Notch signaling through tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells

Abstract

Background: The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, we showed that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1.

Results: In this paper, we show that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. We also demonstrate that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway.

Conclusion: This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch.

Figure 1

Transition in epithelial follicle cells from mitotic cycle to endocycle during Drosophila oogenesis. (A) Signaling from the Notch pathway causes follicle cells surrounding each oocyte to cease mitosis and begin endocycling at stage 6–7. (B) Cyclin A, (C) Cyclin B and (A) PH3 expression through stage 6 indicates the mitotically active follicle cells. (D) Notch protein is also expressed on the apical side of the follicle cells through stage 6 (arrow) and is cleared from the plasma-membrane upon activation. (E) String (stg), as shown by stg-lacZ (6.4 kb) promoter construct and (F) dacapo (dap), as shown by dap-5gm promoter construct, are both expressed in follicle cells prior to the transition and down-regulated in response to Notch signaling. (G) Fizzy-related (fzr), as shown by Fzr-lacZ enhancer trap line fzrG0326, is expressed in follicle cells after the transition. (H and H') Phosphorylated JNK staining (green) is upregulated in the follicle cells prior to the mitotic-to-endocycle transition in WT egg chambers. All cells are labeled by DAPI (red). (I and I') Puc-LacZ (green), a reporter construct for Puckered (puc^A251), expression is upregulated in follicle cells during mitosis. All cells are marked with DAPI (red).

Figure 2

Shaggy (sgg^M1-1) and Disheveled (dsh⁴⁷⁷), components of the wingless pathway, are involved in cell cycle control, but Pygopus (pygo) is not. (A) Quantification of the number of cells in Sgg mutant clones compared to the number in their sister clones shows that they are twice as large. (B) Quantification of the number of cells in Dsh mutant clones compared to the number in their sister clones shows that they are half the size of their twin spots (p = 0.0005). (C) However, quantification of the number of cells in Pygo mutant clones compared to the number in their sister clones shows that there is no affect on cell cycle when Pygo expression is lost. (D, D') Sgg clones (dashed line) show prolonged expression of CycB (red) and PH3 (blue) indicating that mitoses are still occurring in this stage 9 egg chamber. After the mitotic-to-endocycle transition, Sgg mutant clones show upregulation of Notch (E, E') and stg-lac Z (F, F'). (G) Premature expression of pUASpDelta-1 and pUASpDelta-2 causes a 60% and 89% reduction in the number of PH3 positive cells respectively (n = 37 matTub-Gal4; pUASpDl-1; n = 10 pUASpDl-2/matTub-Gal4; n = 30 control). (H) In control ovarioles, PH3 (green) is expressed at stage 6. (I) Driving Delta (red) prematurely in the germline causes all or some of the follicle cells to cease division early (as visualized by an absence of PH3 positive cells). Clones are marked with loss of GFP and indicated by dashed lines.

Figure 3

Tramtrack (ttk) is required in follicle cells for the mitotic-to-endocycle transition. (A) Ttk^1e11 mutant clones marked by loss of GFP (indicated by dashed line) show a decrease in the size of cell nuclei, marked with DAPI (red), indicating a defect in the mitotic-to-endocycle transition of those cells. (B) Quantification of the number of cells in ttk clones compared to the number in their sister clones shows that on average they are twice as large, which implies that ttk mutant cells have undergone an extra round of mitotic division (mean = 2.33, p = 0.0006). (C) ttk mutant clones show prolonged expression of mitotic markers, PH3 (C, C'), Cyc B (D, D') and Cyc A (E, E'; 25% n = 32, 60% n = 30, 50% n = 22, respectively).

Figure 4

Tramtrack affects targets of the Notch pathway involved in the mitotic-to-endocycle transition. Ttk mutant clones past stage 6–7 mitotic-to-endocycle transition show upregulation of stg-lacZ (A, A'; arrow, red, s.10 egg chamber, stg 6.4 kb promoter construct, 37% n = 27) and dap-myc (B, B'; arrow, red, s.8 egg chamber, dacapo5gm promoter construct, 64% n = 25) and down-regulation of Fzr-lacZ (C, C'; red, s.9 egg chamber, fzrG0326, 70% n = 123). All cells are marked with DAPI (blue), GFP is green.

Figure 5

Tramtrack, downstream of Notch, is involved in cell cycle regulation but not in cell differentiation. (A, A') Notch is processed and cleared normally from the apical side of follicle cells in ttk-clones. (B, B') Cut down-regulation is normal in ttk-clones (23/23, except in the most posterior region, Supp Fig. 1; Althauser et al., 2005). N mutant clones (C, C') but not ttk mutant clones (D, D') show ectopic expression of FAS3 after the transition, indicating that Ttk does not act in Notch dependent follicle cell differentiation that involves Fas3. (E, E') N mutant clones (stage 9 egg chamber) show reduction of ttk expression (red), indicating that Ttk is downstream of N. However, this result does not rule out the possibility that Ttk acts in a parallel pathway. All cells are marked with DAPI (blue), GFP is green, clones are marked with dashed lines.

Figure 6

JNK pathway components Basket (Bsk^170B), Puckered (Puc^A251), and Hemipterous (Hep^r75) control cell cycle in follicle cells by promoting mitosis prior to the transition to endocycle. (A, A') Bsk mutant cells (no green GFP) have larger nuclei, marked with DAPI (red), compared to wild-type cells marked with GFP (green). (B, B') Puc mutant cells (no green GFP) appear to be disrupted in mitotic division but the cell nuclei, marked with DAPI (red), are not abnormally large and therefore have not gone into endocycle. (C) Quantification of the number of cells in Bsk mutant clones compared to the number in their sister clones shows that the clones are approximately half the size of their sister clones, indicating a premature exit from mitotic division (mean = 0.58, p = 0.002). (D) Quantification of the number of cells in Hep mutant clones compared to the number in their sister clones shows a similar phenotype (mean = 0.55, p = 0.02). (E) Quantification of the number of cells in Puc mutant clones compared to the number in their sister clones shows no consistent defect in mitotic division (mean = 0,88, p = 0.4, suggesting that the size difference between sister and mutant clones is not statistically significant). (F-H) Bsk mutant cells (no green GFP) compared to wild-type cells marked with GFP (green) in stages prior to the mitotic-to-endocycle show no affects on (F) string expression, marked with LacZ (red), or (G) dacapo expression, marked with c-Myc tag (red). (H, H') Bsk mutant cells (no green GFP) also do not show premature fizzy-related expression, marked with LacZ (red). All cells are marked with DAPI (blue).

Figure 7

The JNK pathway promotes and maintains mitosis until the mitotic-to-endocycle transition point. At the transition, the Notch pathway acts through ttk on the cell cycle components stg, dap, and fzr in follicle cells to stop mitotic cycling and induce endocycling. Notch, which is modulated by Dl, dsh, and sgg, also plays a role in cell differentiation without the involvement of ttk, as shown by its effect on FAS3 expression.