Analysis of a Drosophila cyclin E Hypomorphic Mutation Suggests a Novel Role for Cyclin E in Cell Proliferation Control During Eye Imaginal Disc Development

Corresponding author: Helena Richardson, Department of Genetics, University of Adelaide, South Australia, Australia, 5005., herichardson{at}gina.science.adelaide.edu.au (E-mail).

Communicating editor: K. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have generated and characterized a Drosophila cyclin E hypomorphic mutation, DmcycE^JP, that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w⁺lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w⁺lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycE^JP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog, cdc2c, retinoblastoma, or p21^CIP1 homolog dacapo, which encode proteins known to physically interact with Cyclin E, modified the DmcycE^JP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhanced the DmcycE^JP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhanced the DmcycE^JP phenotype without affecting the number of cells entering S phase, but by decreasing the number of cells entering mitosis. Our analysis establishes the DmcycE^JP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycE^JP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development.

THE coordination of cell proliferation with morphogenesis is important during the development of metazoans. Studies in Drosophila melanogaster have revealed that at different developmental stages, different molecular mechanisms are used to control cell proliferation (reviewed by EDGAR and LEHNER 1996 ). Early in Drosophila development, regulation largely occurs at the G2 to M phase (mitosis) transition, whereas later in development, regulation at the G1 to S phase (DNA replication) transition becomes more important. Studies in cultured mammalian cells have shown that in G1 phase, external signals determine whether a cell will enter S phase or exit the cell cycle and quiesce or initiate differentiation. This behavior is also seen in developing Drosophila tissues. For example, in the developing eye imaginal disc at the third instar larval stage, a band of cells anterior to a furrow, known as the morphogenetic furrow (MF), arrest in G1 in response to developmental signals (reviewed by WOLFF and READY 1993 ; THOMAS et al. 1994 ; HEBERLEIN et al. 1995 ). Posterior to this furrow a subset of cells, known as the precluster cells, terminally differentiate to form photoreceptor cells. The remaining cells enter a synchronous S phase, then either complete the cell cycle and differentiate or undergo apoptosis (BAKER and RUBIN 1992 ). This spatial arrangement of cell cycle phases in the developing eye imaginal disc presents an excellent system in which to study the regulation of the G1 to S phase progression.

Progression through the cell cycle is controlled by the activity of the cyclin-dependent ser/thr protein kinases (Cdks) associated with their regulatory cyclin partners (reviewed by PINES and HUNTER 1991 ; REED 1992 ). Cyclins contain a conserved region known as the cyclin box, required for binding to their Cdk partner (JEFFREY et al. 1995 ). In addition, many cyclins vary dramatically in level during the cell cycle because of the presence of destruction signals that target them for ubiquitin-dependent degradation at specific stages of the cell cycle (reviewed by KING et al. 1996 ). During the G2 to M phase transition, Cdk1 (Cdc2) associates with Cyclin B or Cyclin A to form active kinases that phosphorylate substrates and initiate the events of mitosis. In the G1 to S phase transition of mammalian cells, Cdk4(6) associates with Cyclin D while Cdk2 associates with Cyclin E to form active kinases required for the G1 to S phase transition. In mammalian cells, Cyclin A also associates with Cdk2 to form an active kinase that plays a role in entry into or passage through S phase.

In mammalian cells Cyclin D, Cyclin E, and Cyclin A appear to play different essential roles in entry into or through S phase (reviewed by DESDOUETS et al. 1995 ; REED 1996 ). In Drosophila, Cyclin E is essential for the G1 to S phase transition during embryogenesis and is down-regulated in G1-arrested cells (RICHARDSON et al. 1993 , RICHARDSON et al. 1995 ; KNOBLICH et al. 1994 ). A role for Cyclin A in S phase in Drosophila is less clear. Cyclin A interacts specifically with the Drosophila G2/M phase Cdk, Cdc2 (STERN et al. 1993 ) and not the G1/S phase Cdk, Cdc2c (SAUER et al. 1995 ). Cells from cyclin A mutant embryos exhibit an apparent G2 phase arrest and enter into ectopic endoreplicative S phases mediated by Cyclin E (LEHNER et al. 1991 ; KNOBLICH et al. 1994 ), suggesting that Cyclin A is not normally required for S phase in Drosophila. Conversely, however, ectopic expression of cyclin A can induce S phases (LEHNER et al. 1991 ; DONG et al. 1997 ; SPRENGER et al. 1997 ). The role of Drosophila Cyclin D is as yet uncharacterized.

All three of these G1 cyclins have been shown to be rate-limiting for entry into S phase in mammalian cells, since ectopic overexpression results in premature entry into S phase (reviewed by REED 1996 ). Likewise, in Drosophila, ectopic overexpression of Cyclin E or Cyclin A can induce G1 cells to enter S phase (LEHNER et al. 1991 ; KNOBLICH et al. 1994 ; RICHARDSON et al. 1995 ; DONG et al. 1997 ; SPRENGER et al. 1997 ), although this is yet to be shown for Cyclin D. Although all three G1 Cyclin/Cdks in mammalian cells can be rate-limiting for S phase, their substrates appear to be different (HIGASHI et al. 1995 ; KITAGAWA et al. 1996 ; reviewed by REED 1996 ). Cyclin D/Cdk4 is thought to function primarily by phosphorylating and inactivating the tumor suppressor protein retinoblastoma (Rb) (reviewed by REED 1996 ). Rb functions by binding to and inactivating the E2F/DP transcription factor required for the transcription of S phase genes (reviewed by BARTEK et al. 1996 ). Binding and phosphorylation of Rb by Cyclin D/Cdk4 in mid-G1 inactivates Rb allowing the E2F/DP transcription factor to function. Phosphorylation of Rb by Cyclin A, and perhaps Cyclin E, later in the G1/S phase may also play a role in inactivating Rb (RESNITZKY et al. 1995 ; ROSENBERG et al. 1995 ; KITAGAWA et al. 1996 ). In Drosophila, although homologs of Rb (RBF), E2F (dE2F), and DP (dDP) have been isolated (DYNLACHT et al. 1994 ; OHTANI and NEVINS 1994 ; DU et al. 1996 ), it is unclear how their activity is regulated by Cyclin D or Cyclin A. However, Cyclin E appears to be involved in a mutually activating regulatory loop with E2F (DURONIO and O'FARRELL 1994 , DURONIO and O'FARRELL 1995 ; DURONIO et al. 1995 , DURONIO et al. 1996 ; SAUER et al. 1995 ), and there is genetic and biochemical evidence suggesting that Cyclin E may act to phosphorylate and inhibit RBF activity (DU et al. 1996 ).

In addition to activation of Cdk activity by cyclin association, Cdks are regulated by phosphorylation and by the binding of inhibitory proteins (reviewed by SHERR and ROBERTS 1995 ; LEW and KORNBLUTH 1996 ). During the G2 to M phase transition, dephosphorylation of the Cdk1 tyr 15 and thr 14 residues by Cdc25 (String in Drosophila) can be the rate-limiting step for entry into mitosis (EDGAR and O'FARRELL 1989 , EDGAR and O'FARRELL 1990 ). In mammalian cells, Cdc25 homologs have also been implicated in dephosphorylating and activating Cyclin E/Cdk2 and Cyclin A/Cdk2 in the G1 to S phase transition (GU et al. 1992 ; JINNO et al. 1994 ; HOFFMAN et al. 1994 ; STEINER et al. 1995 ). However, the existence and importance of this control in the activation of Drosophila Cyclin E/Cdc2c or Cyclin A/Cdc2 is not known.

