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First published online 1 September 2005
doi: 10.1242/dev.02015

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Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells

Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA

View larger version (29K): [in a new window]   Fig. 1. Switch of cell-cycle programs and the Cut expression pattern in follicle cells. (A) During Drosophila oogenesis, somatically derived follicle cells undergo two cell-cycle switches: (1) the mitotic cycle to endocycle switch and (2) the endocycle to gene-amplification switch. From the germarium (G) to stage (S) 6, follicle cells undergo unsynchronized mitotic cycles. During stages 7 to 10A, these cells go through three rounds of endoreplication and thereafter switch to a localized amplification pattern characteristic of chorion gene amplification. (B) Cut expression in follicle cells. Expression (shown in red) began in region 2b of the germarium. It persisted in all follicle cells, including the polar cells and the interfollicular stalk cells, until about stage 6 and diminished afterwards, concurrent with the first cell-cycle switch. PH3 (shown in green) was used to mark the M phase of the mitotic cycle. Between stages 7 and 10A of oogenesis, Cut expression ceased in all follicle cells except the polar cells. At about stage 10B, Cut expression resumed in the columnar follicle cells that surround the oocyte. DAPI (in blue) was used to reveal the nuclei.

View larger version (110K): [in a new window]   Fig. 2. Cut was downregulated by Notch signaling in follicle cells that undergo endoreplication. Mutant cells are marked by the lack of GFP (green in A-D). Cut staining is shown in red in A-D and in white in A'-D'. DAPI is shown in blue in A. (A) Follicle cells surrounding a Dl germline clone continued to express Cut in a stage 8 egg chamber. (B-D) Notch (B,B'), psn (C,C'), or Su(H) (D,D') follicle-cell clones (outlined) caused Cut to be continuously expressed in the mutant clones beyond stage 6 in a cell-autonomous manner.

View larger version (88K): [in a new window]   Fig. 3. Cut and cell cycle regulation. ct mutant cells are marked by the absence of histone-GFP (hGFP; green in A,B,D,E and white in A'',D''). DAPI is shown in blue (A,B,B',E,e). (A,A',A'') No Cut expression (shown in red in A and white in A') was detected in ctdb7 mutant follicle-cell clones (arrow). The sister clones (outlined and marked by higher levels of GFP) had higher levels of Cut expression than the heterozygous ctdb7 follicle cells. (B,B',B'') ctdb7 mutant follicle cells had larger nuclei and less CycA expression. In a stage 6 egg chamber, DAPI staining showed that ctdb7 mutant follicle cells (outlined) had larger nuclei than the wild-type cells. In the clone, CycA expression is absent (red in B and B''), but in the neighboring wild-type cells about half of the cells were CycA positive. (C) Number of nuclei in ctdb7 mutant clones (blue bars) compared with that in their associated sister clones (purple bars) in stage 10 egg chambers. The x-axis represents the clone number, and the y-axis represents the number of cells per clone or corresponding sister clone. (C') On average, the number of nuclei in the clonal area was one third of that in the associated sister clone. (D,D',D'') CycE (red in D and white in D') had normal oscillating expression pattern in the clone cells (outlined). (E,E') ctdb7 mutant follicle cells properly switched to the gene-amplification stage. A foci-like pattern of BrdU incorporation (red in E and white in E') was found in the cut-mutant follicle cells in a stage 10B egg chamber. (e,e') The circled areas in E and E' show four dots of BrdU incorporation foci (red in e and white in e') in each nucleus.

View larger version (75K): [in a new window]   Fig. 4. Prolonged Cut expression during mid-oogenesis caused defects in the mitotic cycle/endocycle switch. Cut misexpression and Cut-String double misexpression were achieved by the flip-out Gal4/UAS technique and are marked by co-expression of GFP (green in A-G). All the clones were also marked by Cut staining (not shown in the figure), except those in F. DAPI is shown in blue (A-C,G) and white (A'). (A,A') Continuous expression of Cut during mid-oogenesis caused the follicle cells to be more densely distributed and their nuclei to be slightly smaller than those of the wild type. (B,B') Cyclin A (shown in red in B and white in B') was found to be expressed in some of the follicle cells misexpressing Cut in a stage 9 egg chamber, whereas CycA was no longer present in wild-type cells. (C,C') ß-gal expression (red in C and white in C') from a fzr enhancer trap line fzrG0326 was downregulated in follicle-cell clones with overexpression of Cut during mid-oogenesis. (D,D') ß-gal expression (red in D and white in D') from stg-lacZ showed no change in cut misexpressing clones during mid-oogenesis. (E,E') PH3 (red in D and white in D') staining was found occasionally during mid-oogenesis in follicle cells misexpressing both Cut and Stg. (F,F',F'') Follicle-cell clones overexpressing Cut in stage 10B egg chambers did not switch to the amplification stage. BrdU incorporation (red in F and white in F') and Orc2 staining (blue in F and white in F'') were not present in the clonal cells (outlined). (G,G') Overexpression of Cut in a stage 10B egg chamber affected the uniform Cyclin E expression pattern. No Cyclin E expression (red in G and white in G') was detected in the clone misexpressing Cut (outlined).

