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The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis

The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis The EMBO Journal Vol.18 No.4 pp.916–925, 1999 The role of RBF in the introduction of G regulation during Drosophila embryogenesis 1,2,3 1 progression through the cell cycle and changes in cell Wei Du and Nicholas Dyson cycle composition occur in precise temporal and spatial MGH Cancer Center, Building 149, 13th Street, Charlestown, patterns that have been described in great detail (reviewed MA 02129 and Ben May Institute for Cancer Research and Center for in Foe et al., 1993). The synchrony and reproducibility Molecular Oncology, University of Chicago, JFK R314, 924 E. 57th of this process has facilitated investigations into the Street, Chicago, IL 60637, USA regulatory mechanisms responsible for these transitions. Corresponding author How are different types of cell cycle regulation e-mail: [email protected] imposed? The best understood transition is the introduction of G regulation. The appearance of G in cell cycle 14 The first appearance of G during Drosophila embryo- 2 2 results from the degradation of maternally supplied string, genesis, at cell cycle 17, is accompanied by the down- and a subsequent requirement for de novo string synthesis regulation of E2F-dependent transcription. Mutant (Edgar and O’Farrell, 1989, 1990; Edgar and Datar, 1996). alleles of rbf were generated and analyzed to determine The string-encoded phosphatase promotes M-phase entry the role of RBF in this process. Embryos lacking both by activating Cdc2-containing kinases. String synthesis is maternal and zygotic RBF products show constitutive regulated developmentally in a complex pattern, and cycles expression of PCNA and RNR2, two E2F-regulated 14, 15 and 16 vary considerably in length between genes, indicating that RBF is required for their tran- cell types. scriptional repression. Despite the ubiquitous expres- The mechanisms responsible for the imposition of G sion of E2F target genes, most epidermal cells enter 1 regulation are less well understood, and it is unclear G normally. Rather than pausing in G until the 1 1 how many factors are required for this process. Two appropriate time for cell cycle progression, many of different mutants have been described in which cells these cells enter an ectopic S-phase. These results fail to arrest in G at the appropriate time: dacapo indicate that the repression of E2F target genes by (dap) (de Nooij et al., 1996; Lane et al., 1996) and RBF is necessary for the maintenance but not the fizzy-related (fzr) (Sigrist and Lehner, 1997). In wild- initiation of a G phase. The phenotype of RBF- type embryos, cells of the epidermis leave mitosis of deficient embryos suggests that rbf has a function that cell cycle 16 relatively synchronously and enter a is complementary to the roles of dacapo and fizzy- sustained period of quiescence, the G phase of cell related in the introduction of G during Drosophila cycle 17 [G (17)]. These cells do not normally enter embryogenesis. S-phase until the embryo has hatched and the larva has Keywords: cell cycle/embryogenesis/Drosophila/RBF/ begun to feed. In dap mutant embryos, epidermal cells transcription factor E2F do not arrest following mitosis of cell cycle 16 but continue through an additional cycle (cell cycle 17) before arresting in G of cell cycle 18 (de Nooij et al., Introduction 1996; Lane et al., 1996). dap encodes a cyclin-dependent kinase (cdk) inhibitor with homology to human p21 In mice and humans, most somatic cells progress through and p27 cdk inhibitors. It has been proposed that the a cell cycle in which DNA synthesis (S-phase) and mitosis additional cycle results from a failure to inactivate the (M-phase) are separated by two gap phases (G and G ). 1 2 cyclin E–Cdc2c kinase. Several lines of evidence support However, cycles that differ from the G /S/G /M cycle also 1 2 the idea that inactivation of cyclin E is important for occur in many species, including mammals. Polyploid G to be established. Cyclin E is broadly expressed in cells, for example, have been observed in a wide variety the early embryo but is down-regulated as cells reach of plants, animals and ciliates (for examples, see Nagl, G (Richardson et al., 1993; Knoblich et al., 1994). 1978; Brodsky and Uryvaeva, 1984; MacAuley et al., Moreover, the ectopic expression of cyclin E drives 1998). In Drosophila, non-G /S/G /M cell cycles are 1 2 cells from G (17) into S-phase (Knoblich et al., 1994; widespread throughout development. The first 13 cell Richardson et al., 1995). cycles, that occur synchronously and rapidly in the embryo, In fzr mutant embryos, like dap mutant embryos, consist only of alternating S-phases and M-phases without epidermal cells progress through an additional cell cycle any significant gap phases. G phase first appears in cell following mitosis of cell cycle 16 (Sigrist and Lehner, cycle 14, but G regulation is not apparent until the 1997). Fizzy (fzy) and fzr promote the destruction of completion of mitosis of cell cycle 16 (Foe and Alberts, A- and B-type cyclins (Dawson et al., 1995; Sigrist 1983; Foe, 1989; Edgar and O’Farrell, 1990; Smith and et al., 1995). Studies of mutant embryos lacking Fzy Orr-Weaver, 1991). Endoreduplication cycles, consisting of alternating gap and S-phases, have been observed in and/or Fzr indicate that Fzr is required specifically for