Cdk inhibitory proteins appear to play a major role in the regulation of Cdk activity in the G1 to S phase transition (reviewed by SHERR and ROBERTS 1995 ). The p21^CIP1 class of inhibitors binds to Cyclin D/Cdk4(6), Cyclin E/Cdk2, and Cyclin A/Cdk2 and inhibits their activity leading to G1 arrest. A recently identified Drosophila homolog of p21^CIP1, dacapo, appears to be important for inhibiting Cyclin E/Cdc2c activity during exit into a terminal G1 arrest prior to differentiation (DE NOOIJ et al. 1996 ; LANE et al. 1996 ). However, in the transient G1 arrest that occurs anterior to the MF during eye development, dacapo is not expressed, implicating another mechanism in this G1 arrest.

Recent studies have shown that the roughex (rux) gene is an important regulator of the developmental G1 arrest during eye development. In the rough eye mutant roughex (rux), cells fail to arrest in G1 prior to the MF (THOMAS et al. 1994 ). Recent studies have shown that Rux acts by negatively regulating the activity of Cyclin A/Cdc2 (SPRENGER et al. 1997 ; THOMAS et al. 1997 ). Conversely, Cyclin A appears to be stabilized by Rca1 during eye development (DONG et al. 1997 ). Thus Rux and Rca1 act antagonistically on Cyclin A to control its function (DONG et al. 1997 ; SPRENGER et al. 1997 ; THOMAS et al. 1997 ). Cyclin E/Cdc2c has been shown to phosphorylate and inactivate Rux, leading to activation of Cyclin A/Cdc2 (SPRENGER et al. 1997 ; THOMAS et al. 1997 ). Thus Cyclin A can act to induce entry into S phase in Drosophila, but is normally inhibited by Rux until after S phase initiation, mediated by Cyclin E/Cdc2c. A negative regulator of Cyclin E/Cdc2c activity in the developmental G1 arrest in eye development has yet to be discovered.

Although much is known about the molecular basis of the G1 to S phase transition in metazoans from studies in mammalian cultured cells, genetic analysis in Drosophila presents a means to discover novel genes and mechanisms that control cell proliferation during development. In this article we describe a hypomorphic allele of Drosophila Cyclin E, DmcycE^JP, that results in a rough eye phenotype due to insufficient cells entering S phase during eye development. We show that DmcycE^JP represents a sensitive system for the identification of interacting genes by their ability to act as dominant enhancers or suppressors of this phenotype. We demonstrate genetic interactions at the S phase level between DmcycE^JP and the G1/S regulatory genes, Drosophila cdc2c, RBF, dE2F, and human p21^CIP1. Interestingly, we show that the G2/M phase regulatory genes, Drosophila cyclin A and string (cdc25 phosphatase), genetically interact with DmcycE^JP without affecting S phases but by affecting mitoses. This result suggests a novel role for DmcycE, besides its role in the G1 to S phase transition, during Drosophila development.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of 14.11G and 14.11GX20:
P[w⁺m-lac] 14.11G was isolated in a P-element screen for dominant enhancers of the eye mutant rough; however, with passage, the mild enhancement initially observed was lost. 14.11G (DmcycE^14.11G) flies were out-crossed to remove the third chromosome carrying rough, and this stock was used for all subsequent analyses. For P-element excision, DmcycE^14.11G flies were crossed to w¹¹¹⁸; Adh/SM6a, Cy; P[ry⁺2-3] Sb/TM6b, or w¹; +/CyO, Cy; Sb e¹ P[ry⁺2-3 99B]/TM6 and DmcycE^14.11G/Cy; P[ry⁺2-3] Sb/+ males were crossed to Sco/CyO or Star/CyO females. White-eyed revertant flies containing the DmcycE^14.11G chromosome were isolated and stocks generated. One of these white^- revertant lines, 14.11GX20 (DmcycE^JP), exhibited a rough eye phenotype when transheterozygous with DmcycE null alleles. Homozygous viable DmcycE^JP flies exhibited small, rough eyes and occasional wing defects. P-element excision from DmcycE^JP was achieved as follows: w¹¹¹⁸; Adh/SM6a, Cy; P[ry⁺2-3] Sb/TM6b flies were crossed to b DmcycE^JP bw, and from the progeny of this cross, males of the genotype DmcycE^JP/SM6a; P[ry⁺2-3]Sb/+ or DmcycE^JP/Adh; P[ry⁺2-3]Sb/+ were crossed to b DmcycE^JP cn bw females. The progeny of this cross were scored for reversion of the rough eye phenotype to wild type. Reversion to wild type occurred at a frequency of ~70% if DmcycE^JP/SM6a males were used and at ~90% if DmcycE^JP/Adh males were used. The reason for the increase in reversion frequency with the Adh chromosome is presumably due to chromosome pairing and gene conversion after P-element excision, which is inhibited by the SM6a balancer chromosome (ENGELS et al. 1990 ). Stocks were generated from a number of the DmcycE^JP revertants for further analysis.

Fly strains and genetic interaction analysis:
For the genetic interaction analysis, DmcycE^JP was made isogenic for the second and third chromosomes to minimize any variation in phenotype due to genetic background. In all cases a consistent phenotype was observed when flies were maintained at 25°. To test mutations in other cell cycle genes for dominant genetic interactions with DmcycE^JP, stocks were generated that contained DmcycE^JP (either heterozygous over CyO or homozygous) together with the test allele over a balancer chromosome. For test alleles on the second chromosome (dDP, dacapo, rca1, cdc2, and cyclin B), recombinants with DmcycE^JP were generated using marked DmcycE^JP stocks and the recombinant stock maintained over a CyO or a CyO, Tb second chromosome balancer. For test alleles on the X chromosome (RBF and rux), the stock was balanced over the FM7C w^a, y, B chromosome. Test alleles on the third chromosome were maintained over TM6B, Tb, Hu or TM2, Ubx balancer chromosomes.

For analysis of genetic interactions with DmcycE^JP, stocks were outcrossed to DmcycE^JP at 25°, and progeny that were homozygous for DmcycE^JP and heterozygous for the test allele (at least 50 progeny) were scored for their eye phenotype compared with DmcycE^JP. For bromo-deoxyuridine (BrdU)-labeling experiments, stocks containing the test allele on the third chromosome were balanced over the TM6B, Tb chromosome, while those containing the test allele on the second chromosome were balanced over a CyO, Tb chromosome. Flies were outcrossed to DmcycE^JP and nontubby larvae (those that were homozygous for DmcycE^JP and heterozygous for the test allele) were selected for analysis. The cyclin A⁵ allele could not be maintained over the TM6B balancer and was therefore established as a stock over TM2, Ubx for analysis by BrdU-labeling. To obtain DmcycE^JP larvae containing one copy of the RBF deficiency Df(1)Su(s)83 chromosome, for BrdU-labeling, Df(1)Su(s)83, white⁺/FM7C, white^a; DmcycE^JP females were crossed to white⁺; DmcycE^JP males, and from this cross female larvae with yellow Malpighian tubules (white⁺ homozygotes) were selected.

