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First published online March 1, 2004
doi: 10.1242/10.1242/dev.01019

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Drosophila Hfp negatively regulates dmyc and stg to inhibit cell proliferation

¹ Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne, VIC 3002 Australia
² ARC Centre of Excellence in Biotechnology and Development, University of Melbourne

View larger version (97K): [in a new window]   Fig. 1. Hfp negatively regulates cell cycle progression. (A) A wild-type third instar larva (left) alongside an hfpEP/hfpEP late third instar larva (right). (B) A wild-type pharate adult (left) alongside a hfpEP/hfpEP pharate adult (terminal phenotype; right). (C) Time line of the developmental delay observed in hfpEP/hfpEP animals compared with wild type. The vertical bar indicates the stage at which hfp mutants arrest in development and die. (D) Northern blot of poly(A)+ RNA isolated from the developmental stages shown, and probed with the hfp cDNA, then stripped and re-probed with the ribosomal protein rp49 cDNA as a loading control. (E) Northern blot of poly(A)+ RNA isolated from wild-type and hfp mutant larvae, probed with the hfp cDNA and Actin5c cDNA as a loading control. (F-N) Wing imaginal discs from wandering third instar larvae. Posterior is to the right, and the left margin of the ZNC is marked with a yellow bar. Discs shown are representative samples of at least 30 discs examined for each condition. (F,G) Wild type disc co-stained with anti-Geminin antibody (F) and anti-Hfp antibody (G). Geminin is present in late S-phase and G2 cells, but absent from G1-arrested cells (Quinn et al., 2001). (H) Anti-Hfp antibody staining of a hfpEP/hfpEP larval wing disc. (I-N) Wing discs from wild type (I-K) and hfpEP/hfpEP (L-N) larvae co-labelled with BrdU (I,L), anti-phosphohistone H3 antibody (PH3) (J,M) or merged (K,N). (O-T) Third instar eye imaginal discs from wild-type (O-Q) and hfpEP/hfpEP (R-T) larvae co-labelled with BrdU (O,R), PH3 (P,S) or merged (Q,T). The morphogenetic furrow (MF) is indicated by a yellow bar and arrows indicate the normal position of the S-phase band posterior to the MF. (U,V) Cell size visualized by spectrin staining of wild type (U) and hfpEP/hfpEP (V) wing discs. (W,X) TUNEL staining of wild-type (W) and hfpEP/hfpEP (X) wing discs, revealing elevated apoptosis in hfp mutant tissue.

View larger version (102K): [in a new window]   Fig. 2. Overexpression of hfp inhibits cell cycle entry in the developing eye and wing. (A,B) Scanning electron micrographs of adult eyes of wild type (A) and GMR-GAL4, UAS-hfp/+; UAS-hfp/+ (B). Scale bar equals 200 µm. (C-F) Eye imaginal discs from wild type (C,E) and GMR-GAL4,UAS-hfp/+; UAS-hfp/+ (D,F) third instar larvae, co-labelled with BrdU (C,D) and anti-phosphohistone H3 antibody (E,F). Posterior is to the left. Yellow bars indicate the MF. (G-J) Adult wings mounted in Canada balsam (G-I) or fresh (J) from en-GAL4,UAS-GFP (G) and en-GAL4,UAS-GFP/+,UAS-hfp/+ (H-J) flies. (K-N) Third instar wing discs from en-GAL4,UAS-GFP (K,M) and en-GAL4,UAS-GFP/+,UAS-hfp/+ (L,N) flies, co-labelled using GFP antibody staining to mark the posterior region of the wing disc (K,L) and BrdU (M,N). The ZNC is marked with an arrow, and in (N) the GFP-positive region is outlined in white.

View larger version (75K): [in a new window]   Fig. 3. hfp mutation suppresses the GMR-p21 eye phenotype by promoting cell cycle entry. (A-C) Scanning electron micrographs of adult eyes from wild-type (A), GMR-p21/+ (B) and GMR-p21/+; hfpEP/+ (C) flies. Scale bar equals 200 µm. (D-L) Eye imaginal discs from wild-type (D,G,J), GMR-p21/+ (E,H,K) and GMR-p21/+; hfpEP/+ (F,I,L) larvae, co-labelled with BrdU (D-F) and PH3 antibody (G-I). Merged images are shown (J-L). Posterior is to the left. The MF is indicated by a yellow bar and arrows indicate the normal position of the S-phase band posterior to the MF. (M-R) Scanning electron micrographs of adult eyes, to show genetic interactions between p21/Dacapo and dMyc; (M) GMR-p21/+ males, (N) dmycP0/+;GMR-p21/+ females, (O) dmycP0/y; GMR-p21/+ males, (P) GMR-GAL4/+, UAS-dacapo/+, (Q) GMR-GAL4/+; UAS-dmyc/+, (R) GMR-GAL4/+, UAS-dacapo/+; UAS-dmyc/+.