View larger version (76K): [in a new window]   Fig. 5. Prolonged expression of Cut maintains follicle cells at an immature stage. Cut-misexpressing cells are marked with co-expression of GFP (green in A,B,D,F) or Cut staining (green in E,G). (A,A') FasIII (red in A and white in A') continued to be expressed in follicle cells overexpressing Cut (outlined) in stage 9 egg chamber, whereas neighboring wild-type follicle cells no longer expressed FasIII, except in the polar cells (arrowhead) and the stalk cells (not shown). (B,B') Big brain (Bib, red in B and white in B'), a stalk-cell marker, was expressed in the stalk cells (arrow) but not in the main-body follicle cells even if Cut was overexpressed. (C) The Eya expression pattern during early oogenesis. Eya protein (shown in red) was expressed in the germarium and in all follicle cells except the polar cells up until stage 8, but the expression was lower after stage 6. PH3 (shown in green) was used to indicate the M phase of the mitotic cycle. (D,F) Eya (red in D,F and white in D',F') was continuously expressed in follicle cells overexpressing Cut after stage 7 and until stage 10. Eya was not expressed in stage 12 egg chambers (F,F' with increased background). (E,G) Polar-cell fate marker A101 (stained with anti-ß-gal, red in E,G and white in E',G') was only expressed in polar follicle cells (arrowhead in E and E') in the middle stage, whereas it was expressed in main-body follicle cells overexpressing Cut after stage 10.

View larger version (89K): [in a new window]   Fig. 6. Loss of cut function during early oogenesis is sufficient to drive the follicle cells to differentiate. ct mutant cells are marked by the absence of GFP (green in A,B,E,F). FasIII staining is shown in red (A-D) and white (A'-D'). DAPI is shown in blue (A-E). (A,B) FasIII expression was downregulated and was restricted to the apical-lateral regions (arrowheads in B and B') in ctdb7 clones (outlined) in early stages. (C,D) Different FasIII expression patterns before and after stage 7 in the wild type. During stage 8 (S8), FasIII expression in the basal lateral region was downregulated, whereas the apical lateral region retained FasIII expression (C,C'). In contrast, FasIII was expressed in all lateral regions of the follicle cells at stage 6 (D,D'). The apical (Ap) and basal (Ba) surfaces of the follicle cells are outlined (C',D'). (E,F) Eya (red in E, blue in F, and white in E',F') was also downregulated in ctdb7 follicle-cell clones (outlined) in early stages, whereas wild-type follicle cells, except the polar cells (arrow in F), expressed Eya. ctdb7 follicle-cell clones did not express the polar cell marker, PZ80-lacZ (red in F).

View larger version (18K): [in a new window]   Fig. 7. A schematic drawing showing how Notch might regulate cell-cycle transition and cell differentiation through Cut. In early oogenesis, Cut is expressed in all follicle cells and maintains the normal mitotic cycle partially by suppressing the APC/C adaptor, Fzr (Fizzy-related); it maintains the follicle cells in the undifferentiated cell fate. During the cell-cycle transition from the mitotic cycle to the endocycle, upregulated Dl (Delta) from germline cells binds to Notch receptor and activates Notch signaling in follicle cells. Activated notch signaling activates gene X and indirectly turns off Cut through X, which results in follicle-cell differentiation from the immature state to the mature state. At the same time, lack of Cut expression derepresses fzr. Activated notch signaling somehow downregulates Stg (String), a G2/M promoter, and prevents the follicle cell from going into M phase. A high level of Fzr can activate APC/C E3 ligase to degrade G2 cyclins and allow follicle cells in G2 phase to by-pass the M phase and get into G1 phase directly.