1. Volume 12, Issue 4, Pages 631-643 (April 2007)
A Double-Assurance Mechanism Controls Cell Cycle Exit upon Terminal Differentiation in Drosophila 
Laura A. Buttitta, Alexia J. Katzaroff, Carissa L. Perez, Aida de la Cruz, Bruce A. Edgar 
Developmental Cell 
Volume 12, Issue 4, Pages (April 2007)
DOI: /j.devcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Cell Cycle Exit in the Drosophila Eye and Wing
(A and B) Phosphorylated histone H3 (PH3) staining was used to visualize mitoses, and FACS was used to determine DNA content in wild-type (A) eyes and (B) wings at the indicated time points (hours after pupa formation, hr APF). By 24 hr APF, almost 100% of cells in the eye and wing are arrested with a G1 DNA content (2N).
(C and D) Wings were dissected at the indicated time points (x axis) and were examined for levels of (C) positive or (D) negative cell cycle regulators by qRT-PCR. The average ΔCt (equivalent to log2 of the fold difference from −10 hr APF) is shown on the y axis. Error bars indicate the range of three biological replicates. Dotted lines indicate a ΔCt of 1, equivalent to a 2-fold difference.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Ectopic E2F or Cyclin/Cdk Activity Delays, but Does Not Prevent, Cell Cycle Exit
(A–L) GFP-marked clones expressing the indicated cell cycle regulators were generated by using hs-FLP tub>Gal4/UAS, tub-Gal80TS and were examined for ectopic mitoses by staining for PH3 (red). Wings were also stained for Elav (blue, A′–D′ and H′–K′) or F-actin (blue, E′–G′). PH3 is seen in clones at 24–36 hr APF (A, D, G, and J), but not 36–40 hr APF (B, E, H, and K). FACS was used to examine the DNA content of overexpressing cells (green) and control cells (GFP-negative, black) in quiescent wings at 44 APF (C, F, I, and L).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Requirements for Negative Cell Cycle Regulators upon Exit Are Tissue Specific
(A–H′) Null mutant clones for dap or rbf1 were generated by using mitotic recombination. Mitoses are detected by PH3 staining (red). Elav labels neurons (blue). (A–B) No ectopic PH3 is observed in dap mutant clones (GFP-negative) at any stage examined (C–D) or in rbf1−/− clones after 28 hr APF (LacZ in green; clones are LacZ negative). (E–F) GFP-positive rbf1−/− cells overexpressing CycE/Cdk2 were generated by using MARCM. (E) Mitoses are evident at 40 hr APF in the eye in ∼1.5% of cells and in Elav+ neurons of the eye and wing margin (insets E–F; arrows indicate mitotic neurons), but not in the epithelial wing blade (F). (G–H) GFP-positive dap−/− cells overexpressing E2F were generated by using MARCM. (G) Mitoses are evident at 40 hr APF in the eye (in ∼0.8% of cells), (H) but not the wing.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Cell Cycle Exit Is Bypassed by Simultaneous Activation of CycE and E2F
(A–H′) Cells expressing cell cycle regulators during pupal stages were generated using (A–D) Ap-Gal4/UAS, tub-Gal80TS, (E–H) hs-FLP tub>Gal4/UAS, tub-Gal80TS, or (F) GMR-Gal4/UAS, tub-Gal80TS. Late pupal stages, 44 hr APF in (A–D) and (G–H) or 40 hr APF in (E–F) were examined for PH3 (red). Ectopic PH3 was evident in cells expressing the indicated regulators in the eye and wing at all stages examined. Terminal differentiation proceeded in cycling cells, as indicated by (E′) expression of Elav in the eye (blue), (F) GFP+ axonal projections to the optic stalk (GFP white), and (H′) formation of actin-rich wing hairs (white). (B and D) FACS of overexpressing (GFP-positive, green) and control cells (black) in wings shows some DNA overreplication in cells that bypass exit (>G2 DNA content).
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5 Cell Cycle Exit Is Reversible
(A–G) Cells were induced to express the indicated cell cycle regulators after cell cycle exit at 28 hr APF by combining (A–F) Ap-Gal4/UAS or (G) GMR-Gal4/UAS with tub-Gal80TS. (A–E) FACS of overexpressing cells (green) and control cells (black) in wings at 48 hr APF shows that some cells expressing CycE/Cdk2, CycD/Cdk4, or E2F enter S and G2 phases. (F and G) Cells simultaneously overexpressing E2F, CycE, and Stg re-enter the cell cycle and proceed through mitosis (PH3, red). Nonoverlapping GFP-labeled clones expressing the indicated regulators and Drosophila IAP to prevent apoptosis were induced after cell cycle exit in wings at (H–K) 28 or (L) 36 hr APF. Wings were fixed at (H–K) 48–54 or (L) 80 hr APF, and cells/clone were counted. (H and I) Clones expressing CycE or E2F contained one cell. (J and K) In contrast, 60% of clones expressing E2F+CycE or 85% expressing E2F+CycE+Stg contained > 2 cells/clone. (L) Clones expressing E2F+CycE+Stg that were induced 36 hr APF and observed at 80 hr APF contained up to 8 cells; (L′) nuclei were observed at various stages in the cell cycle (arrows). (L″) Bright-field image showing the 80 hr APF wing epithelium and cuticle with the GFP signal overlayed.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6
Cyclin/Cdk Activation of E2F-Dependent Transcription Decreases after Exit
(A–E′) Clones expressing the indicated regulators were generated by using the hs-Flp act>Gal4/UAS system and were examined at the indicated times. (A–E) Clones were detected by the absence of CD2 staining (white). (A′–E′) E2F activity was detected by using a PCNA-GFP reporter amplified by α-GFP antibody (white). (A–B) CycE/Cdk2 does not activate the reporter in pupal wings after exit, while CycD/Cdk4 activates the reporter during the initial exit period (C) 24–36 hr APF, (D) but not later (38 hr APF). (E) E2F strongly activates the reporter at all stages (40 hr APF shown).
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 7
E2F-Independent Mechanisms Regulate CycE/Cdk2 Activity after Exit
(A) Wings with high E2F activity during pupal stages (Ap-Gal4/UAS-E2F/DP, tub-Gal80TS, solid lines) and controls (Ap-Gal4/UAS-GFP, tub-Gal80TS, dotted lines) were examined at the indicated time points for expression of cell cycle regulators by qRT-PCR. The average ΔCt is shown. Error bars indicate the range of three biological replicates. E2F activity increases cycE, cdk2, and pcna, but does not induce dap. (B–C) GFP-labeled clones overexpressing E2F during pupal stages were generated with the hs-FLP tub>Gal4/UAS, tub-Gal80TS system and were examined for CycE protein and CycE/Cdk2 activity. (B) E2F expression increases CycE levels at 40–44 hr APF in eyes, (C) but does not generate MPM2+ nuclear foci. (D) In contrast, CycE/Cdk2 overexpression does induce foci (arrows).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

9. Figure 8 A Double-Assurance Mechanism for Cell Cycle Exit
(A) Feedback between E2F and CycE (B) is lost after terminal differentiation (gray dotted arrows), ensuring that cell cycle exit induced by inhibition of CycE or E2F is robust.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions