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First published online 2 June 2004
doi: 10.1242/dev.01172

Development 131, 3169-3181 (2004)
Published by The Company of Biologists 2004
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The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions

Halyna R. Shcherbata*, Cassandra Althauser*, Seth D. Findley{dagger} and Hannele Ruohola-Baker{ddagger}

Department of Biochemistry, University of Washington, J591, HSB, Seattle, WA 98195-7350, USA



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  		Fig. 1. Cell cycle transitions in epithelial follicle cells. (A) Follicle cells in Drosophila oogenesis undergo two cell cycle transitions: from a mitotic cell cycle to an endocycle and from endocycle to amplification (A, parts I-III, drawing of the stages of oogenesis). From the germarium (g) to stage (st) 6, somatically derived follicle cells undergo mitotic cycles, which are not synchronized (A, part I). At stage 7 they switch to endocycles. From stage 7 to stage 10A, these cells undergo three rounds of endoreplication (A, part II) and thereafter switch to the localized replication pattern characteristic of chorion gene amplification (A, part III). These three replication patterns are observed in BrdU incorporation analysis (B). The staining of mitotic markers Cyclin B (CycB) (red arrows), Phospho-Histone 3 (PH3) (green arrows) (C) and stg (6.4) promoter construct (D) show expression in mitotic follicle cells, whereas Fzr enhancer trap line, fzrG0326 (ß-gal expression, red arrow) shows Fzr expression in endocycling cells (E). Green, PH3; red, CycB (C); Anti-ß-gal (D,E); blue, DAPI (C-E).

 


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  		Fig. 2. Fzr is partially sufficient to induce precocious endocycles, dMyc to accelerate endocycles. Ectopic expression of fzr (hsFlp; UAS-fzr; UASGFP act<FRT-CD2-FRT<Gal4) reduces CycA and B levels, stops mitosis (A,B) and allows the formation of large nuclei (B) indicative of precocious endocycles prior to stage 6 in oogenesis. The diagram in C presents the percentage of the wild-type and mutant follicle cells that show positive staining to mitotic markers during the mitotic stage. fzr (D,F,J) and myc (E,G,K) overexpression (hsFlp; UAS-fzr or UAS-myc; UASGFP act<FRT-CD2-FRT<Gal4) generate enlarged nuclei, which is indicative of abnormal endocycles but do not affect the amplification stage because normal BrdU-incorporation pattern at stage 10B-12 (red arrows) is observed in cells overexpressing fzr (F) and myc (G). (H-K) FACS profiles of DNA content in UAS-fzr (J) and UAS-myc (K) mutant nuclei compared with these in WT nuclei (H) show the appearance of a cell population that has 32n DNA, also observed in Rbf mutants (I) (Bosco et al., 2001Go). Green, GFP (A,D,E), Fzr Ab (F); red, CycB (A), DAPI (D,E), BrdU (F,G); blue, DAPI (A,F,G).

 


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  		Fig. 3. The Cyclin E levels are dynamic during cell cycle transitions; constant CycE levels halt endocycles and amplification. (A) Wholemount in situ hybridization indicates equal level of cyclin E mRNA in the follicle cells throughout oogenesis (arrows). (B,C) Cyclin E antibody staining pattern (green) between stages 5 (B) and 8 (C) shows a clear downregulation of the protein after the transition to the endocycle, when cells stop expressing PH3 (red). Cells overexpressing CycE (hsFlp;;UAS-cycE/UASGFP act<FRT-CD2-FRT<Gal4) express mitotic cyclins B and A past stage 6 in oogenesis (D-E, arrow), have smaller nuclei (F,F') and show highly reduced BrdU incorporation during endocycling (G,G') and amplification (H,H') stages, suggesting that cells ectopically expressing cyclin E fail to undergo S-phase and are halted at G2. Green, CycE (B,C), GFP (D-F), BrdU (G,H); red, PH3 (B,C), CycB (D), CycA (E), CycE (F-H); blue, DAPI.

