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Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells

¹ Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA
² Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
³ Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA
⁴ Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (50K): [in a new window]   Fig. 1. E2F2 interacts with DP in a yeast two hybrid assay. DP was used as bait to investigate its ability to interact with full-length E2F2 and various C-terminal deletions of E2F2 constructed in the pACT2 activation domain vector. The DNA binding, dimerization and pRB interaction domains of E2F2 are indicated by hatched, shaded and black boxes, respectively. An interaction between DP and different E2F2 constructs was scored as the ability to grow on minimal media lacking adenine.

View larger version (86K): [in a new window]   Fig. 2. Embryonic expression of E2f2. The expression of E2f2 was analyzed by whole-mount in situ hybridization of wild-type embryos of different developmental stages using an antisense E2F2 cDNA probe. Embryos are oriented with anterior towards the left and dorsal towards the top. (A) Maternal E2f2 mRNA can be detected in syncytial stage embryos. (B) A cellularizing blastoderm embryo, at which time maternal message has been destroyed. (C) A stage 10 germ band extended embryo. E2f2 is expressed throughout the embryo during the post-blastoderm divisions, but its expression is not coupled to the cell cycle. (D) A stage 13 germband retracted embryo. E2f2 expression is confined to dividing cells of the CNS (arrowhead) and endoreduplicating cells such as the gut (arrow). (E) A stage 15 germband retracted embryo. E2f2 expression is still confined to dividing and endocycling cells, and its expression is still not coupled to a particular cell cycle phase. (F) A stage 14 Df(2L)DS8/Df(2L)DS8 embryo. Zygotic E2f2 expression is lacking, indicating that this deletion removes the E2f2 gene.

View larger version (17K): [in a new window]   Fig. 3. The E2f2 locus and construction of E2f2 mutations. (A) The top line is a low resolution schematic of the E2f2 locus located at 39B2-3 on the left arm of chromosome 2. Triangles represent the location of lethal P-element insertions in the region. The broken line indicates the extent of Df(2L)1129, a 12 kb deletion generated by transposase-mediated excision of l(2)k07215. This deletion uncovers the genes CG9246, CG9247, CG9248, CG9249, CG9250 (Mpp6) and CG1071 (E2f2). The second line is a higher resolution schematic of a 6.4 kb HindIII (H) restriction fragment isolated from a cosmid clone containing the E2f2 locus. The intron-exon structure of E2f2 and three other genes identified within this restriction fragment are shown beneath this line. Mpp6 (CG9250) is divergently transcribed from E2f2, and its translational start site lies 900 bp upstream of the E2f2 translational start site. We have not recovered Mpp6 cDNAs, and the arrow represents a 450 bp open reading frame that predicts a 148 residue protein highly similar to mammalian M-phase phosphoprotein-6 (Matsumoto-Taniura et al., 1996). Only the first exon and intron of CG9248 are contained in the HindIII fragment shown. The next line indicates the extent of Df(2L)E2f2329, which was generated by excision of l(2)16402a. The next series of lines each indicate a region of genomic DNA included in a P-element transgene used for constructing E2f2 mutant flies (E2f2–; Mpp6+ and E2f21-188; Mpp6+), or for rescuing E2f2 mutant phenotypes (E2f2+; Mpp6+ and E2f2+; Mpp6–). R and S indicate the RsrII and SacII restriction sites used for constructing the P[E2f2+; Mpp6–] and P[E2f2–; Mpp6+] transgenes, respectively. (B) Northern blot hybridization of total RNA extracted from dissected ovaries and simultaneously hybridized with E2f2 and rp49 probes. rp49 encodes a ribosomal protein and is used as a loading control. Lane 1, yw67 wild type; lane 2, Df(2L)E2f2329/E2f21-188; Lane 3, Df(2L)E2f2329/Df(2L)E2f2329.

View larger version (131K): [in a new window]   Fig. 4. Decreased chorion gene amplification and thin eggshells caused by loss of E2f2. (A) Photomicrograph of a yw67 wild-type egg from the dorsal perspective. All other panels show anterior towards the left and dorsal towards top. (B-D) Eggs laid by a Df(2L)E2f2329/Df(2L)E2f2329 mutant females. Note that the chorions are more translucent compared with wild type. (E) Egg laid by a Df(2L)E2f2329/Df(2L)E2f2329; P[E2f2+; Mpp6+] female. (F) Southern hybridization was used to measure chorion gene amplification in stage 13 egg chambers dissected from yw67 wild type (lanes 1, 2, 3) and Df(2L)E2f2329/Df(2L)E2f2329; P[E2f2–; Mpp6+]/+ (lanes 4, 5, 6) females. Genomic DNA (1 µg, lanes 1 and 4; 2.5 µg, lanes 2 and 5; 5 µg, lanes 3 and 6) was simultaneously hybridized with a rosy gene probe and a third chromosome chorion gene cluster probe. The intensity of the 7.8 kb rosy fragment and the 3.8 kb chorion fragment were compared for each lane using a PhosphoImager. A 50% decrease in chorion gene amplification is reproducibly observed in E2f2 mutants.