Fly stocks containing the specific mutations that were tested are listed below. cyclin A (LEHNER and O'FARRELL 1989 ): cyclin A⁵/TM2 (P-element insertion), Df(3L)vin3 (68C5-6; 68E3-4)/TM3, Sb (deletion); string (EDGAR and O'FARRELL 1989 ; EDGAR et al. 1994 ): stg⁵/TM3, Sb (strong EMS-generated allele); stg¹/TM6B (weak EMS-generated allele); stg^AR2/TM3, Sb (transcriptional null generated by P-element excision); Df(3R)3450 (98A1-7; 99A6-8)/TM6B (deletion); dE2F (DURONIO et al. 1995 ): dE2F⁹¹/TM3, Sb, Ubx-lacZ (EMS-generated allele); dE2F^RM729/TM3, Sb, Ubx-lacZ (P-element insertion); dDP (DURONIO et al. 1998 ): dDP^vr10/CyO (EMS-generated allele, semilethal); dacapo (DE NOOIJ et al. 1996 ; LANE et al. 1996 ): dap⁴ (intragenic deletion generated by imprecise excision of the dap¹ P element); cyclin B (KNOBLICH and LEHNER 1993 ): Df(2R)59AB/CyO (deletion); cyclin B3 (SIGRIST et al. 1995 ): Df(3R)XTAI (96B; 96D) th¹, st¹, ri¹, roe¹, p¹/Dp(3;3) (94D; 96E) SuM(3)w¹³, st¹, e¹ (deletion); cdc2 (LEHNER and O'FARRELL 1990B ): cdc2^E1-9/CyO (EMS-generated allele); cdc2c (STERN et al. 1993 ): Df(3R)H81 (92E9; 92E15)/TM2 (deletion); Df(3R)H-B79 (92B2-3; 92F13-93A1)/TM2 (deletion); cdc2c^JS (EMS-generated allele)/TM6B (H. RICHARDSON, J. SECOMBE and R. SAINT, unpublished data); Dmcks (H. RICHARDSON, P. KYLSTEN, B. J. JENNINGS, P. H. O'FARRELL, S. I. REED and R. SAINT, unpublished data): Df(3R)P14 (90C2-D1; 91A1-2) sr¹/T(2:3)ap^Xa (deletion); cyclin C (LEOPOLD and O'FARRELL 1991 ): Df(3R)ry506-85C (87D1-2; 88E5-6)/Dp(3:Y), ry (deletion); RBF (DU et al. 1996 ): Df(1)Su(s)83 (1B10; 1D6-E1), y¹, cho¹, ras¹, v¹/Dp(1:Y)y², sc/C(1)DX, y¹, f¹ (deletion); roughex (THOMAS et al. 1994 ): y¹, cho¹, rux⁸/FM7c (X-ray-generated allele, small deletion); y¹, rux⁷/FM7C (X-ray allele, rearrangement); and rca1 (DONG et al. 1997 ): rca1³³⁰⁰/CyO (P-element allele). Previously described stocks of DmcycE mutations used in this study were the null allele DmcycE^AR95/CyO (KNOBLICH et al. 1994 ), the deletions Df(2L)TE35D-1/CyO and Df(2L)TE35D-3/CyO, the hypomorphic EMS-generated alleles DmcycE^P28/CyO and DmcycE^P41/CyO (H. RICHARDSON, unpublished data), and the partial female sterile allele DmcycE^PZ01672/CyO (LILLY and SPRADLING 1996 ).

The cdc2c allele, cdc2c^JS, was generated by EMS mutagenesis and isolated as an enhancer of the DmcycE^JP rough eye phenotype (H. RICHARDSON, J. SECOMBE and R. SAINT, unpublished data). This mutation was shown to be an allele of cdc2c by its failure to complement the cdc2c deficiencies Df(H81) and Df(HB-79) and by rescue of its lethality by a genomic cdc2c transgene (obtained from C. LEHNER, unpublished data).

Stocks for increasing the dosage of genes were, for RBF (DU et al. 1996 ): P[w⁺ GMR-RBF⁴ (homozygous for the GMR-RBF² transgene on the third chromosome)]; for roughex (THOMAS et al. 1997 ): P[ry⁺ 6.0C1 (third chromosome)]/TM6B; for dacapo (DE NOOIJ et al. 1996 ): GMR-dacapo⁵⁶ (third chromosome); and human p21^CIP1 (DE NOOIJ and HARIHARAN 1995 ): GMR-p21^CIP1 (third chromosome). Fly stocks were obtained from the Bloomington stock center or relevant laboratories.

Molecular analysis:
For analysis of P-element reversion, genomic DNA prepared from DmcycE^14.11G, DmcycE^14.11G white^- revertant, DmcycE^JP and DmcycE^JP revertant adult flies was analyzed using polymerase chain reaction (PCR), using P-element end and DmcycE genomic DNA primers. PCR was carried out using 0.1 µg of genomic DNA and 0.1 µg of each of the primers, in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.01% gelatin, 0.2 mM of each dNTP, and 0.025 units Taq polymerase (Perkin-Elmer, Branchburg, NJ) for 40 cycles of 94° for 30 sec, 48° for 1 min, 72° for 1 min in a thermal sequencer (Corbett Research). DmcycE genomic primers spanning the insertion site were as follows: 5' primer,5'-CGTTTTAGTACTGCGCTC-3'; 3' primer, 5'-CGGAATTCGTATGTACGTAGATTGTGGGTAAGG-3' (the underlined region at the 5' end represents a nonhybridizing sequence containing an EcoRI site for cloning); and the P-element end primer was 5'-CGACGGGACCACCTTATG-3'.

BrdU-labeling, DmcycE antibody staining and Western analysis:
For BrdU-labeling, larval tissues were dissected and incubated with 60 µg/ml BrdU in Schneider's tissue culture medium for 60 min at 25°. Tissues were fixed in ethanol, rehydrated, hydrolyzed in 2 N HCl in phosphate-buffered saline (PBS) for 60 min at 25° and BrdU-labeled cells were detected, after incubations with a mouse monoclonal anti-BrdU antibody (Becton-Dickinson, San Jose, CA) followed by anti-mouse-biotin (Jackson ImmunoResearch Labs., Inc., West Grove, PA) and Vectastain (Vector Labs, Inc., Burlingame, CA) streptavidin-Horse Radish Peroxidase (HRP), by HRP staining (as described in RICHARDSON et al. 1993 ). Detection of DmcycE in embryos and larval tissue was carried out using a DmcycE mouse polyclonal antibody (RICHARDSON et al. 1995 ). Mitotic cells in larval eye imaginal discs were detected using the mitosis-specific mouse monoclonal MPM-2 antibody (DAKO Corp., Carpinteria, CA). Fixation and antibody staining of embryos was carried out as described previously (RICHARDSON et al. 1995 ). Larval tissues were fixed in 4% formaldehyde, 0.113 M PIPES, 2.25 mM MgSO₄, 1.125 mM EGTA, 1.125% NP-40, pH 6.9 for 30 min on ice. Blocking and antibody staining of larval tissues was carried out in PBS containing 0.3% Triton X-100, 5 mg/ml BSA. Antibody staining was detected by incubation with an anti-mouse-biotin antibody (Jackson ImmunoResearch Labs.) followed by streptavidin-FITC (Jackson ImmunoResearch Labs.) or Vectastain (Vector Labs, Inc.) streptavidin-HRP and HRP staining. DNA was stained for 5 min with 10 µg/ml Hoechst 33258 in PBS with 0.1% Triton X-100. Fluorescent-labeled samples were mounted in 30% glycerol with 1% p-phenylenediamine p1519 (Sigma Chemical Co., St. Louis) and viewed using a MRC 1000 Confocal microscope with a x60 oil immersion objective or using an Olympus Provis AX70 microscope (Olympus Corp., Lake Success, NY) mounted with a Photometrics (Tucson, AZ) Nu200 CCD camera, with a x40 oil immersion objective.

Western analysis was carried out on protein extracts prepared from dissected eye discs (20 pairs per sample), and the immunoblot was probed with a DmcycE antibody (ascites fluid from the 8B10 monoclonal line; RICHARDSON et al. 1995 ) or a Cdc2c mouse monoclonal antibody (obtained from C. LEHNER), detected by incubation with an anti-mouse-HRP secondary antibody (Jackson ImmunoResearch Labs.) followed by enhanced chemiluminescence (Amersham Inc., Buckinghamshire, UK). Heat-shock induced hsp70-DmcycE type I and hsp70-DmcycE type II samples were prepared by heat-shocking larvae for 60 min at 37° followed by immediate dissection.