many, if not most, larval and adult tissues (Spradling and the down-regulation of cyclins A, B and B3 in G Orr-Weaver, 1987). During Drosophila embryogenesis, when epidermal cells cease to proliferate, or in G 916 © European Molecular Biology Organization Role of RBF in G regulation in Drosophila embryogenesis preceding salivary gland endoreduplication (Sigrist and carrying deletions of the 1B–2A region were analyzed Lehner, 1997). and two deficiencies were identified that delete rbf In both dap and fzr mutant embryos, epidermal cells [Df(1)AD11 and Df(1)su(s)83]. Consistent with the complete only one additional cycle before entering G , mapping data, deficiencies that extend from 1E to 2B suggesting that other regulatory mechanisms can over- [Df(1)A94 and Df(1)S39] leave rbf intact. Previously, ride the proliferative stimulus to these cells. The identity we have shown that the overexpression of dE2F and of the other regulators is unclear. One potential target dDP in the developing eye generates a rough eye of this regulation is the E2F transcription factor. dE2F phenotype that is suppressed by the co-expression of and dDP, two components of E2F, are broadly expressed RBF (Du et al., 1996a). We found that the two in Drosophila embryos (Duronio et al., 1995; Hao deficiencies that delete rbf caused a moderate enhance- et al., 1995). However, the expression of RNR2 and ment of the GMRdE2FdDP eye phenotype (Figure 1). PCNA, two genes whose transcription requires dE2F While individual ommatidia are relatively normal in the and dDP, is down-regulated in wild-type embryos as eyes of GMRdE2FdDP flies (Figure 1C and D), the cells enter G (Duronio and O’Farrell, 1994; Duronio 1 introduction of either Df(1)AD11 or Df(1)su(s)83, that et al., 1995). It is unclear whether this decline in E2F remove one copy of rbf, resulted in abnormal, variably activity is important in establishing G control, or shaped ommatidia and, in places, additional bristles simply a consequence. Although ectopic expression of (Figure 1E and F, and data not shown). Expression of dE2F and dDP can drive cells from G (17) into S- human p21 in the eye (GMRp21) blocks the second phase (Duronio and O’Farrell, 1995; Duronio et al., mitotic wave during eye development, resulting in 1996), the analysis of dDP and dE2F mutant embryos abnormal eyes that are characterized by missing cone shows that regulated S-phase entry can occur in the cells, pigment cells and bristles (de Nooij and Hariharan, absence of measurable expression of E2F target genes 1995). Interestingly, these deficiencies that delete the (Royzman et al., 1997). rbf gene strongly suppressed the eye phenotypes caused The abundance and activity of E2F complexes are by the expression of human p21 (Figure 1K and L). subject to multiple levels of control. Studies of E2F in These genetic interactions suggest that this region mammalian cells have illustrated how E2F activity is contains an important negative regulator of eye cell altered by changes in E2F gene expression, subcellular proliferation. Although both Df(1)AD11 and Df(1)su(s)83 location, phosphorylation, ubiquitination and by protein are large deletions and are likely to remove many association (reviewed in Dyson, 1998). In particular, genes, rbf represented the most likely candidate for the pRB family proteins act to repress E2F-dependent critical gene. We sought mutants within this region that transcription and are thought to provide an important specifically affect rbf using these observations. level of regulation. Mammalian cells contain at least three pRB family members, and the analysis of cells Generation of mutant alleles of RBF lacking these proteins has been complicated by evidence Pre-existing mutants that had been mapped to the 1B– that there is extensive functional overlap and/or functional 1DE interval were obtained, but none enhanced the compensation between family members in knockout GMRdE2FdDP phenotype. Overlapping cosmids of the cells (Mulligan and Jacks, 1998). cytological region 1B–1DE were obtained from the RBF, a Drosophila protein with homology to the European Genome Mapping project and used to charac- pRB family of proteins, has a sequence and structural terize the rbf genomic locus further. Colony hybridization organization that is intermediate between that of human identified two overlapping cosmids (158H9 and 26B3) pRB, p107 and p130, raising the possibility that RBF that map to cytological region 1C and contain rbf might represent the archetypal family member (Du sequences (data not shown; see Figure 6 for a diagram). et al., 1996a). RBF associates with dE2F/dDP and Lines carrying P-elements inserted in the 1C region inhibits the effects of dE2F/dDP overexpression (Du were obtained from the Bloomington Stock Center and et al., 1996b). Here, we have generated mutant alleles the Berkeley Drosophila Genome Project. To identify of rbf and used these to investigate the role of RBF P-elements inserted in the vicinity of the rbf gene, in the embryonic cell cycles. The phenotype of mutant probes corresponding to the genomic sequences flanking embryos lacking both maternal and zygotic RBF products the P-element insertion sites were generated by an reveals that RBF is dispensable for the early cell cycles inverse PCR approach (Dalby et al., 1995) and screened but plays an essential role in the introduction of G for hybridization to the 158H9 and 26B3 cosmids. By control during development. The cell cycle