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First published online 9 July 2008
doi: 10.1242/dev.021766

Development 135, 2707-2716 (2008)
Published by The Company of Biologists 2008
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The Ecdysone-inducible zinc-finger transcription factor Crol regulates Wg transcription and cell cycle progression in Drosophila

Naomi Mitchell1, Nicola Cranna1, Helena Richardson2 and Leonie Quinn1,*

1 Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Melbourne, Australia.
2 Peter MacCallum Cancer Centre, East Melbourne 3002, Melbourne, Australia.


Figure 1
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  		Fig. 1. Crol interacts with the Wg-inducible cell cycle inhibitor Hfp. (A) Hfp is upregulated by Wg and inhibits G1- to S-phase transition by downregulating dm and mitosis via Stg. (B-D) Halving the dose of crol enhances the hfp overexpression wing phenotype. Adult wings for en-GAL4/+ control (B), en-GAL4, UAS-hfp/+ (C) and en-GAL4, UAS-hfp/crol[k05205]; (D). A tracing of the posterior compartment from en-GAL4, UAS-hfp/+ has been superimposed on the en-GAL4, UAS-hfp/crol[k05205] wing. (E-G) Crol antibody (red) on wild-type third instar wing disc, merged with DNA (blue, F) and with Wg (G, green). (H-J) UAS-crol;UAS-p35 flip-out clones marked with GFP (H), with Crol antibody (red, I) and merged with DNA (blue, J).

 

Figure 2
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  		Fig. 2. crol is required for cell cycling. (A-N,P-S) Clones generated using MARCM (positively marked with GFP). (A,B) 120-hour wild-type clones; (C,D) 120-hour crol-/- clones. (E) 120-hour crol mutant clones expressing UAS-crol. (F-J) crol clones die by apoptosis. (F) Apical section of 96-hour crol clones. (G) Basal section of the disc in H, counterstained with propidium iodide (red). (H) Higher magnification to show pyknotic nuclei with DAPI (blue) and (I) merged with GFP. (J) 120-hour crol-/- clones overexpressing UAS-p35. In B and D, discs have been co-stained with phalloidin-TRITC to distinguish the hinge from the pouch; the pouch is marked by a white line in A,C,E-G,J. (K-T) Cell cycle analysis of crol mutant clones in the UAS-p35 background. (K-N) 96-hour discs 48 hours after heat-shock. BrdU labelling (red) in control clones (K) and crol-/- clones (L) or PH3 staining (purple) in control (M) and crol mutant clones (N). (P,Q) crol-/- clones in 120-hour discs with BrdU (P) and a higher magnification (Q) to show cells flanking the ZNC, which corresponds to the white square in P. (R,S) crol-/- clones in 120-hour discs with PH3 and a higher magnification (S) to show mitotic cells adjacent to the ZNC, which corresponds to the white square in R. (O,T) Quantification of BrdU (O) and PH3 (T) in 96-hour discs. Counts are from equivalent clone areas (three sets of 70,000 pixels) from control (BrdU 488.6±24.3, PH3 52.0±3.2), crol-/- (BrdU 84.24±5.2, PH3 7.02±0.6) and crol-/-; UAS-p35 (BrdU 131.61±7.8, PH3 22.14±2.4). A statistically significant decrease was observed between crol-/- and control clones for BrdU (P<0.0001) and PH3 (P<0.0001), and for crol-/-; UAS-p35 and control clones BrdU (P<0.0001) and PH3 (P<0.0002).

 

Figure 3
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  		Fig. 3. Overexpression of Crol promotes cell cycle. (A,B) BrdU (red) of control en-GAL4, UAS-GFP/+. (C,D) BrdU of en-GAL4, UAS-GFP/UAS-Crol. (E,F) PH3 (purple) of control. (G,H) PH3 of the same disc for which BrdU is shown in C and D. In A-H, the arrow indicates the ZNC. (I,J) TUNEL labelling (red) of the basal layer of an en-GAL4, UAS-GFP/UAS-Crol disc and counterstained with DAPI (blue) in I. (K) en-GAL4, UAS-GFP/+;UAS-p35/+ control and (L) en-GAL4, UAS-GFP/UAS-Crol;UAS-p35/+. Discs in K and L are both taken at 10x magnification and stained with PH3 (red) and DAPI (blue). (M-P) 120-hour en-GAL4, UAS-GFP/UAS-Crol;UAS-p35/+ taken at 20x magnification compared with 40x for discs in A-H. (M) BrdU (red), (O) PH3 (purple). The merge with GFP and DNA (blue) is shown with BrdU in N and PH3 in P. In M-P, a white line marks the AC of the pouch. (Q-X) Cell cycle analysis of 120-hour discs with flip-out clones 72 hours after induction. (Q) UAS-p35 with BrdU (red), (R) UAS-crol;UAS-p35 with BrdU (red) and merged with DAPI in S. (T) Quantification of BrdU in 120-hour discs. Counts are from equivalent GFP-positive areas (three sets of 70,000 pixels) for UAS-p35 (249.7±14.2) and UAS-crol;UAS-p35 (509.8±10.9). (U) UAS-p35 with PH3 (purple), (V) UAS-crol;UAS-p35 with PH3 (purple) and merged with DAPI in W. (X) Quantification of PH3 (as above) from UAS-p35 (39.9±0.5) and UAS-crol;UAS-p35 (67.9±7.7). A statistically significant increase was observed between UAS-crol;UAS-p35 and UAS-p35 clones for BrdU (P<0.0001) and PH3 (P<0.0033).