Preparation of adult eyes for scanning electron microscope analysis and sectioning:
Flies were prepared for scanning electron microscopy by dehydration in acetone, followed by air drying. Eyes were viewed without coating using a field emission scanning electron microscope at 1 kV accelerating voltage. Sectioning was carried out as described in RICHARDSON et al. 1995 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of an adult viable cyclin E P-element allele:
The insertion site of an adult viable P[w⁺m-lac]-element allele, 14.11G, was localized to the cyclin E gene DmcycE by chromosome in situ hybridization (data not shown). Genomic Southern analysis and DNA sequence analysis of the genomic DNA flanking the P-element insertion site revealed that the 14.11G P[w⁺m-lac] element is inserted ~14 kb 5' to the transcriptional start site of the zygotic DmcycE transcript (L. JONES, W. WINNALL, R. SAINT and H. RICHARDSON, unpublished results).

Homozygous DmcycE^14.11G flies were found to be viable, fertile, and indistinguishable from wild type in their rate of development, but a minor eye defect was observed (Figure 1B). Eyes from DmcycE^14.11G appeared slightly reduced in size on the ventral side and occasionally contained regions of mild disorganization. To obtain more severe phenotypes, the 14.11G P[w⁺m-lac] element was mobilized from DmcycE^14.11G flies mediated by 2-3 transposase, and white^- revertant progeny were selected (see MATERIALS AND METHODS). Approximately 3% of progeny carrying the insertion chromosome were viable white^- revertants, due to precise excision of the P element as assayed using PCR analysis (data not shown). At low frequency (0.18%), homozygous lethal revertants were obtained that were also lethal over DmcycE null alleles, suggesting that loss of the P element in these cases was accompanied by deletion into essential regions of the DmcycE gene. One white^- revertant allele, 14.11GX20, renamed DmcycE^JP, was found to give a strong rough eye phenotype over DmcycE null alleles (Figure 1C). Genomic Southern analysis of the DmcycE^JP allele using P-element end probes revealed that there was a single copy of the P element in the genome (data not shown). In addition, probing genomic Southerns with DmcycE probes showed that the P element was still present in the same position but had undergone an internal deletion of ~4 kb, removing the white gene while maintaining the amp^R and lacZ genes and the P-element ends (L. JONES, W. WINNALL, R. SAINT and H. RICHARDSON, unpublished results). The presence of the lacZ gene was confirmed by LacZ antibody staining, with both DmcycE^JP and DmcycE^14.11G showing a Cyclin E-like pattern of LacZ expression in embryos, with the exception that, unlike DmcycE^14.11G, DmcycE^JP gave no expression of LacZ in third instar larval tissues (data not shown).

View larger version (169K): In this window In a new window Download PPT slide   Figure 1. DmcycEJP flies have rough eyes and a disorganized array of ommatidia. Scanning electron micrographs of adult eyes from (A) wild type, (B) DmcycE14.11G, (C) DmcycEJP, (D) DmcycEJP/DmcycEAR95, (E) DmcycEJP/TE35D-3 (DmcycE deficiency), and (F) DmcycEJP/DmcycEP41 show enhancement of the DmcycEJP rough eye phenotype when the dosage of DmcycE is decreased further using the amorphic DmcycEAR95 allele or the TE35D-3 deficiency or suppression when DmcycE dosage increased using the hypomorphic DmcycEP41 allele. DmcycE14.11G shows only slight hints of roughening and a slight reduction in the size of the eye on the ventral side. Eyes are oriented with anterior to the right and dorsal side up. Transverse sections of adult eyes from (G) wild type and (H) DmcycEJP show the fusion of ommatidia due to the loss of pigment cells (stained black). The arrows (in H) point to examples of ommatidia containing reduced complements of R cells.

To confirm that DmcycE^JP phenotype is caused by the presence of the internally deleted P element, 2-3 transposase was used to mobilize the P element from DmcycE^JP, and individuals were scored for reversion of the rough eye phenotype when crossed back to the DmcycE^JP allele (see MATERIALS AND METHODS). Reversion of DmcycE^JP rough-eyed flies to wild type, mediated by 2-3 transposase, occurred at high frequency, indicating that the internally deleted P element was responsible for the rough eye phenotype of DmcycE^JP. Furthermore, PCR analysis of a number of these revertants revealed that the P element had excised precisely. Thus, the DmcycE^JP rough eye mutant phenotype is caused by the internal deletion of the P element. It is curious that an internal deletion within a P element can create a new phenotype. It is possible that the internal deletion permits the P element to more efficiently sequester particular DmcycE enhancers or enhancers that are inaccessible to the w⁺ P-element allele, DmcycE^14.11G, resulting in greater disruption of DmcycE transcription in particular tissues. The change in the lacZ expression pattern observed for DmcycE^JP compared with DmcycE^14.11G is consistent with these possibilities.

The rough eye phenotype of DmcycE^JP is caused by a reduction in S phases during eye development:
DmcycE^JP flies are homozygous viable and fertile but exhibit small, rough eyes (Figure 1C). The eye is the only consistently affected tissue in DmcycE^JP flies, although at low frequency, wing defects such as small notches at the posterior/distal side of the wing and a shortening of the fifth vein were also observed (data not shown). In addition, a slight delay in development time was observed (11 days vs. 10 days to eclosion at 25°), indicating that there may be other effects on cell proliferation in DmcycE^JP mutants.

DmcycE^JP adult eyes showed disorganization in the arrangement of ommatidia, blistering on the surface of the eye and defects in bristle number and arrangement. The degree of disorganization of the eyes from DmcycE^JP homozygotes, however, was not as severe as that seen in eyes of individuals transheterozygous for DmcycE^JP and the severe allele DmcycE^AR95, or the DmcycE deficiencies Df(2L)TE35D-3 or Df(2L)TE35D-1 (Figure 1D and E; and data not shown). To test whether the converse was true, the dosage of DmcycE was increased by generating flies transheterozygous for DmcycE^JP and weak alleles of DmcycE. The first of these, DmcycE^PZ01672, is a homozygous viable, partial female sterile P-element allele that shows very mild eye roughening when placed over DmcycE null alleles (LILLY and SPRADLING 1996 ; data not shown). Two other weak alleles, DmcycE^P28 and DmcycE^P41, were also tested. DmcycE^P28 is an EMS-generated allele that is homozygous viable, female sterile and has a milder rough eye phenotype than DmcycE^JP (data not shown). Flies transheterozygous for DmcycE^JP and DmcycE^PZ01672 had essentially wild-type eyes (data not shown), whereas DmcycE^JP/DmcycE^P41 and DmcycE^JP/DmcycE^P28 flies showed a milder rough eye phenotype than homozygous DmcycE^JP flies (Figure 1F; and data not shown). The eyes of these flies were larger, the arrangement of ommatidia appeared more even, and there was less blistering. Thus the DmcycE^JP phenotype is capable of being suppressed by increasing the dosage of DmcycE and enhanced by decreasing the dosage of DmcycE. These results suggest that the DmcycE^JP allele is affecting the expression of DmcycE in the eye, but is not a null allele in the eye, and that the degree of disorganization of DmcycE^JP adult eyes is sensitive to the dose of DmcycE.

To examine the cellular basis of the eye disorganization, sections were taken of adult eyes from DmcycE^JP (Figure 1H). The most common defect observed in DmcycE^JP was the reduction or absence of pigment cells surrounding the photoreceptor cells, resulting in the fusion of the ommatidia. The number and arrangement of photoreceptor (R) cells within each ommatidium were generally normal (Figure 1H compared with G). Occasionally, more severe defects were observed where there were less than seven R cells per ommatidium (Figure 1H; and data not shown). The reduction in pigment cells, one of the last types of cells to be recruited during eye development, suggests that DmcycE^JP eye imaginal discs are defective in the cell cycle that occurs posterior to the MF. To examine the effect that the DmcycE^JP allele was having on cell proliferation during eye development, S phases were monitored by BrdU-labeling (Figure 2, A–C). While S phases in antennal discs from DmcycE^JP third instar larvae are similar to wild type, the effect of the DmcycE^JP allele on eye imaginal disc S phases was clearly apparent. In DmcycE^JP homozygous eye discs, the number of cells entering S phase posterior to the MF, and the number of S phase cells in the asynchronously proliferating undifferentiated cells anterior to the MF, were dramatically reduced (Figure 2C; Table 1 and Table 2). In addition, DmcycE^JP eye discs were clearly smaller than wild type, indicating that a decrease in cell proliferation of the undifferentiated eye disc cells was also occurring earlier in development. The reduction in eye imaginal disc S phases accounts for the disorganization and the reduction in size observed with DmcycE^JP adult eyes. The number of S phases from DmcycE^14.11G eye discs was not significantly altered compared with wild type, consistent with the adult eye phenotype (Figure 2B compared with A).