defects wd1 this method, one P-element line, P[w]cx31A.2 (see observed in RBF-deficient embryos are strikingly differ- Materials and methods), generated probes that hybridized ent from those described previously in dap and fzr with the cosmid 26B3 but not with 158H9 (data not mutant embryos, and suggest that Dacapo, Fizzy-related shown; see Figure 6 for a diagram of the rbf genomic and RBF provide distinct functions that are required locus). This P-element insertion is not lethal and fails for the timely cessation of cell cycle progression. to enhance the GMRdE2FdDP phenotype. Western blot analysis showed that the P-element does not alter the Results level of RBF protein (data not shown), and Southern Chromosomal deletions in the 1B–2A region blot analysis indicated that the P-element lies at least modify eye phenotypes that result from altered 20 kb 3 to the RBF coding sequences (data not shown). cell proliferation A local hopping strategy was used to generate P- The rbf gene was mapped by in situ polytene element insertions in the rbf locus. The P-element in wd1 hybridization to the cytological region 1CD. Stocks P[w]cx31A.2 was mobilized by the introduction of 917 W.Du and N.Dyson Fig. 1. rbf mutants enhance the phenotypes of GMRdE2FdDP and suppress the phenotypes of GMRp21. Scanning electron micrographs of adult eyes. (A), (C), (E), (G), (I) and (K–N) were at the same magnification, the white bar in (A) corresponding to 100 μm. (B), (D), (F), (H) and (J) were at the same magnification, the white bar in (B) corresponding to 10 μm. Genotypes: (A and B) wild-type; (C and D) GMRdE2FdDP/; 120a 14 (E and F) Df(1)su(s)83/;GMRdE2FdDP/;(G and H) P[w]wd /Y; GMRdE2FdDP/;(I and J) rbf /;GMRdE2FdDP/;(K) GMRp21/; 120a 14 (L) Df(1)su(s)83/;GMRp21/;(M) P[w]wd /;GMRp21/; and (N) rbf /;GMRp21/. Note that the GMRdE2FdDP phenotype (in C and D) is enhanced in (E), (G) and (I), and in (F), (H) and (J), whereas the GMRp21 phenotype (in K) is suppressed in (L), (M) and (N). a Δ2-3 transposase, and two complementary screening lines were established and rbf was analyzed by Southern strategies were used to identify insertions in the vicinity blot analysis (see Figure 2C and D for an example). rbf , 14 16 of rbf as described in Materials and methods. A total of rbf and rbf are three alleles obtained by this method that 15a 38a three new P-element lines (P[w]wd , P[w]wd , contain complete deletions of the RBF coding sequence. 120a and P[w]wd ) were obtained. 15a 38a 120a Analysis of P[w]wd , P[w]wd and P[w]wd RBF mutations enhance a phenotype caused by revealed that in each case the P-element was inserted 5 the overexpression of dE2F/dDP and suppress a to the rbf open reading frame. This is illustrated by the phenotype caused by the overexpression of p21 PCR analysis shown in Figure 2. PCR reactions using a Mutant alleles of rbf were assessed for interactions with the primer from the P-element and a primer from the rbf 5- GMRdE2FdDP and GMRp21 phenotypes as predicted from untranslated region (Figure 2A) specifically amplified the interactions observed using the large deficiencies. As DNA fragments from DNA prepared from each of these shown in Figure 1, reducing the gene dosage of rbf by either lines (Figure 2B). The size of these fragments indicates 14 120a the null allele RBF or the viable weak allele P[w]wd that the P-elements lie ~700 bp from the translation start both strongly suppressed the GMRp21 phenotype (Figure codon. Flies homozygous for any one of these new 1K–N). In addition, reducing the gene dosage of rbf also insertions were viable, and Western blot analysis showed enhanced the GMRdE2FdDP phenotype (Figure 1C–J). that RBF levels varied between 50 and 100% of wild- 120a 15a These interactions indicate that P[w]wd indeed type (data not shown), indicating that P[w]wd , 38a 120a behaves like a viable weak allele of RBF, and showed that P[w]wd and P[w]wd are not null alleles of rbf. the activity of ectopic dE2F/dDP is limited by endogenous To generate null alleles of rbf, these new P-element RBF protein and that the p21-mediated cell cycle arrest lines were used to carry out imprecise excision. After crossing with flies carrying the transposase Δ2-3, new depends on RBF. These results strongly suggest that RBF 918 Role of RBF in G regulation in Drosophila embryogenesis Fig. 2. PCR and Southern blotting analysis illustrating P-element insertion in the rbf locus and its subsequent excision to generate mutant alleles of 120a rbf.(A) Diagram showing the relative position of the P-element insertion in P[w]wd and the position of primers used for PCR. P1, P2 and P3 are primers from the ends of the P-element; a and b indicate the primers from the RBF 5-untranslated region. Arrows indicate the direction of the primers from 5 to 3. ATG indicates the start of translation. (B) PCR products from the P-element insertion lines. DNA isolated from the P-element 120a 38a 15a lines P[w]wd , P[w]wd and P[w]wd are in lanes 1–6, 7–12 and 14–19, respectively. Lane 13 is a molecular weight marker. Primer pairs: (P3 and a) are used in lanes 1, 7 and 14; (P2 and a) in lanes 2, 8 and 15; (P1 and a) in lanes 3, 9 and 16; (P3 and b) in lanes 4, 10 and 17; (P2 and b) in lanes 5, 11 and 18; and (P1 and b) in lanes 6, 12 and 19. Note that PCR products were generated from each of the lines by the 120a combination of primers P1 and b. (C) Diagram showing the position of HindIII sites at the rbf locus in P[w]wd and FM6.In FM6 (as in