild-type sequences. Lanes 1-7, sequence-specific binding of Ci protein to the Ci1 site; lane 1, Ci1 wild-type probe and no protein; lanes 2-6, Ci1 wt probe + Ci protein and no competitor (lane 2), 10x specific competitor (Ci1 wt oligo) (lane 3), 100x specific competitor (lane 4), 10x nonspecific competitor (Ci1 mut oligo) (lane 5), and 100x nonspecific competitor (lane 6). Lane 7, Ci1 mut oligo + Ci protein; lanes 8-14, sequence-specific binding of Brk protein to the CMB element; lane 8, CMB wt probe and no protein; lanes 9-13, CMB wt probe + Brk protein and no competitor (lane 9), 10x specific competitor (CMB wt oligo) (lane 10), 100x specific competitor (lane 11), 10x nonspecific competitor (Brk mut oligo) (lane 12), 100x nonspecific competitor (lane 13). Lane 14, Brk mut probe + Brk protein; lanes 15-22, sequence specific binding of Mad protein to the CMB element; lane 15, CMB wt probe and no protein; lanes 16-22, CMB wt probe + Mad protein and no competitor (lane 16), 10x specific competitor (CMB wt oligo) (lane 17), 100x specific competitor (lane 18), 10x nonspecific competitor (Mad dmut oligo) (lane 19), 100x nonspecific competitor (lane 20). Lane 21-22, Mad protein + Mad dmut probe (lane 21) or Mad smut probe (mutation is in Mad2 site) (lane 22). Lanes 23-27, Brk protein binding to CMB wt probe (lane 23), C mut probe (lane 24), M2 mut probe (lane 25), BM2 mut probe (lane 26) and MM mut (lane 27).

 


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  		Fig. 4. A D-h repression element attenuates activity of the activation element. (A-F) Comparison of endogenous Hairy [red in A,D (arrowhead indicates endogenous D-h stripe)] with D-h-GFP [green in B (arrowhead indicates D-h-GFP stripe)] and HHRE-GFP (green in E) expression in prepupal legs, 3 hours APF. (C) Merge of images in A and B. (F) Merge of images in D and E. en expression (red in G-J) relative to D-h-GFP (green in H) and HHRE-GFP (green in J) in prepupal legs 5 hours APF.

 


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  		Fig. 5. Dpp signals through a D-h repression element and defines the domain of D-h expression. D-h-GFP (A) and HHRE-GFP expression (B) in wild-type leg imaginal discs. The insets in A, B and I show GFP expression in the basitarsal segment of a 4-hour prepupal leg. (C-E) Comparison of D-h-GFP expression (C) with dpp-lacZ expression (D). (E) Merge of images in C and D. (F,G) In legs with reduced dpp function (dppd6/dppd12), D-h-GFP expression (F) is significantly compromised, while HHRE-GFP expression appears normal (G). (H) GFP expression in a UAS-GFP/+; dpp-Gal4/+ leg imaginal disc. (I) In UAS-tkvQD/+; dpp-Gal4/+ legs, Dpp signaling is elevated within dpp-expressing cells, resulting in partial ventral expansion and widening of the D-h-GFP stripe.

 


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  		Fig. 6. Brk is a potential D-h repressor. (A) D-h-GFP expression (green) relative to brk-lacZ expression (red) in a wild-type leg disc. (B,C) brk-lacZ expression in a wild-type leg disc (B) and a dpp hypomorph (dppd6/dppd12) leg disc (C). (D,G) Overexpression of brk, in UAS-brk/+; dpp-Gal4/+ legs, results in drastic reduction of D-h-GFP expression (D) and D-h-C-GFP (G), while HHRE-GFP (E) and D-h-CB-GFP are still expressed (F). It appears that in legs overexpressing brk, there are fewer HHRE-GFP and D-h-CB-GFP-expressing cells. This is probably due to compromised growth of leg discs when Brk levels are elevated near the AP boundary (Jazwinska et al., 1999Go).

 


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  		Fig. 7. Brk represses D-h expression. Clones lacking brk function were examined in a prepupal leg, 3 hours APF (A-C) and in a third instar leg disc (D-F). brk– clones exhibit ectopic GFP expression anterior to the D-h-GFP stripe (A-C) and in the ventral leg (D-F). (A,D) D-h-GFP expression (small nuclei, green). Clones are outlined in white in all panels. White line marks the anterior boundary of D-h-GFP expression in A-C. Large GFP-expressing nuclei (arrowheads) probably correspond to adepithelial cells. (B,E) myc expression (red). brk– clones are marked by the loss of myc expression. (C) Merge of images in A and B. (F) Merge of images in D and E.

 


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  		Fig. 8. A Brk response site in the D-h repression element is not sufficient for D-h repression. (A) Sequence of CMB element and CMB mutants: Mad2 (M2), Brk/Mad2 (BM2), Mad1/Mad2 (MM), Mad1/Brk/Mad2 (MBM), CRE/Brk (CB), CRE (C) and Mad1/CRE/Mad2 (MCM). Mutagenized bases are shown in lower case. (B-G) D-h-GFP directed by D-h-CMB site mutants: M2 (B), BM2 (C), MM (D), MBM (E), CB (F) and C (G). The insets in all panels show GFP expression in the basitarsal segment of a 4-hour prepupal leg.

 


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  		Fig. 9. Model for D-h regulation. Ci acts through the HHRE to activate expression in a broad AP boundary stripe that can be divided into zones 1 (blue) and 2 (red). The intact D-h element directs expression only in zone 1. Brk and perhaps a second factor, X (see text), act through the CMB to repress D-h expression in zone 2. In zone 1, Dpp signaling prevents Brk repression of D-h expression. Dpp function might be restricted to repression of brk expression in zone 1. However, it also possible that Mad acts more directly through the Mad-binding sites.

 

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