View larger version (60K): In this window In a new window Download PPT slide   Figure 2. DmcycEJP eye imaginal discs have a decreased number of S phase cells and reduced DmcycE expression. S phase cells in eye imaginal discs, labeled with BrdU, from (A) wild-type, (B) DmcycE14.11G, and (C) DmcycEJP third instar larvae, show the decrease in S phases in DmcycEJP eye imaginal discs. Confocal micrographs of DmcycE protein levels in eye imaginal discs from (D) wild-type and (E) DmcycEJP third instar larvae show low levels of DmcycE protein in DmcycEJP eye imaginal discs relative to wild type. DmcycE protein was detected using an anti-DmcycE antibody and visualized using an FITC-conjugate. D and E are shown at a higher magnification relative to A–C. The arrows indicate the morphogenetic furrow (MF). Eye imaginal discs are oriented with anterior to the right.

The expression of DmcycE in DmcycE^JP homozygotes was examined during embryogenesis and in various tissues during larval development by DmcycE antibody staining (data not shown; Figure 2D and Figure E). During embryogenesis, DmcycE expression did not differ significantly from wild type in mitotically proliferating or endoreplicating cells. In DmcycE^JP third instar larval brain lobes and wing, haltere, leg, and antennal imaginal discs, the level of DmcycE protein appeared to be slightly lower than wild type, although this did not appear to significantly affect S phases in these tissues (data not shown). In contrast, in DmcycE^JP larval eye imaginal discs where the number of S phase cells was significantly reduced (Figure 2C), there was a marked reduction in the level of DmcycE and in the number of cells expressing DmcycE compared with wild type (Figure 2E compared with D). This was confirmed by Western analysis, which showed that DmcycE^JP eye-antennal discs contained levels of DmcycE protein significantly lower than those of DmcycE^14.11G and wild-type eye-antennal discs (Figure 3). Thus it appears that the Dm-cycE^JP phenotype results from a reduction of DmcycE expression that primarily affects the developing eye.

View larger version (43K): In this window In a new window Download PPT slide   Figure 3. Western analysis of DmcycE protein levels in DmcycEJP eye-antennal imaginal discs. Protein samples, prepared from dissected eye-antennal discs from third instar larvae, were analyzed by immunoblotting with DmcycE mouse monoclonal sera (upper panel) or with anti-Cdc2c sera (detecting a doublet at ~34 kD; KNOBLICH et al. 1994 ) as a loading control (lower panel), and detected using enhanced chemiluminescence. Tracks are as follows: track 1, wild type; track 2, DmcycEJP; track 3, DmcycE14.11G; track 4, heat-shocked hsp70-DmcycE type I; track 5, heat-shocked hsp70-DmcycE type II. Tracks 4 and 5 are shown at a much shorter exposure time than tracks 1–3. Heat-shocked hsp70-DmcycE type I and type II serve as controls for the unmodified forms of DmcycE and give rise to bands at ~70 kD and ~95 kD, respectively, as well as lower molecular weight breakdown products. The DmcycE antisera detected protein bands at ~70 kD that comigrate with the heat-shocked hsp70-DmcycE type I, as well as a number of higher molecular weight bands that migrate between 97 kD and 116 kD in wild-type and DmcycE14.11G eye-antennal disc extracts (tracks 1 and 3; see RICHARDSON et al. 1995 ). Protein bands at these sizes were also detected in DmcycEJP eye-antennal disc extracts (track 2), but the levels were significantly reduced compared with wild-type and DmcycE14.11G eye-antennal disc extracts. Molecular weight markers are indicated.

The DmcycE^JP rough eye phenotype represents a sensitive system for analyzing DmcycE genetic interactors:
As described above, the DmcycE^JP rough eye phenotype is sensitive to the dosage of DmcycE. To determine whether DmcycE^JP represents a genetically sensitive system capable of responding to the dosage of interacting genes, we sought to test whether altering the dosage of genes known to interact with Cyclin E would modify the rough eye phenotype of DmcycE^JP.

First we tested the effect of halving the dosage of the cdc2c gene, encoding the Cdk partner of Cyclin E (KNOBLICH et al. 1994 ). Surprisingly, when we used the available deficiencies of cdc2c to decrease the dosage of cdc2c, the DmcycE^JP rough eye phenotype was suppressed rather than enhanced as may have been expected (data not shown). The reason for this may be because of the codeletion of a gene that acts as a dosage-sensitive suppressor of DmcycE^JP. We have recently isolated a specific cdc2c allele, cdc2c^JS, in a genetic screen using the DmcycE^JP allele, as an enhancer of the rough eye phenotype (H. RICHARDSON, J. SECOMBE and R. SAINT, unpublished data; see MATERIALS AND METHODS). As shown in Figure 4B, this allele enhances the DmcycE^JP rough eye phenotype. BrdU-labeling analysis revealed that the enhancement was a result of effects on S phase, because there were fewer cells labeled in eye imaginal discs from DmcycE^JP cdc2c^JS/+ flies than in DmcycE^JP discs (Figure 5B; Table 1 and Table 2). Importantly, altering the gene dosage of the G2/M regulatory protein kinase Cdc2, which does not physically interact with DmcycE (LEHNER and O'FARRELL 1990B ; KNOBLICH et al. 1994 ), did not alter the eye phenotype or alter the S phase pattern in the eye imaginal disc of DmcycE^JP (Figure 4C and Figure 5C; Table 1 and Table 2).

View larger version (170K): In this window In a new window Download PPT slide   Figure 4. Altering the dose of cdc2c or RBF, but not cdc2, and ectopic expression of human p21CIP1 modifies the rough eye phenotype of DmcycEJP. Scanning electron micrographs of adult eyes from (A) DmcycEJP, (B) DmcycEJP; cdc2cJS/+, (C) DmcycEJP, cdc2E1-9/DmcycEJP, +, (D) Df(1)Su(s)83 (RBF deficiency)/+; DmcycEJP, (E) GMR-RBF, (F) DmcycEJP; GMR-RBF, (G) GMR-p21CIP1, and (H) DmcycEJP; GMR-p21CIP1 show that reducing the dosage of cdc2c or increasing the dosage of RBF or p21CIP1 enhances the rough eye phenotype of DmcycEJP, while reducing the dosage of RBF suppresses the DmcycEJP rough eye phenotype. Reducing the dosage of cdc2 has no effect on the rough eye phenotype of DmcycEJP. GMR-RBF by itself results in a mild rough eye phenotype and GMR-p21CIP1 alone results in a severe eye roughening, but both of these phenotypes are enhanced by DmcycEJP. Eyes are oriented with anterior to the right and dorsal side up.

View larger version (81K): In this window In a new window Download PPT slide   Figure 5. Altering the dose of cdc2c and RBF, but not cdc2, affects S phases in DmcycEJP eye imaginal discs. BrdU-labeling, to reveal S phase cells, in eye imaginal discs from (A) DmcycEJP, (B) DmcycEJP; cdc2cJS/+, (C) DmcycEJP, cdc2E1-9/DmcycEJP, +, (D) Df(1)Su(s)83 (RBF deficiency)/+; DmcycEJP, (E) DmcycEJP; GMR-RBF, and (F) GMR-RBF shows that decreasing the dosage of cdc2c or increasing the dosage of RBF reduces the number of S phase cells posterior to the MF in DmcycEJP eye imaginal discs, while decreasing the dosage of RBF increases the number of S phases posterior to the MF in DmcycEJP eye imaginal discs. Reducing the dosage of cdc2 has no effect on the number of S phases relative to DmcycEJP. GMR-RBF by itself results in a slight reduction of S phase cells posterior to the MF (see Table 2). Anterior is to the right. Arrows point to the MF.