wild- 120a type), the RBF cDNA hybridizes to two HindIII fragments (fragment II and III). The P-element insertion in P[w]wd (labeled 120a) introduces 120a a new HindIII site, resulting in the appearance of an additional short fragment I. Thus the two fragments that hybridize to RBF in P[w]wd are fragments I and II, and the two fragments that hybridize to RBF in FM6 are fragments II and III. (D) Southern blot analysis illustrating a P-element 14 16 insertion in the RBF locus and its subsequent excision in RBF and RBF . The genotypes of the DNA samples: lane 1, FM6; lane 2, 120a 14 16 P[w]wd /FM6; lane 3, RBF /FM6; and lane 4, RBF /FM6 as indicated. The full-length cDNA RBF was used as a probe. Note that the 120a 14 16 additional HindIII fragment (fragment I) introduced in P[w]wd is deleted in RBF and RBF . In addition, the intensity of fragment II is also reduced by 50% in lanes 3 and 4 compared with lane 2, whereas the fragment III that is derived solely from the FM6 chromosome is unchanged (compare lanes 2, 3 and 4). Lane 1 contains less DNA than lanes 2–4. normally acts as a negative regulator of cell proliferation RBF is an essential regulator of E2F-dependent during eye development. transcription in the embryo Previous work has demonstrated that the transcription of Expression of RBF cDNA rescues the lethality of PCNA and RNR2, two genes that are coordinately RBF mutants expressed as cells enter S-phase, requires both dE2F and Mutations resulting in the complete deletion of rbf were dDP and is induced by ectopic expression of dE2F/dDP lethal as homozygotes, hemizygotes or trans-heterozy- (Duronio and O’Farrell, 1994, 1995; Duronio et al., 1995, gotes. To demonstrate that the lethality of these rbf alleles 1996, 1998; Royzman et al., 1997). In wild-type embryos, is due to the lack of RBF activity, rescue experiments RNR2 is expressed uniformly during early stages but is were carried out by expressing the RBF cDNA under the down-regulated following cell cycle 16 in cells entering control of a heat shock promoter. The lethality of a trans- G , the first time that G regulation is apparent (Duronio 1 1 11 14 heterozygous combination of rbf and rbf , two null and O’Farrell, 1994). Periodic expression of RNR2 is seen alleles in which the RBF coding sequence is completely in the gut and peripheral nervous system (PNS) as cells deleted, was rescued efficiently by expression of the RBF enter S-phase. RNR2 expression persists in the CNS that cDNA from the heat shock-regulated transgene hsRBF contains actively dividing cells, but RNR2 is not re- (see Materials and methods). Similarly, the hsRBF trans- expressed in epidermis of the embryo following mitosis gene also allowed rescue of viable males carrying rbf of cell cycle 16 as these cells remain quiescent until the or rbf . For these experiments, vials were incubated at first larval instar. 11 14 37°C for 1 h/day throughout development. No viable rbf / While no rbf /rbf trans-heterozygous adult flies were 14 11 14 rbf females, or rbf or rbf male adult flies were found in the absence of the hsRBF transgene, egg counts 11 14 observed without heat shock treatment or by heat shock showed that most rbf /rbf trans-heterozygous embryos in the absence of the hsRBF transgene. Thus, rbf is the hatched (data not shown). Embryos homozygous for 14 11 only essential function missing in rbf and rbf . mutant alleles for rbf were assayed by in situ hybridization 919 W.Du and N.Dyson using a probe for RNR2 expression, but no abnormalities deficient embryos examined, BrdU incorporation was were apparent. In particular, RNR2 expression was observed in only a subset of the epidermal cells. These repressed after mitosis 16 in rbf mutant embryos in a observations suggest that epidermal cells initially enter manner that appeared identical to the wild-type embryos G following mitosis 16 but, in the absence of RBF, a (data not shown). These observations might suggest that significant proportion of the cells were unable to remain RBF is neither required for the repression of RNR2 in G and entered S-phase. In situ hybridization with a expression nor has an essential function during embryogen- probe for cyclin E showed that the level of cyclin E esis; alternatively, RBF may have essential functions that mRNA is significantly elevated in the epidermis of RBF- can be performed by maternally supplied products. To test deficient embryos (Figure 4G and H). In these embryos, this, we generated rbf mutant embryos that were derived cyclin E expression is only partially deregulated, consistent from germline clones and lacked both maternal and zygotic with previous evidence that dE2F and dDP provide only RBF. As described below, such RBF-deficient embryos one of several activities that regulate cyclin E expression displayed a variety of phenotypes that reveal essential (Duronio and O’Farrell, 1995; Royzman et al., 1997; roles for RBF in E2F regulation and cell cycle control Duronio et al., 1998). Interestingly, cyclin E expression during embryogenesis. was slightly higher in epidermal cells near the segment In situ hybridization experiments show that the expres- boundaries, in a pattern that resembled the pattern of sion of RNR2 is strongly deregulated in the RBF-deficient BrdU incorporation. It is possible that RBF is more embryos. In these embryos, RNR2 was expressed ubiquit- important for cyclin E regulation in these cells. Alternat- ously and uniformly in both early stages of