 


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  		Fig. 4. Notch-dependent repression of the Cyclin E inhibitor Dacapo is required for mitotic-to-endocycle transition. (A) The dacapo mRNA in situ pattern indicates transcriptional downregulation after transition to endocycles (light arrow, st. 7). (B) A similar expression pattern was observed in a dap5gm construct, which includes the entire gene and a part of the promoter region fused with myc-epitope tag (arrow indicates follicle cells staining prior to st. 7). (C,C') Prolonged follicle cell expression of dap5gm in st. 9 Delta germ line clone shows that dap is controlled by Notch signaling. (D) Overexpression of dap (hsFlp; UAS-dap; UASGFP act<FRT-CD2-FRT<Gal4) inhibits endoreplication: follicle cells overexpressing dacapo have smaller nuclei (D') than the neighboring wild-type cells and they fail to incorporate BrdU during both the endocycling (E,E') and the amplification (F,F') stages, indicating that these cells could not undergo the S-phase and probably are blocked at the G1/S transition. Green, GFP (C,D), BrdU (E,F); red, CycE (D), Dap (E,F); blue, DAPI.

 


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  		Fig. 5. Ago is dispensable for mitosis but is required for endocycles in follicle cell epithelium. (A) The ago mRNA is expressed in both mitotic and endocycling follicle cells (arrows). (B) Ago is responsible for CycE degradation; follicle cells in ago (hsFlp;; ago FRT80B/Ubi-GFP FRT80B) clones show increased level of CycE protein at all stages during oogenesis. Ago loss-of-function does not affect the mitotic cycles in follicle cells: ago clones showed normal expression pattern of stg (C; expression prior to st. 6, red arrow, but not past st. 6, black arrow) and the ratio between the number of cells in sister versus mutant clone was 1:0.9 (D). However, ago clones halt the transition to endocycles: prolonged CycB expression (E,E'), small nuclei size (F,F'; clones marked with elevation of CycE levels) and highly reduced BrdU incorporation during both endocycles (G,G') and amplification (H,H') are observed in ago clones. Green, GFP (B,C,E), BrdU (G,H); red, CycE (B, CycE level marks the clones in F-H), ß-gal (C), CycB (E); blue, DAPI.

 


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  		Fig. 6. The Notch pathway regulates Fzr independently from String and Dacapo. (A) Although cells ectopicly expressing dacapo (arrowhead) do not undergo endocycles and have smaller nuclei (arrowhead in A''), they express normal levels of Fzr (arrowhead in A''') compared with wild-type cells (arrow) (hsFlp/fzrG0326; UAS-dap; UASGFP act<FRT-CD2-FRT<Gal4). (B) The normal Fzr expression is observed in most cells overexpressing dacapo (hsFlp/fzrG0326; UAS-dap; UASGFP act<FRT-CD2-FRT<Gal4) or cyclinE (hsFlp/fzrG0326; UAS-cycE/UASGFP act<FRT-CD2-FRT<Gal4). (C) Premature expression of fzr (arrowhead in C') causes precocious endocycles (large nuclei, arrowhead in C'') but does not affect string expression (C''', arrowhead indicates UAS-fzr cells, arrow – wild-type cells). (D) No effect on string or dacapo expression was observed upon premature expression of fzr (the diagram shows that UAS-fzr and wild-type follicle cells show close to equal percentage of cells with Dacapo and String staining, hsFlp; UAS-fzr/UASGFP act<FRT-CD2-FRT<Gal4;6.4-string-lacZ or dap5gm). Green, GFP; red, ß-gal; blue, DAPI.

 


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  		Fig. 7. The mitotic-to-endocycle transition in Drosophila follicle cells is executed by Notch-dependent control of cell cycle regulators. (A) A proposed scheme showing that the Notch pathway induces a switch from the mitotic-to-endocycle by controlling the activity of key cell-cycle regulators; cyclin-dependent kinases that control M- and S-phase entry and the APC/ubiquitination complex that regulates degradation of cyclins. Notch activity controls these regulators by downregulating String and Dacapo, and activating Hec1/CdhFzr. (B) The molecular model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks M-phase, activation of Hec1/CdhFzr allows G1 progression and repression of Dacapo assures entry into S-phase.

 

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© The Company of Biologists Ltd 2004