We then tested two other genes that encode proteins that physically interact with DmcycE. The tumor suppressor protein, retinoblastoma (RBF), may physically interact with Drosophila Cyclin E because DmcycE, associated with its kinase partner Cdc2c, is able to phosphorylate RBF in vitro (DU et al. 1996 ). Furthermore, Drosophila RBF has been shown to genetically interact with DmcycE, as decreasing the dosage of DmcycE by half enhances the rough eye phenotype that results from GMR (glass-responsive enhancer)-induced ectopic expression of RBF posterior to the MF in eye imaginal discs (DU et al. 1996 ). Another cell cycle regulator known to physically interact with Cyclin E is the Cdk inhibitor p21^CIP1, which acts to inhibit Cyclin/Cdk activity and prevent entry into S phase (SHERR and ROBERTS 1995 ). Ectopic expression of mammalian p21^CIP1 using the GMR enhancer in the Drosophila eye imaginal disc has been shown to inhibit entry of cells posterior to the MF into S phase, resulting in a rough eye phenotype (DE NOOIJ and HARIHARAN 1995 ). This effect is overcome by overexpression of DmcycE (I. HARIHARAN, personal communication), suggesting that the inhibition of S phase entry is caused by the inhibition of Cyclin E/Cdc2c activity. A Drosophila homolog of mammalian p21^CIP1, dacapo, has recently been isolated and shown to inhibit Cyclin E/Cdk2 (DE NOOIJ and HARIHARAN 1995 ; LANE et al. 1996 ). To determine whether RBF, p21^CIP1, or dacapo also interacted with DmcycE^JP, the dosage of these genes was increased or decreased in a DmcycE^JP background and the effect on the eye phenotype examined by scanning electron microscopy.

Decreasing the dosage of RBF resulted in suppression of the rough eye phenotype of DmcycE^JP, whereas ectopic overexpression of RBF, using the GMR enhancer that drives expression in cells posterior to the MF, enhanced the eye roughening observed in DmcycE^JP flies or GMR-RBF flies, which exhibit a slight eye roughening (Figure 4, D–F compared with A; Table 1). Likewise, DmcycE^JP increased the severity of the rough eye phenotype of GMR-p21^CIP1 (Figure 4H compared with G; Table 1). Furthermore, the DmcycE^JP rough eye phenotype was substantially enhanced by two copies of GMR-dacapo, which alone exhibited only a very mild eye roughening (Table 1; data not shown; I. HARIHARAN, personal communication). Conversely, DmcycE^JP flies homozygous for the dacapo null mutant dap⁴ showed strongly suppressed eyes (Table 1; data not shown). However, in this case, halving the dosage of dacapo was not sufficient to achieve suppression of the DmcycE^JP rough eye phenotype (data not shown).

To examine whether these interactions were occurring at the level of S phase induction, BrdU-labeling experiments were carried out (Figure 5, D–F compared with A; Table 2). Reducing the dosage of RBF in a DmcycE^JP background increased the number of S phases throughout the eye disc (Figure 5D; Table 2). Increasing the dosage of RBF in cells posterior to the MF, decreased the number of S phases in DmcycE^JP eye imaginal discs, relative to DmcycE^JP or GMR-RBF eye discs (Figure 5E compared with F and A; Table 2). Ectopic expression of human p21^CIP1 in cells posterior to the MF, using GMR-p21^CIP1 results in a significant decrease in S phase cells posterior to the MF (DE NOOIJ and HARIHARAN 1995 ; I. HARIHARAN, personal communication; Table 2; and data not shown). In DmcycE^JP; GMR-p21^CIP1 flies these S phase cells posterior to the MF were almost completely abolished (Table 2). This decrease in post-MF S phases, as well as the reduction of S phases anterior to the MF, probably accounts for the increased eye roughening observed with DmcycE^JP; GMR-p21^CIP1 flies compared with GMR-p21^CIP1 flies.

These results show that the DmcycE^JP rough eye phenotype is sensitive to the dosage of the known G1 regulators cdc2c, RBF, dacapo, and human p21^CIP1. Therefore, the DmcycE^JP allele provides a unique dosage-sensitive phenotype whereby genetic interactions with DmcycE can be explored.

Genetic interaction of DmcycE with other cell cycle regulatory genes:
To assess the ability of other previously identified cell cycle genes to interact with DmcycE^JP, flies homozygous for DmcycE^JP and heterozygous either for specific mutations (where possible, two different alleles) or for deficiencies removing these genes (Table 1) were generated. Genes known to have a role in the G1 to S phase transition, or required for the G2 to M phase transition in Drosophila, as well as genes with currently unspecified function in the cell cycle in Drosophila, were tested. The G1/S phase regulators tested were the S phase transcription factor genes dE2F and dDP, which are required for DmcycE transcription in endoreplicating tissues during embryogenesis (DURONIO et al. 1995 ; ROYZMAN et al. 1997 ; DURONIO et al. 1998 ). Drosophila G2 to M phase genes tested were cyclin A, cyclin B, cyclin B3, and the Schizosaccharomyces pombe cdc25 mitotic inducer homolog, string. Two other cell cycle genes that have poorly defined functions in the Drosophila cell cycle were also tested. The first of these is the Drosophila homolog of the suc1/CKS1/p13 (cks) gene (Dmcks) (H. RICHARDSON, P. KYLSTEN, B. J. JENNINGS, P. H. O'FARRELL, S. I. REED and R. SAINT, unpublished results). Cks binds to Cdk proteins and has been shown to be required at both the G1 to S phase transition and the G2 to M phase transition in budding yeast, but is also capable of inhibiting entry into mitosis when overexpressed (reviewed by PINES 1996 ). The second of these was cyclin C, which appears to play a role in general transcription (LECLERC et al. 1996 ). The effect of altering the dosage of these genes on the DmcycE^JP rough eye phenotype was analyzed by scanning electron microscopy, and the results are summarized in Table 1.

Decreasing the dosage of dE2F resulted in an enhancement of the DmcycE^JP rough eye phenotype (Figure 6B; Table 1). A similar result was obtained when the dosage of dDP was reduced (Table 1; data not shown). BrdU-labeling experiments confirmed that the enhancement of DmcycE^JP eye roughening by halving the dosage of dE2F resulted from effects on S phase, since fewer S phase cells were observed both anterior and posterior to the MF in eye imaginal discs (Figure 7B; Table 2). These results show that dE2F genetically interacts with DmcycE to promote the G1 to S phase transition.

View larger version (166K): In this window In a new window Download PPT slide   Figure 6. Altering the dose of dE2F, cyclin A, string (stg), roughex (rux), and rca1, but not cyclin B, modifies the DmcycEJP rough eye phenotype. Scanning electron micrographs of adult eyes from (A) DmcycEJP, (B) DmcycEJP; dE2F91/+, (C) DmcycEJP, Df(2R)59AB (cyclin B deficiency)/DmcycEJP, +, (D) DmcycEJP; cyclin A5/+, (E) DmcycEJP; stringAR2/+, (F) DmcycEJP, rca13300/DmcycEJP, +, (G) roughex7/+; DmcycEJP, and (H) DmcycEJP; P[ry+]roughex, show that reducing the dosage of dE2F, cyclin A, string, or rca1 or increasing the dosage of roughex enhances the rough eye phenotype of DmcycEJP, while decreasing the dosage of roughex suppresses the DmcycEJP rough eye phenotype. Decreasing the dosage of cyclin B has no significant effect on the rough eye phenotype of DmcycEJP. Eyes are oriented with anterior to the right and dorsal side up.