embryogenesis ively, these cells may be more sensitive to deregulation and also in later stage embryos following germband of E2F, and the elevated level of cyclin E expression may retraction (Figure 3E–H, and data not shown). The expres- be caused by S-phase entry. sion of PCNA was altered similarly. In RBF-deficient To determine whether ectopic S-phases resulted in cell embryos, the level of PCNA expression in the epidermis proliferation, RBF-deficient embryos were stained with is equivalent to that seen in the PNS, where PCNA staining an antibody to phosphorylated histone H3 that detects is detected in wild-type embryos at this stage (Figure 3A– mitotic chromosomes (de Nooij et al., 1996). No extra D). Using RNR2 and PCNA expression as a measure of mitotic cells were found in the epidermis in RBF-deficient E2F activity, these results suggest that E2F-dependent embryos (data not shown), indicating that the epidermal transcription is constitutively elevated in the absence of cells that entered S-phase did not complete a mitotic cell RBF. Thus, RBF is required for the developmental down- cycle. The disparity between the S-phase and M-phase regulation of E2F that occurs when G regulation is markers prompted us to assess the level of apoptosis introduced during Drosophila embryogenesis. in these embryos. Wild-type embryos show a low but significant level of apoptosis in the epidermis that can be Defective G regulation in the absence of RBF detected by the TUNEL assay. In contrast, extensive Crosses between females bearing rbf germline clones apoptosis was found in the epidermis of stage 13 RBF- and wild-type males gave viable females, indicating that deficient embryos (Figure 4E and F). Although the majority expression of the wild-type rbf gene from the paternal X of cells incorporating BrdU were located near the segment chromosome was sufficient for viability. As the first 13 boundaries, TUNEL-positive cells were distributed cell cycles are driven exclusively by maternally encoded throughout the segment. This difference suggests that, in products, the paternal rescue indicates that RBF has no many cells, induction of apoptosis is unlinked to S-phase essential functions during these early stages. entry. Taken together, these observations indicate that Bromodeoxyuridine (BrdU) incorporation was used to epidermal cells initially stop in G following mitosis of monitor DNA synthesis in the RBF-deficient embryos. cell cycle 16, but that these cells are unable to maintain Aberrant cell cycle control was observed in cells of the this arrest effectively. With time, an increasing proportion midgut in stage 12–14 embryos. In wild-type embryos, of cells enter S-phase. In addition, many cells are elimin- pulses of BrdU incorporation are observed in the midgut ated by apoptosis. due to differential timing of G (17) in subsets of cells. In RBF-deficient embryos, the pulses of BrdU incorporation Discussion that normally distinguish the central and anterior midgut were weaker, and BrdU incorporation occasionally was RBF-deficient embryos provide a third example, in addi- seen throughout the midgut region (data not shown). tion to dap and fzr mutants (de Nooij et al., 1996; Lane However, the most dramatic changes in DNA synthesis et al., 1996; Sigrist and Lehner, 1997), where epidermal were seen in the epidermis. In wild-type embryos, cells cells are unable to stop cell cycle progression following of the epidermis complete cell cycle 16 in late stage 11; mitosis of cell cycle 16. Although the same cells enter an these cells enter G of cell cycle 17 and no longer ectopic S-phase in each case, the phenotypes of these incorporate BrdU (Figure 4A and C). DNA synthesis is embryos are quite different. First, in dap and fzr mutants, evident in mid-stage 12 in the PNS cells that lie just epidermal cells entering an ectopic S-phase complete a below the epidermis (Figure 4A). No defect was observed mitotic cell cycle; in RBF mutants, no additional mitotic in the pattern of S-phases of RBF-deficient embryos until cells were detected. A second distinction lies in the mid-stage 12 when the germband was partially retracted. persistence of ectopic S-phases. In dap and fzr mutants, In these embryos, ectopic S-phase cells were first observed ectopic S-phases were seen for a short time window; once in the dorsal epidermis. In stage 13 and stage 14 embryos, epidermal cells complete an additional cycle, they remain ectopic S-phases had become more abundant and extended arrested in G . In RBF-deficient embryos, ectopic S-phases to the ventral epidermis (Figure 4B and D). In all RBF- persisted in the epidermis and even increased as the 920 Role of RBF in G regulation in Drosophila embryogenesis Fig. 3. Deregulation of E2F activity in embryos lacking RBF. Endogenous E2F activities were detected by in situ hybridization with antisense probes to PCNA (A–D) or RNR2 (E–H); the same patterns of expression were detected for these two genes. Two different stage of embryos are shown. (A–D) Embryos at the beginning of germband retraction; (E–H) embryos with a completely retracted germband. RNR2 and PCNA are expressed ubiquitously at high levels at each of the stages shown in RBF-deficient embryos. (A and B) A wild-type embryo at the beginning of germband retraction; the image was focused on the epidermis in (A) and on the midline in (B). (C and D) An RBF maternal and zygotic null embryo; the image was focused on the epidermis in (C) and on