The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles

Abstract

The endocycle is a developmentally programmed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. In Drosophila, the endocycle is driven by the oscillations of Cyclin E/Cdk2 activity. How the periodicity of Cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. Here, we demonstrate that the p21(cip1)/p27(kip1)/p57(kip2)-like cyclin-dependent kinase inhibitor (CKI), Dacapo (Dap), promotes replication licensing during Drosophila endocycles by reinforcing low Cdk activity during the endocycle Gap-phase. In dap mutants, cells in the endocycle have reduced levels of the licensing factor Double Parked/Cdt1 (Dup/Cdt1), as well as decreased levels of chromatin-bound minichromosome maintenance (MCM2-7) complex. Moreover, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with a reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S-phase. Finally, genetic interaction studies suggest that dap functions to promote replication licensing in a subset of Drosophila mitotic cycles.

Figure 1

dap^−/− egg chambers have abnormal nurse cell nuclei. (A) Wild-type and (B, C) egg chamber containing a dap^−/− germ-line clone stained with the DNA dye DAPI. Boxes in (A) and (C) are blow-ups of the indicated nurse cells. Note that dap^−/− nurse cells have a condensed chromatin structure. Arrowhead in (B) denotes an example of a dap^−/− nurse cell with an apparent DNA content lower than the anterior nurse cell indicated by an arrow.

Figure 2

The endocycle S-phase is lengthened in dap^−/− mutants. The FLP/FRT technique was used to generate dap^−/− clones in a wild-type (dap^+/−) background. (A–D) Egg chambers containing (A, B) wild-type and (C, D) dap^−/− germ-line clones labeled with (A, C) DAPI and (B, D) the nucleotide analog BrdU. (E–G) dap^−/− follicle cell clone from a stage 7 egg chamber labeled with (E) αBrdU and (F) αGFP. In (F) and (G), dap^−/− clones are identified by the absence of αGFP staining. (G) An overlay of αBrdU (red), αGFP (green) and DAPI staining (blue). (H) Wild-type and (I) dap^−/− nurse cell nuclei stained with DAPI. Arrows indicate the primarily heterochromatic fourth chromosome.

Figure 3

Dap differentially affects the accumulation of Dup/Cdt1 in endocycling versus mitotic cells. The FLP/FRT technique was used to generate dap^−/− clones in a wild-type (dap^+/−) background. (A–I) Ovaries containing dap^−/− clones were stained with (A, D and I) DAPI, (B) α-β-gal, (E, F, G, I) αGFP and (C, F, H, I) αDup/Cdt1 antibodies. In (B), dap^−/− clones are identified by the absence of α-β-gal staining, whereas in (E, F, G, and I), dap^−/− clones are identified by the absence of αGFP staining. (A–C) An egg chamber containing a dap^−/− germ-line clone, arrowhead, flanked by two wild-type egg chambers. Note the low levels of Dup/Cdt1 in the polyploid nurse cells of the dap^−/− cyst. (D–F) In contrast, Dup/Cdt1 is present in dap^−/− germ-line clones undergoing the mitotic cyst division in region 1 of the germarium (F, arrowhead). (G–I) dap^−/− follicle cell clones, marked by the absence of (G) αGFP, have reduced levels of (H) αDup/Cdt1 staining compared with adjacent wild-type cells. (J–L) The FLP/Gal4 system was used to clonally express Dap with β-gal. Follicle cells overexpressing Dap, marked by (J) α-β-gal staining, have higher levels of (K) α-Dup/Cdt1 staining than neighboring wild-type cells.

Figure 4

dap^−/− nurse cells have reduced levels of the chromatin-bound MCM2–7 complex. (A, B) Wild-type and (C, D) dap^−/− egg chambers stained with (A, C) αMCM2–7 antibody and (B, D) DAPI, and washed with low salt to reveal the nucleoplasmic pool of MCM2–7. (E, F) Wild-type and (G–H) dap^−/− egg chambers stained with (E, G) αMCM2–7 antibody and (F, H) DAPI and washed with high salt to reveal chromatin-bound MCM2–7.

Figure 5

dap^−/− mutants accumulate increased α-γ-H2Av staining upon entry into the endocycle. Wild-type (A, B) mitotic and (C, D) endocycling follicle cells stained with (A, C, white; B, D, blue) DAPI and (B, D, red) α-γ-H2Av antibody. (A, B) Wild-type mitotic follicle cells have little α-γ-H2Av staining, whereas (C, D) α-γ-H2Av staining accumulates near the chromocenter upon entry into the endocycle (C, D, arrows). (E, F) Two dap^−/− germ-line cysts marked by the absence of (F) α-GFP staining have increased levels of (E, F) γ-H2Av staining compared with a neighboring older wild-type cyst. (G–I) A stage 9 egg chamber containing a dap^−/− clone of follicle cells that have entered the endocycle. In the dap^−/− cells (G), marked by the absence of α-GFP staining (H, I), γ-H2Av staining is often increased and not restricted to the region near the chromocenter.

Figure 6

Reducing the dosage of dup/cdt1 enhances the dap^−/− phenotype in several polyploid cell types. (A–C) DAPI staining of (A) wild-type, (B) dap^−/− and (C) dap⁻, dup^a1/dap⁻, + ovarioles. (D–F) Magnification of (D) wild-type, (E) dap^−/− and (F) dap⁻, dup^a1/dap⁻, + nurse cell nuclei. (G–J) Thorax of (G) wild-type, (H) dap⁻/CyO, (I) dap^−/− and (J) dap⁻, dup^a1/dap⁻, + flies. dap^−/− mutant macrochaetae (I, arrowhead) are often thinner than the control (G, H, arrowheads). A single copy of dup^a1 mutation dominantly enhances the dap^−/− reduced macrochaetae phenotype (J, arrowhead).

Figure 7

dap⁻, dup⁻/dap⁻, + flies have rough eyes. (A–C) Scanning electron micrographs of (A) wild-type, (B) dap^−/− and (C) dap⁻, dup^a1/dap⁻, + eyes. (C) Note that the dap⁻, dup^a1/dap⁻, + compound eye has an irregular pattern of ommatidia relative to both wild-type and dap^−/− eyes. Tangential sections (D–F) of (D) wild-type, (E) dap^−/−, and (F) dap⁻,dup^a1/dap⁻, + eyes, stained with toluidine blue. Note the missing photoreceptor cells and irregularly shaped and oriented ommatidia in dap⁻,dup^a1/dap⁻, + (F, arrows). (G–I) Eye antennal imaginal discs from (G, G′) wild-type, (H, H′) dap^−/− and (I, I′) dap⁻, dup^a1/dap⁻, + stained with (G–I′, red) α-cleaved caspase 3^*, (G′–I′, green) α-Cyclin B antibodies and (G′–I′, blue) DAPI. Anterior is on the left. (I, I′) Discs from dap⁻, dup^a1/dap⁻, + have increased numbers of cleaved caspase 3^*-positive cells relative to both (G, G′) wild-type and (H, H′) dap^−/−, indicating increased levels of cell death.