View larger version (161K): In this window In a new window Download PPT slide   Figure 7. Altering the dose of dE2F, but not cyclin A, string (stg), roughex (rux), or rca1 affects S phases in DmcycEJP eye imaginal discs. BrdU-labeling to reveal S phase cells in eye imaginal discs from (A) DmcycEJP, (B) DmcycEJP; dE2F91/+, (C) DmcycEJP; cyclin A5/+, (D) DmcycEJP; stringAR2/+, (E) DmcycEJP, P[ry+]roughex, and (F) DmcycEJP, rca13300/DmcycEJP, + shows that decreasing the dosage of dE2F reduces the number of S phase cells posterior to the MF in DmcycEJP eye imaginal discs, while reducing the dosage of cyclin A, string or rca1 or increasing the dosage of roughex has no significant effect. Eye imaginal discs are oriented with anterior to the right. Arrows point to the MF.

As summarized in Table 1, halving the dosage of the G2 to M phase genes cyclin B or cyclin B3, using deficiencies, did not affect the DmcycE^JP rough eye phenotype, nor did deficiencies that remove cyclin C or Dmcks (Figure 6C; and data not shown). In contrast, mutations in cyclin A and the cdc25 mitotic inducer, string, were found to be dominant enhancers of the DmcycE^JP rough eye phenotype (Figure 6D and Figure E). This result was initially surprising, given that cyclin A and string mutants arrest before mitosis in Drosophila embryogenesis (EDGAR and O'FARRELL 1989 ; LEHNER and O'FARRELL 1989 ). However, there is evidence that Cyclin A and String can play a role in passage into or through S phase during eye development, since cyclin A and string mutations dominantly suppress mutations in rux, which is required to establish the G1 arrest during eye development (see Introduction). The genetic interaction of string or cyclin A with rux is consistent with their enhancement of the DmcycE^JP rough eye phenotype.

Given the interaction we had observed between DmcycE^JP and cyclin A or string mutations, we were interested to test whether the DmcycE^JP rough eye phenotype was also sensitive to the dosage of the Cyclin A regulators, rux and rca1 (see Introduction). Fly stocks homozygous for DmcycE^JP and heterozygous either for rca1 or rux mutations, or homozygous for a rux genomic rescue construct, P[ry⁺] rux, were generated and adult eyes examined. Decreasing the dosage of rca1 or increasing the dosage of rux resulted in an enhancement of the DmcycE^JP rough eye phenotype, while decreasing the dosage of rux resulted in suppression of this phenotype (Figure 6, F–H). These genetic interactions are consistent with those observed with cyclin A or string mutations with DmcycE^JP.

The suppression of the rux rough eye phenotype by halving the dosage of string or cyclin A occurs by decreasing the number of ectopic S phases (DONG et al. 1997 ). Considering this evidence that Cyclin A and String are capable of functioning in entry into or through S phase, it was important to determine whether the interactions observed between string or cyclin A alleles and DmcycE^JP were due to an effect on S phases. BrdU-labeling experiments were carried out on larvae homozygous for DmcycE^JP and heterozygous for cyclin A, string, rux, or rca1 mutations, or containing P[ry⁺] rux (Figure 7, C–F; Table 2). Contrary to expectations, BrdU-incorporation revealed that decreasing the dosage of cyclin A or string in DmcycE^JP homozygotes had no significant effect on the number of S phase cells posterior to the MF (Figure 7C and Figure D compared with A; Table 1). Likewise, manipulating the dosage of rux or rca1 also had no significant effect on the number of S phases posterior to the MF in DmcycE^JP eye discs (Figure 7E and F; Table 2). In addition, although more difficult to accurately quantitate, no significant effect on DmcycE^JP S phases in the anterior asynchronously proliferating region was observed when the dosages of cyclin A, string, rca1, or rux were altered (Figure 7, C–F). Thus, unlike the genetic interaction observed between rux and cyclin A, string, or rca1 (DONG et al. 1997 ), the genetic interaction observed between DmcycE^JP and cyclin A, string, rux, or rca1 does not appear to be occurring by affecting the number of S phase cells.

Considering their established role in entry into mitosis, we examined whether decreasing the dosage of cyclin A or string was affecting mitoses in DmcycE^JP eye imaginal discs. The monoclonal antibody MPM-2 that recognizes an M phase phosphorylated epitope (DAVIS et al. 1983 ; WESTENDORF et al. 1994 ) has been widely used to examine mammalian mitotic cells. To confirm that it was also able to recognize mitotic cells in Drosophila tissues, eye imaginal discs were costained with the MPM-2 antisera and the DNA stain Hoechst 33258 (Figure 8, A–C). A subset of cells anterior and posterior to the MF stained with the MPM-2 antibody (Figure 8A and Figure D). These MPM-2-stained cells contained condensed DNA and mitotic figures as revealed by Hoechst 33258 staining (Figure 8B and C; and data not shown) consistent with cells in mitosis. Thus MPM-2 also stains mitotic cells in Drosophila tissues. To examine the effect on mitoses when the dosage of cyclin A or string was halved in DmcycE^JP eye imaginal discs, MPM-2 staining was carried out on DmcycE^JP, DmcycE^JP; cyclin A⁵/+, DmcycE^JP; stg^AR2/+, and wild-type eye imaginal discs. Interestingly, MPM-2 staining revealed that cells from eye imaginal discs homozygous for DmcycE^JP were larger than from wild-type imaginal discs (Figure 8E and Figure F compared with D). This is consistent with a delay in G1 phase expected for DmcycE^JP where cells can continue to grow. Despite the lower number of S phase cells, DmcycE^JP eye imaginal discs showed only a slightly lower number of M phase cells compared with wild type (Figure 8E compared with D; Table 3). This result is not surprising, given that during eye development only G2 phase cells associated with the photoreceptor clusters appear to be driven into M phase (BAKER and RUBIN 1982; KYLSTEN and SAINT 1997 ). Thus in a normal eye disc, more cells enter S phase than are required to complete the cell cycle. Due to this excess of G2 phase cells, only a small decrease in the number of mitoses may be expected posterior to the MF in DmcycE^JP eye discs relative to wild-type discs, despite the effect on S phases.

View larger version (123K): In this window In a new window Download PPT slide   Figure 8. The effect of halving the dosage of cyclin A or string on mitoses in DmcycEJP eye imaginal discs. To demonstrate the specificity of the mammalian mitosis-specific MPM-2 antibody, eye imaginal discs from wild-type larvae were stained with (A) MPM-2 antisera using FITC (green) and with the (B) DNA stain Hoechst 33258 (false colored red). The merge of the two stainings (C) shows that cells in mitosis with condensed DNA are stained with the MPM-2 sera. An example of a cell in mitosis, stained with the MPM-2 antibody, is indicated by the arrrowheads in A–C. Staining with MPM-2 antiserum was used to compare the number of mitotic cells in (D) wild-type, (E) DmcycEJP, (F) DmcycEJP; cyclinA5/+, and (G) DmcycEJP; stringAR2/+, eye imaginal discs, showing that decreasing the dosage of cyclin A or string reduces the number of mitotic cells posterior to the MF (arrows). Anterior is to the right.