the midline in (D). (E and F) A germband-retracted wild-type embryo; the image was focused on the epidermis in (E) and on the midline in (F). Note that the anterior and posterior midgut staining in (F) is out of focus in (E). (G and H)A germband-retracted RBF maternal and zygotic null embryo; the image was focused on the epidermis in (G) and on the midline in (H). embryos aged. Third, in RBF-deficient embryos, most of E2F-dependent transcription (Duronio and O’Farrell, epidermal cells initially enter G (17). Because epidermal 1994). As the overexpression of dE2F and dDP is able to cells of dap mutant embryos enter S-phase synchronously drive cells from G into S-phase, it appeared likely that and rapidly following mitosis of cell cycle 16, it has been this inhibition of E2F activity would be essential for the unclear whether these cells enter G (17) or whether they appearance of G . The analysis of RBF-deficient embryos 1 1 progress directly from mitosis to S-phase (de Nooij et al., argues against this conclusion. RNR2 and PCNA, the two 1996; Lane et al., 1996). In mid-stage 12 RBF-deficient genes that have been used most widely in previous studies embryos, only a subset of cells incorporate BrdU, indicat- to provide a measure of endogenous E2F activity, are ing that the majority of cells initially remained in G (17). constitutively expressed in RBF-deficient embryos. Never- Cells of the dorsal epidermis were already post-mitotic theless, the majority of epidermal cells are able to enter by this stage in control embryos. G , and it is only as the embryo ages that large numbers We infer from these observations that RBF plays an of these cells enter ectopic S-phases. Thus, most epidermal important role in the imposition of G regulation during cells require neither RBF nor the repression of E2F- Drosophila development. It is well established that the dependent transcription to enter G . Instead RBF’s role appearance of G (17) correlates with the down-regulation appears to lie in the maintenance of the G phase. 1 1 921 W.Du and N.Dyson Fig. 4. Ectopic S-phases and increased apoptosis in RBF-deficient embryos. BrdU incorporation of wild-type embryos (A and C) or RBF maternal and zygotic null embryos (B and D). (A and B) Germband partially retracted stage 12 embryos. Cells in the epidermis were not labeled with BrdU in wild-type embryos (A); the cells that are labeled with BrdU in the middle of each segment are PNS cells just below the epidermis. In contrast, cells in the epidermis in RBF-deficient embryos were labeled with BrdU (B); note that the cells that are labeled are on the dorsal epidermis and are adjacent to the segment boundary. (C and D) Germband completely retracted embryos. Cells in the epidermis were not labeled with BrdU in wild- type embryos (C); the dark shadow in the middle of the embryo is due to BrdU staining of midgut and hindgut. CNS cells at the ventral side of the embryo are labeled strongly; these cells are still proliferating at this stage. Cells in the epidermis in RBF-deficient embryos were labeled with BrdU (D); note that cells in the ventral epidermis also incorporate BrdU. CNS cells are below this focal plane. (E and F) TUNEL staining of a wild-type embryo shown in (E) and an RBF-deficient embryos shown in (F). Note that the number of TUNEL-stained cells is significantly increased; however, these cells do not appear to have the same pattern of BrdU-labeled cells (compare B, D and F). (G and H) Cyclin E expression was detected by whole-mount in situ hybridization. A germband-retracted wild-type embryo is shown in (G); cyclin E transcripts were not detected in the epidermis. A similar staged RBF-deficient embryo is shown in (H); a significant level of cyclin E RNA was detected in the epidermis. Note that cyclin E expression is elevated in cells adjacent to the segment boundary, resembling the pattern of BrdU incorporation in these embryos. The properties of dap mutant embryos and RBF- exit the cell cycle (de Nooij et al., 1996; Lane et al., deficient embryos are highly consistent with a model in 1996). Our results suggest that the repression of cyclin E which the complementary roles of RBF and Dacapo in and other E2F-regulated genes by RBF is not important G control are due to their complementary roles in the at this stage, but becomes essential later, as the expression regulation of cyclin E activity (Figure 5). In this model, of Dacapo declines. In RBF-deficient embryos, the elev- G is triggered by the inhibition of the cyclin E–Cdc2c ated expression of cyclin E in cells that lack Dacapo will kinase. Dacapo is required for this process (de Nooij et al., lead to the accumulation of an active kinase, and eventually 1996; Lane et al., 1996), although it is not certain that it to S-phase entry. In dap mutants, however, RBF repression is the only mechanism involved. Previous studies have of cyclin E expression would limit its ability to drive shown that Dacapo is only transiently expressed as cells epidermal cells through multiple cycles. 