View larger version (80K): [in a new window]   Fig. 4. hfpEP dominantly suppresses the dmyc mutant ovary phenotype and dmyc expression is increased in hfp mutant clones. (A-T) All ovarioles are oriented with the most mature/posterior egg chamber to the right. nc=nurse cells, af=actin filament bundles, fc=follicle cells. (A-B) Ovaries stained with phalloidin (red) to show filamentous actin and (C-H) with phalloidin and the DNA stain Oligreen. Egg chamber genotypes and stages: wild-type stage 10 (A), dmycP0/dmycP0 stage 10 (B), wild-type stage 11 (C), dmycP0/dmycP0 stage 11 (D), dmycP0/dmycP0; hfpEP/+ stage 11 (E), wild-type stage 14 (F), dmycP0/dmycP0 arrested at stage 11 (G), dmycP0/dmycP0; hfpEP/+ stage 14 (H). (I-N) Ovarioles containing stage 10B egg chambers, labelled with BrdU (green) to visualize chorion gene amplification and counterstained with the DNA stain propidium iodide (red in L-N). Genotypes: wild type (I,L), dmycP1/dmycP1 (J,M), dmycP1/dmycP1; hfpEP/+ (K,N). (O-T) Ovarioles containing stage 10 egg chambers, showing in-situ hybridization to dmyc mRNA (red) and counterstained with Oligreen. Genotypes: wild type (O,R), dmycP0/dmycP0 (P,S), dmycP0/dmycP0; hfpEP/+ (Q,T). (U-Z) Analysis of dmyc mRNA in third instar wing discs, the ZNC is marked with a yellow bar. (U) wild-type dmyc in-situ pattern, (V-Z) hs-FLP/+; FRT80BhfpEP/FRT80B Tb-GFP, (V) hfpEP/hfpEP clones marked by the absence of GFP antibody staining and outlined in white, (W) dmyc mRNA expression in hfp mutant clones, (X) merged image. (Y,Z) high power images of hfpEP/hfpEP mutant clones; (Y) GFP antibody staining and (Z) dmyc in situ.

View larger version (126K): [in a new window]   Fig. 5. Hfp negatively regulates G2-M progression. (A-F) Wild type, (G-L) GMR-GAL4/+, UAS-dmyc/+ and (M-R) GMR-GAL4/+, UAS-dmyc/hfpEP. Cells posterior of the morphogenetic furrow stained with the nuclear stain PI in red (A,G,M) and for cell size with spectrin in green (B,H,N). Scanning electron micrographs showing ommatidia at high power (C,I,O) and the overall size of the adult eye (D,J,P). Analysis of cell cycle progression posterior of the MF in third instar larval eye discs using BrdU (E,K,Q) and anti-phosphohistone H3 (F,L,R). The MF is indicated with a yellow bar.

View larger version (82K): [in a new window]   Fig. 6. Hfp negatively regulates Stg. (A-H) Cycle 16 embryos stained with anti-phosphohistone H3 in green to detect mitotic cells and anti-Actin in red to show cell cortex. (A,B) wild type, (C,D) hfpEP mutant embryos (E,F) stgAR2 mutant embryos and (G,H) hfpEP, stgAR2 double-mutant embryo. (I-N) Wing discs from third instar hs-FLP; FRT80BhfpEP/FRT80BTb-GFP flies. (I,L) hfp mutant clones are marked by the absence of GFP and outlined in white, (J) in-situ hybridization of stg mRNA, (K) in situ merged with GFP, (M) staining with anti-stg antibody and (N) stg-antibody staining merged with GFP.

View larger version (108K): [in a new window]   Fig. 7. Activation of the Wg pathway causes induction of Hfp in wing discs. Hfp expression in wing discs from larvae of the following genotypes: en-GAL4/+,UAS-GFP/+ (A-C), en-GAL4/+, UAS-GFP/+;UAS-hfp/+ (D-F), en-GAL4/+,UAS-GFP/+;UAS-sggDN/+ (G-I). (A,D,G), GFP (green) marks the posterior region of the wing disc, (B,E,H) anti-Hfp antibody staining (red), and (G,F,I) are merged images. (J-L) Hfp expression in axin mutant clones from third instar wing discs, (J) clones marked by the absence of GFP, (K) anti-Hfp antibody staining and (L) merged image. (M) Hfp expression and GFP in the ZNC of control C96-GAL4/+, UAS-GFP/+ wing discs, (N) Hfp expression in C96-GAL4/+, UAS-GFP/+, UAS-TCFDN/+ and (O) merge with GFP.

View larger version (24K): [in a new window]   Fig. 8. Model for Wg signalling through Hfp during Drosophila development. The Wg signalling pathway has a role in tissue patterning and is also required to downregulate dmyc expression and limit cell proliferation in the ZNC during wing development via repression of dmyc expression (Johnston et al., 1999). Our results suggest that Hfp may link Wg signalling to the control of cell growth and proliferation by repressing dMyc expression (see text). Wg signalling is also required to induce the domain of G2-arrested cells in the ZNC, via upregulation of achaete and scute, which in turn downregulate stg (Johnston and Edgar, 1998). Our data is consistent with Wg signalling upregulating Hfp, which would then play a role in negatively regulating stg post-transcriptionally and thereby leading to a G2-arrest.