Significantly, halving the dosage of cyclin A or string reduced the number of mitotic cells posterior to the MF in DmcycE^JP eye imaginal discs (Figure 8F and Figure G compared with E; Table 3). No effect on mitoses was observed by halving the dosage of cyclin A or string in a wild-type background (data not shown). These results suggest that cyclin A or string mutations enhance the DmcycE^JP rough eye phenotype by decreasing the number of mitoses during eye development.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We report here the characterization of a hypomorphic allele of Drosophila cyclin E, DmcycE^JP, that results in a rough eye phenotype caused by the presence of an internally deleted P[w⁺ m-lac] element inserted in the first intron of the DmcycE type II transcription unit. The DmcycE^JP rough eye phenotype results largely from the failure to form the full quota of pigment and bristle cells surrounding each ommatidium, resulting in fusion of ommatidia and general disorganization. This phenotype is similar to that observed when the human Cdk inhibitor p21^CIP1 is expressed in the posterior region of the eye disc from the glass response element (DE NOOIJ and HARIHARAN 1995 ) and suggests that there are too few cells for proper eye development in homozygous DmcycE^JP eye imaginal discs. During eye development, the precluster photoreceptor cells (R8, R2–R5) arise in the MF and sequentially recruit the R1, R6, and R7 cells, cone cells, pigment cells, and bristle cells from cells that have undergone the post-MF cell division (WOLFF and READY 1993 ). DmcycE^JP eye imaginal discs have much lower levels of Cyclin E and, as a consequence, fewer S phase cells in the band of proliferating cells posterior to the MF. This would be expected to reduce the number of cells that can be recruited into the developing ommatidia. In addition, DmcycE^JP third instar larval eye imaginal discs are smaller than wild type, indicating a general proliferation defect in the eye imaginal disc during larval development. Consistent with this, DmcycE^JP eye imaginal discs have fewer S phases in the anterior undifferentiated proliferating region. Thus the small and rough eye exhibited by DmcycE^JP can be accounted for by the reduction of S phases in the undifferentiated region and in the region posterior to the MF, respectively.

Interactions of DmcycE^JP with other cell cycle genes:
We have shown that the DmcycE^JP rough eye phenotype is responsive to the level of DmcycE, establishing the DmcycE^JP allele as a sensitive system for examining interacting genes. We have tested this system using the Drosophila cdc2c, retinoblastoma (RBF), dacapo (dap), and human p21^CIP1 genes for which there is already evidence for interaction with DmcycE in Drosophila (KNOBLICH et al. 1994 ; DE NOOIJ and HARIHARAN 1995 ; DE NOOIJ et al. 1996 ; DU et al. 1996 ). Modifying the dosage of these genes altered the DmcycE^JP rough eye phenotype consistent with their previously defined roles in the cell cycle. In addition, halving the dosage of the S phase transcription factor genes dE2F or dDP enhances the rough eye phenotype of DmcycE^JP by decreasing the number of S phases in DmcycE^JP eye imaginal discs, indicating that in the eye, as in some embryonic tissues, dE2F and dDP interact with DmcycE to promote the G1 to S phase transition. Whether dE2F/dDP acts predominantly upstream or downstream of DmcycE in the eye imaginal disc will require further experiments.

Implications of the genetic interaction of DmcycE with cyclin A and string:
cyclin A and string (cdc25) have been shown to be involved in entry into mitosis during Drosophila embryogenesis (EDGAR and O'FARRELL 1989 ; LEHNER and O'FARRELL 1989 ), but can also function in entry into or through S phase during eye development (THOMAS et al. 1994 ; DONG et al. 1997 ). We found that halving the dosage of cyclin A, string, or rca1, but not other genes required for entry into mitosis (cdc2, cyclin B, or cyclin B3), enhanced the DmcycE^JP rough eye phenotype. In contrast, halving the dosage of Rux, an inhibitor of Cyclin A required to establish G1 arrest in the eye imaginal disc, suppressed the DmcycE^JP rough eye phenotype. However, unlike the suppression of rux by cyclin A, string, or rca1 (THOMAS et al. 1994 ; DONG et al. 1997 ), these genes did not modify the DmcycE^JP phenotype by altering the number of S phases. Rather, they led to a decrease in M phase cells posterior to the MF. These interactions are, however, unlikely to be caused by a G2/M defect imposed upon a G1/S defect, since reducing the dosage of other G2/M genes, such as cyclin B, does not enhance the DmcycE^JP rough eye phenotype and, conversely, halving the dosage of string in a cyclin E⁺ background has no effect on mitoses. The genetic interactions between DmcycE^JP and cyclin A, rca1, or rux are consistent with the mechanism for the activation of Cyclin A by the phosphorylation and inactivation of Rux by Cyclin E/Cdc2c (DONG et al. 1997 ; SPRENGER et al. 1997 ; THOMAS et al. 1997 ).

Existing mechanisms do not explain the genetic interaction between DmcycE and string that we observed or between roughex and string (THOMAS et al. 1994 ). In Drosophila embryos at least, phosphorylation of the tyr 15 and thr 14 residues of Cdc2 in Cyclin A/Cdc2 complexes inhibits Cdk activity (SPRENGER et al. 1997 ). String (Cdc25) acts to dephosphorylate these residues and activate Cdk activity. In mammalian cells Cyclin E/Cdk2 phosphorylates and activates the Cdc25A phosphatase in the G1 to S phase transition (HOFFMAN et al. 1994 ). One possible explanation, therefore, is that DmcycE acts to phosphorylate and activate String phosphatase activity, leading to the activation of Cyclin A/Cdc2 activity.

Why is there a specific genetic interaction between DmcycE and cyclin A, but not cyclin B, if they both are involved in entry into mitosis? The precise role of Cyclin A in the cell cycle is still not well defined, and the specific interaction with DmcycE hints at unique roles for Cyclin A/Cdk in the cell cycle. Based on the fact that Cyclin E, but not Cyclin A, is required for endoreplication cycles (LEHNER and O'FARRELL 1990A ; KNOBLICH et al. 1994 ), a role for Cyclin A in S phase would be expected to be distinct from that of Cyclin E in the initiation of S phase. Endoreplication cycles are unique in that the late-replication of heterochromatic DNA does not occur (reviewed by SPRADLING and ORR-WEAVER 1987 ; LILLY and SPRADLING 1996 ). Since Cyclin A is normally absent in these cycles, Cyclin A/Cdc2 may play a role in the completion of S phase as well as in entry into mitosis in Drosophila. Further analysis is needed to provide evidence for a role for Cyclin A and String in the completion of S phase.

In summary, we have shown that the DmcycE^JP allele provides a dosage-sensitive system in which to examine the role of cell cycle regulatory genes in G1/S phase. Our analysis of genetic interactions between DmcycE^JP and other cell cycle mutations has raised the possibility that Cyclin E controls cell cycle progression by a novel mechanism involving the activation of Cyclin A and String. However, this DmcycE hypomorphic allele is of far greater significance than just analyzing the role of known cell cycle regulatory components. Dosage-sensitive alleles have been used extensively in mutagenic screens to identify novel interacting genes (e.g., see SIMON et al. 1991 ; RAFTERY et al. 1995 ; DONG et al. 1997 ). On the basis of the results presented here, the dosage-sensitive allele DmcycE^JP should be a valuable tool in the identification of novel modifiers of Cyclin E activity operating during the development of the Drosophila eye imaginal disc. We are now carrying out a genetic screen to exploit this system.

	FOOTNOTES

¹ Present address: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

	ACKNOWLEDGMENTS

The authors thank SHELAGH CAMPBELL, BARBARA THOMAS, NICK DYSON, BOB DURONIO, ISWAR HARIHARAN and CHRISTIAN LEHNER for supplying many of the fly strains used in the genetic analysis and BARBARA THOMAS for communicating unpublished data. We thank ANGELA GARDIAKOS for technical help, HELEN RODGERS for help with the eye sectioning, JOHN TERLET and MARILYN HENDERSON for help with the field emission scanning electron microscope and PETER KOLESIK for help with the confocal microscope. Thanks to TONY BRUMBY and JULES HORSFIELD for comments on the manuscript. This work was supported by an Australian Research Council Grant (# A09601106), H.R. was supported by an Australian Research Council Fellowship, J.S. was supported by an Australian Postgraduate Award, and J.P. was supported by the Finnish Academy.

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