922 Role of RBF in G regulation in Drosophila embryogenesis Previous studies have shown that PCNA and RNR2 are not expressed in dE2F or dDP mutant embryos (Duronio et al., 1995, 1998; Royzman et al., 1997). These results suggested that the induction of gene expression that occurs in wild-type embryos as cells enter S-phase was due primarily to transcriptional activation by a dE2F–dDP complex. However, the finding that these genes are con- stitutively expressed in RBF-deficient embryos adds a level of complexity. This result indicates that RBF actively represses the expression of PCNA and RNR2 in G phase cells and suggests that the pulses of gene expression seen as cells enter S-phase could be due largely to the release of repression. If RBF, dE2F and dDP are common compon- ents of a repressor complex, one wonders why PCNA and Fig. 5. RBF and Dacapo cooperate to regulate cyclin E kinase activity RNR2 are not constitutively expressed in dE2F and dDP and to establish G . The cyclin E-associated kinase activity after mutant embryos? One possible explanation is that dE2F mitosis 16 in wild-type, dap or RBF-deficient embryos are drawn in black, blue and red, respectively. Lines on top show the time at which and dDP are not only co-repressors with RBF but are also RBF and Dacapo are important. In this model, we suggest that the required to activate the RNR2 and PCNA promoters. An functions of rbf and dap converge on the regulation of cyclin E, alternative possibility is that RBF repression of PCNA Dacapo acting to inhibit the residual cyclin E kinase activity following and RNR2 expression is not mediated by dE2F/dDP, but mitosis 16, and RBF acting to repress cyclin E expression. In dap by a different RBF-binding protein. The recent discovery mutants, residual cyclin E-associated kinase activity is sufficient to drive cells into S-phase following mitosis 16. Only one additional of dE2F2, that associates with dDP and RBF in Drosophila cycle occurs because cyclin E-associated kinase activity drops below embryos (D.Huen, W.Du, Y.Chen, and N.Dyson, unpub- the threshold due to the repression of cyclin E expression by RBF. In lished observations), opens up a variety of potential RBF-deficient embryos, the expression of Dacapo inhibits the cyclin explanations. It is also evident that RNR2/PCNA and E-associated kinase, initially causing a G arrest. However, in the absence of RBF, cyclin E is mis-expressed and cells accumulate cyclin cyclin E represent two different types of E2F target genes. E kinase activity as Dacapo levels decline and the cyclin E level Although expression of all three genes can be induced by increases. Once the cyclin E kinase activity passes the threshold for ectopic expression of dE2F and dDP, RNR2 and PCNA S phase, these cells will initiate DNA replication. were completely derepressed by the absence of RBF, whereas cyclin E was deregulated in a more subtle manner. The level of cyclin E expression observed in the epidermis Why do the ectopic S-phase cells in RBF-deficient of RBF-deficient embryos remains considerably lower mutants fail to progress to mitosis? We suggest that this than the level of cyclin E expressed in the central nervous may occur for several reasons. The levels of cyclins A system and brain. In some cell types, expression of cyclin and B drop rapidly once cells enter G (Lehner and E is independent of both dE2F and dDP (Duronio and O’Farrell, 1989, 1990). In dap mutant embryos, where O’Farrell, 1995; Royzman et al., 1997; Duronio et al., cells enter S-phase rapidly after mitosis of cell cycle 16, 1998), and it seems likely that the expression of cyclin E, there may still be sufficient A- and B-type cyclins to like that of string and other critical cell cycle regulators, allow these cells to progress to mitosis 17. Given the is under the control of multiple enhancer elements. delay before ectopic S-phase cells appear in RBF-deficient The phenotype of RBF-deficient embryos has several embryos, the levels of mitotic cyclins may be insufficient –/– features in common with the phenotype of Rb mice for cell cycle progression. In wild-type animals, epidermal (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992, cells will leave G (17) to enter an endoreduplication S- 1994). Both mutants are characterized by ectopic S-phases, phase. By the time that ectopic S-phase cells appear the expression of E2F-regulated genes and high levels of in RBF-deficient embryos, these cells may already be apoptosis. Such similarities emphasize that these homolog- committed to an endoreduplication cycle. In addition, ous proteins serve analogous functions. Interpretation of there is evidence that the down-regulation of E2F activity the phenotypes of pRB, p107 and p130 knockout mice is is important for cells to exit S-phase (Krek et al., 1995). complicated by the fact that these proteins share overlap- Potentially, the failure to down-regulate E2F or, alternat- ping functions. Even in studies of double knockout mice, ively cyclin E expression (Follet et al., 1998; Weiss et al., these animals progress through many stages of mouse 1998), may arrest cells either in S-phase or post-S-phase, development before cell cycle defects become apparent in RBF-deficient embryos. Finally, the overexpression of (Cobrinik et al., 1996; Lee et al., 1996; Mulligan and E2F genes induces apoptosis in a variety of experimental Jacks, 1998). It has been unclear whether pRB family systems (Qin et al., 1994; Shan and Lee, 1994; Wu and proteins are unimportant for many cell cycles or whether Levine, 1994; Kowalik et al., 1995; Asano et al., 1996; they have a redundant but critical function. The phenotype Du et al., 1996b). Although our results suggest that S- of RBF-deficient embryos reveals that RBF is important phase entry is not a prerequisite for apoptosis in the RBF- at the very first cell cycle in Drosophila development deficient embryos, cells that enter S-phase inappropriately where G regulation is introduced. Interestingly, RBF is may be especially sensitive to the effects of elevated E2F required for the maintenance of G , but not for its activity and may not survive to complete the cell cycle. initial appearance. By analogy, one of the initial roles of The changes in expression of E2F-regulated genes caused by the absence of RBF provide new insights into mammalian pRB family members may be to allow rapidly the regulation of E2F activity in the Drosophila embryo. proliferating cells to pause in G . 