 

Figure 4
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  		Fig. 4. Crol regulates G1-S and G2-M cell cycle genes. (A) Adult wings overexpressing UAS-crol with en-GAL4 and with the following cell cycle mutants: (B) dmPO, (C) cycEAR95, (D) stgAR2, (E) cycB2, (F) E2F1. A tracing of the PC from en-GAL4/+, UAS-crol/+ has been superimposed to show relative modification of compartment size. (G-I) β-gal (red) of 120-hour wing discs for PL35/+; en-GAL4,UAS-GFP/UAS-crol;UAS-p35/+. (J-L) β-gal (red) on 96-hour wing discs for stg-lacZ 6.4/+; Ptc-GAL4,UAS-GFP/UAS-crol; UAS-p35/+. (M) Control PCNA-GFP in the 120-hour wing disc with the PC marked by En (red). (N) en-GAL4/UAS-crol; UAS-p35/PCNA-GFP, merged with En antibody in red (O).

 

Figure 5
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  		Fig. 5. Crol inhibits Wg transcription. (A) crol overexpression wing phenotype alone and with (B) armYD35/+; (C) AxinE77/+ and (D) Sgg1/+. (E-G) crol mutant clones in the armYD35/+ background, positively marked with GFP and counterstained for DNA (red), compare with Fig. 2C,D. β-gal staining (red) for control en-GAL4, UAS-GFP/wg-lacZ (H-J) and en-GAL4, UAS-GFP/wg-lacZ; UAS-Crol/+ (K-M). (J,M) Merge with DAPI to show cells present in the apical region of the disc. Wg antibody (red) on control clones (N-P) and crol mutant clones generated in the Minute background (Q-S) are marked by the absence (arrows) of βgal (green). (T-Y) Arrows indicate crol clones adjacent to the ZNC (T-V) and the hinge (W-Y), and ectopic Wg in small clones are highlighted with asterisks (W-Y). (Z) Q-RT-PCR for wg mRNA in third instar larval imaginal tissues: Actin-GAL4/+ (1.0±0.21); Actin-GAL4/UAS-crol, (0.34±0.03). PCR was carried out in triplicate and normalized using GAPDH. The level of Wg mRNA is significantly reduced in Actin-GAL4/UAS-crol compared with Actin-GAL4/+ (P=0.0376).

 

Figure 6
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  		Fig. 6. crol overexpression results in downregulation of the cell cycle inhibitor Hfp. (A,C) Hfp antibody (red) on en-GAL4, UAS-GFP/+; (D,F) en-GAL4, UAS-GFP/+; UAS-Crol/+; (B,E) DNA (blue) to show the presence of cells.

 

Figure 7
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  		Fig. 7. Signalling via EcR is required for repression of Wg and cell cycle progression. (A,B) β-gal (red) for UAS-EcRdN flip-out clones in the wg-lacZ background. (C) BrdU (red) for UAS-EcRdN clones and (D) quantification of BrdU in 120 hour larval wings (see above); UAS-EcRdN clones (121.7±14.4) have significantly less BrdU than the control (246±19.7; P=0.0001). (E) PH3 (purple) for UAS-EcRdN clones and (F) quantification of PH3; UAS-EcRdN clones (13.0±4.0) have significantly less PH3 than the control (42.65±5.3; P=0.0001). (G) Model for Crol connecting steroid hormone signalling to cell cycle progression. Crol is upregulated in response to ecdysone signalling and increased Crol results in decreased wg mRNA expression. Reduced Wg signalling results in less Hfp, which leads to increased dm expression to drive S phase and mitosis via increased Stg.

 

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© The Company of Biologists Ltd 2008