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Notch-Delta signaling induces a transition from mitotic cell cycle to endocycle in Drosophila follicle cells

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

View larger version (71K): [in a new window]   Fig. 1. Follicle cells in Drosophila oogenesis undergo a transition from a mitotic cell cycle to an endocycle. (A) Drawing of the stages of oogenesis. From the germarium (g) to stage (st) 6, somatically derived follicle cells undergo mitotic cell cycle. At stage 7 they switch to endocycles. From stage 7 to stage 10A, these cells undergo three rounds of endoreplication. (B) Staining of mitotic markers Cyclin B (CycB, red, red arrow) and Phospho-Histone 3 (PH3, blue, blue arrow) of two egg chambers at stages 6 and 7, showing the transition from mitotic cell cycle to endocycle at these two stages. (C) Whole-mount in situ hybridization shows that string, which encodes a Cdc25 phophatase, is expressed in follicle cells from the germarium to stage 6. string expression in follicle cells is turned off at stage 7 (arrow), suggesting that the transition from mitotic cell cycle to endocycle is regulated at the transcriptional level.

View larger version (105K): [in a new window]   Fig. 2. Delta signals from the germline cells to regulate the transition from mitotic cycle to endocycle in follicle cells. (A) Delta expression is upregulated at stage 6 and reaches its highest at stage 7 in the germline cells (green). This upregulation is coincident with the cell cycle transition (Phospho-Histone 3 staining in red is observed in stage 6 (arrows), but not in stage 7 egg chambers). (B) Delta is not required in the follicle cells for the transition, as no extra cell divisions were observed in Dlrev10 follicle cell clones (PH3, blue in B). (C,D) Delta is required in the germline, as Phospho-Histone 3 (PH3, blue in C, arrows) and Cyclin B (Cyc B, red in D, arrows) are observed in follicle cells of stage 8 and 9 egg chambers carrying germline clones of Delta. Green in B-D shows GFP, which marks the wild-type cells, and cells that lack GFP expression are mutant clones. Epithelial polarity is normal in these egg chambers based on normal Armadillo localization (red in B,C). In later egg chambers, some epithelial defects were observed (data not shown).

View larger version (97K): [in a new window]   Fig. 3. Notch is required in the follicle cells for the mitotic-to-endocycle transition. (A) Notch protein is observed in the apical surface of the follicle cell layer in wild-type egg chambers. (B) Cells in Notch clones (black, outlined by broken white line) have smaller nuclei than their wild-type neighbors. This most probably indicates lack of endocycles in the mutant clones. (C-E) Mitotic markers (PH3, CycB) are observed in cells in Notch mutant clones at stage 8-9, indicating a failure to enter the endocycle. (F) string expression can still be observed at stage 8 in egg chambers with Notch mutant clones. In this case, the mutant clone is unmarked. (G) Quantitation of the number of cells in Notch mutant clones (dark purple bar, the black clone in the figure is marked with an unbroken white line) compared with the sister clones (light purple bar, bright green in the figure marked with broken white line). The results are plotted as a function of sister clone size. (H) Ratio of the size of mutant clone per the size of sister clone in G plotted as a function of sister clone size. More cells are observed in the mutant than in sister clones (mean ratio=1.8). Importantly, the ratio does not change as a function of clone size, suggesting that the defect in Notch clones only occurred after the normal mitotic stage. Notch clones marked with broken lines in B-D and with an unbroken line in G.

View larger version (130K): [in a new window]   Fig. 4. Supressor of Hairless (Su(H)) but not E(spl) is required in the follicle cells for exit from the mitotic cycle. (A) In this stage 12 egg chamber, Su(H)SF8 mutant cells (no green GFP) are smaller compared with the wild-type cells marked by nuclear GFP (green). Armadillo staining (red) outlines the cell boundaries while DAPI, blue, labels all cells. (B,C) In these stage 8 egg chambers, CycB (red) and PH3 (blue), indicate additional mitoses in Su(H)SF8 clones (arrows). (D) The Enhancer of Split (E(spl)) gene complex is not required in follicle cells to regulate the mitotic-to-endocycle transition, as no CycB or PH3 staining was observed in E(spl) mutant clones after stage 8 (mutant clones are black, arrows).