View larger version (111K): [in a new window]   Fig. 5. E2f2 restricts follicle cell DNA replication to gene amplification foci. (A) A wild-type stage 10B egg chambers stained with DAPI. (A') Schematic of a wild-type stage 10B egg chamber. At this stage, the columnar follicle cells that execute chorion gene amplification and that will eventually secrete the eggshell surround the developing oocyte in a single epithelial layer. Dissected ovaries were pulse labeled with BrdU for 1 hour and then immediately fixed. Incorporated BrdU was detected by indirect immunofluorescence. (B-F) Stage 10B egg chambers; (B'-F') stage 13 egg chambers. The left image in each panel is a surface view of the follicle cell epithelium located over the oocyte. The right image in each panel is a high magnification view of a single follicle cell nucleus. (B) yw67 wild type. Distinct foci of BrdU incorporation corresponding with sites of gene amplification are observed in wild type. (C) Df(2L)E2f2329/Df(2L)E2f2329; P[E2f2–; Mpp6+]/+. BrdU incorporation is detected throughout E2f2 null mutant follicle cells. (D) Df(2L)E2f2329/Df(2L)DS8, P[E2f21-188; Mpp6+]. This phenotype is similar to null at stage 13, but slightly weaker at stage 10B. (E) Df(2L)E2f2329/Df(2L)E2f2329; P[E2f2+; Mpp6+]/+. A wild type E2f2 transgene rescues the null mutant phenotype. (F) E2F216402a/Df(2L)DS8, P[E2f21-188; Mpp6+]. In this hypomorphic situation, different classes of replication patterns are apparent (best seen in F'): replication throughout the entire nucleus (large arrow), normal gene amplification foci (large arrowhead), absent replication (small arrowhead), and both amplification foci and genomic replication (small arrow).

View larger version (16K): [in a new window]   Fig. 6. DNA content of E2f2 mutant follicle cells determined by FACS analysis. (A) Nuclei preparations from yw67 wild-type ovaries were stained with propidium iodide and subjected to FACS analysis. Nuclei with 2C DNA content are from mitotically active follicle cells, and the three follicle cell endocycles give rise to the 4C, 8C and 16C nuclei. A small 32C peak is always observed in wild type, probably from other less abundant cell types in the ovary (e.g. nurse cells). (B) FACS profile of nuclei prepared from Df(2L)E2f2329/Df(2L)DS8, P[E2f21-188; Mpp6+] ovaries. The size of the 32C peak varies between preparations, with this particular profile containing the largest peak obtained. (C) Intact follicle cells were prepared from c323:GAL4/+; Df(2L)E2f2329/+; UAS-GFP/+ ovaries and subjected to FACS analysis. The open profile represents signal from the DNA-binding dye Hoechst 33342, and the shaded profile represents GFP-positive cells. The GAL4 driver begins expression during stage 9 and continues until the end of oogenesis. By stage 9, all follicle cells have completed the first endocycle S phase, and therefore GFP-positive cells are only found in the 8C and 16C populations. (D) FACS profile of follicle cell preparations from c323:GAL4/+; Df(2L)E2f2329/Df(2L)DS8, P[E2f21-188; Mpp6+]; UAS-GFP/+ ovaries. Note that the profile is similar to wild type, indicating that E2f2 mutant follicle cells do not achieve a 32C ploidy value. (E) FACS profile of follicle cell preparations from Rbf120/Rbf14 ovaries. Note the population of cells at 32C. (F) Equal amounts of follicle cells prepared from yw67 and Rbf120/Rbf14 ovaries were mixed prior to FACS analysis, because the 16C peak was reproducibly small in the Rbf mutant preparations. Note that five peaks are clearly visible when compared with wild type (C).

View larger version (22K): [in a new window]   Fig. 7. E2f2 mutant follicle cells terminate endocycles on schedule. BrdU-positive nuclei were counted in photomicrographs of stage 9 and stage 10 egg chambers dissected from wild-type and Df(2L)E2f2329/Df(2L)E2f2329 mutant females. Each measurement represents an average of counts from 8-10 egg chambers.

View larger version (176K): [in a new window]   Fig. 8. Replication initiation proteins are mis-localized in E2f2 mutant follicle cell nuclei. Ovaries were dissected, fixed and treated with anti-ORC2 (left panels), anti-ORC5 (middle panels) or anti-CDC45L (right panels) antibodies. An image of a single nucleus from a stage 10B egg chamber is shown in each panel. (A) yw67 wild type. These three proteins are detected in a focus coincident with the third chromosome chorion cluster. (B) Df(2L)E2f2329/Df(2L)E2f2329; P[E2f2–; Mpp6+]/+. In the mutant, these replication proteins are not localized. (C) Df(2L)E2f2329/Df(2L)E2f2329; P[E2f2+; Mpp6+]/+. Mis-localization in the mutant is rescued by a wild-type E2f2 transgene.

View larger version (82K): [in a new window]   Fig. 9. Gene expression analysis in E2f2 mutant follicle cells. Total RNA was isolated from intact follicle cells prepared by trypsin dissociation of dissected ovaries and subjected to RT-PCR analysis with gene specific primers. Left panels are from yw67 wild-type RNA, and right panels are from Df(2L)E2f2329/Df(2L)DS8, P[E2f21-188; Mpp6+] RNA. RT-PCR reactions were sampled at cycle 22 (lane 1), cycle 25 (lane 2), cycle 30 (lane 3) and cycle 40 (lane 4) for ORC2, ORC5, ORC1, RNR2 and PCNA, and cycles 5, 10, 15 and 20, for rp49 (lanes 1-4, respectively).