View larger version (93K): [in a new window]   Fig. 5. Comparison of the phenotypes of Notch and lgl, a tumor-suppressor gene. (A) In Notch mutant clones, follicle cell apical-basal polarity appears to be normal, as shown by localization of the apical marker, Armadillo, a Drosophila homolog of ß-catenin (red). PH3 (blue) indicates additional mitosis. (B) In a lgl mutant clone, additional cell division is also observed after stage 6 (PH3, blue), but is accompanied by loss of follicle cell apical-basal polarity, as shown by mislocalization of Armadillo in the clone (red). (C) lgl mutant clones frequently lead to the formation of multiple layers of follicle cells. (D) Notch protein (red) localization in the apical side of the follicle cells is disrupted in lgl clones (arrows). (E) In small Notch clones, Notch mutant cells can still differentiate, as shown by expression of Broad-Complex proteins, normally observed in all follicle cells beginning at around stage 6. In Notch clones, BR-C expression persists, as in the wild-type neighbors. (F) In small Notch clones, several markers, including Cadherin, Armadillo, Disks-lost and Fasciclin III (F; FasIII, red) are upregulated. In all panels, clones are outlined with a broken white line.

View larger version (55K): [in a new window]   Fig. 6. string expression in follicle cells. (A) The 50 kb genomic region surrounding string is indicated. A thick black arrow indicates the string gene; the string intron is white. Stg15.3 and stg31.6 indicate the genomic fragments tested for rescue. Fragments used to drive lacZ expression in transgenic animals are shown below the genomic region. Cell types in ovary in which expression is driven by these fragments are described in Table 2. (B,D) The 4.9 kb element (D10) supports follicle cell expression in the germarium and stage 1-2 egg chambers. (C,E) The 6.4 kb element (D12) supports expression in stage 4-6 egg chambers. (B,C) X-gal staining, black arrows indicate the follicle cells with X-Gal staining, white arrows indicate lack of staining. (D,E) Anti-ß-gal (green). Yellow arrows indicate the follicle cells with staining, white arrows indicate lack of staining. (F) String is required for follicle cell mitotic divisions, as only 0-2 cell string mutant clones (black) were observed with 2-16 cell sister clones (bright green). On average, the mutant clone is one tenth the size of the sister clone. (H) Quantitation of the size of the string mutant (red bar) and sister (blue bar) clones. (G) Stg15.3 rescue construct shows partial rescue for the clone size (on average, the black mutant clone is half the size of the bright green sister clone). (I) Quantitation of the sizes of string clones with Stg15.3 rescue (red bar) and sister clones (blue bar). DAPI is blue in D,E and red in F,G. GFP is green in F,G. Mutant clones are indicated with unbroken white lines, sister clones with broken white lines. hhhhhfh

View larger version (94K): [in a new window]   Fig. 7. 6.4 kb string promoter region is precociously active in Delta germline clones. ß-Gal expression from 6.4-LacZ string promoter fusion (red) is observed in follicle cells of stage 5 (A) but not stage 9 (B) control egg chambers. Some overlapping expression is observed between CycB (blue) and the 6.4-LacZ string promoter fusion construct (red) at stage 5 egg chambers (A). In contrast to control egg chambers, in Delta germline clones (black germ line) the 6.4-LacZ string promoter fusion construct is still observed in the follicle cells at stage 8-9 egg chambers (C,D). Green in A-D shows GFP, which marks the wild-type cells, and cells that lack GFP expression are mutant clones (C is a fused egg chamber, the border between wild-type cyst and Delta germline clone is marked with arrows). Red in A-D shows ß-gal. Blue is CycB in A and DAPI in B-D.