923 W.Du and N.Dyson Materials and methods Fly stocks 1 1 1 1 Deficiency lines Df(1)AD11/FM7 and Df(1)su(s)83, y cho ras v / 2 1 1 Dp(1;Y)y sc/C(1)DX, y f , the P-element stock P1318 (P[w] cx31A.2) and the FLP, FRT lines for germline clonal analysis were obtained from the Bloomington Stock Center. P[w; hsp70-RBF] was generated by subcloning of the RBF full-length cDNA (Du et al., 1996a) into pCaSpeR-hs vector. A P[w; hsp-RBF] on the X chromosome was used for the rescue experiments. Characterization of the RBF chromosomal region Cosmid contigs between the 1B and 1DE polytene region were obtained from the European Genome Mapping Project, and were hybridized using the RBF cDNA as a probe. Two overlapping cosmids, 158H9 and 26B3, contain RBF sequences and were used to identify P-elements in the vicinity of rbf. P-element lines were obtained from the Bloomington Stock Center and the Berkeley Drosophila Genome Project. An inverse PCR method was used to make probes from the genomic sequences adjacent to the P-element insertions. These probes were hybridized to the two cosmids that contained RBF sequences. Stock number P1318 (P[w]cx31A.2) gave probes that hybridize to the cosmid 26B3 but not to 158H9. Since this stock contains two P-element insertions on the X chromosome, this stock was crossed with w , and the resulting two different classes of lighter eye color flies were established. One such wd1 line, P[w]cx31A.2 , was shown to retain the P-element in the vicinity of rbf and was used in subsequent experiments. Embryo analysis In situ hybridization. Digoxigenin-labeled antisense RNA probes were prepared by in vitro transcription reaction. Properly aged embryos were collected and fixed. Hybridization was carried out at 70°C as described (Duronio and O’Farrell, 1994). BrdU staining. Properly aged embryos were collected, dechorionated in 50% Chlorox, permeabilized in octane, and incubated in 1 mg/ml BrdU in Schneider’s medium for 20 min. Embryos were fixed in an equal volume of 4% formaldehyde and heptane. BrdU was detected using a mouse anti-BrdU antibody (Becton Dickenson, 1:100). TUNEL assay. Collected and fixed embryos were treated with proteinase K (10 μg/ml) for 5 min, washed twice with PBT and post-fixed with 4% formaldehyde for 20 min. These embryos were processed further using the Apotag kit from ONCOR (Gaithersberg, MD) according to the instructions provided. Generation of RBF mutants The scheme used for hopping is shown in Figure 6. Females from 20 different lines (one female each line) were pooled for inverse PCR to Fig. 6. The scheme for hopping. Two different crosses were used to generate probes. These probes were hybridized to the 26B3 and 158H9 generate rbf mutants. A transposase Δ2-3 was crossed to the cosmid DNA digested with NotI. A total of 1600 lines were established wd1 P[w]cx31A.2 line. Resulting males were either crossed to a first and tested using cross A (Figure 6); one line was found to have a chromosome balancer FM6 to establish lines in (A) or crossed to new P-element insertion in cosmid 158H9. Four hundred lines were GMRdE2FdDP to identified flies with slightly enhanced eye established using cross B (Figure 6) and two lines were found to have phenotypes in (B). new P-element insertions in cosmid 158H9. Southern and PCR analysis showed that all three P-elements were inserted in the 5-untranslated region of the RBF cDNA, ~700 bp from the start codon ATG. To generate null alleles of RBF, these three P-element lines isolated 15a 38a 120a (P[w]wd , P[w]wd and P[w]wd ) were crossed to the transposase Δ2-3 and new lines were established. The deletions of the progeny from this cross are expected to be the mutant class; however, rbf coding sequences were determined by Southern analysis. All lines the FM7 male is usually under-represented and, taking this into account, with deletions of the rbf coding sequences were found to be lethal. complete rescue of the mutant class could provide up to 30% of the adults. Generation of germline clones rbf mutants were recombined onto an X chromosome carrying an FRT Acknowledgements site inserted at 14AB. Germline clone females were generated as described (Soto et al., 1995). Virgin females with rbf germline clones We thank the Bloomington Stock Center and the Berkeley Drosophila were collected and were crossed with males carrying Eve-lacZ on the X Genome Project for the fly stocks used in this study, and the European chromosome. Embryos with the wild-type rbf gene were identified by Genome Mapping Project for the cosmid contigs. We are grateful to anti-β-gal staining. Iswar Hariharan for stimulating discussions and for the GMRp21 stocks. We thank Kristin White for advice on TUNEL staining, Ed Seling for Rescue of homozygous RBF mutants help with the SEM, and Simon Boulton and Iswar Hariharan for For the rescue experiments, rbf ,P[w; hsp70-RBF]/DP(1:Y) males comments on the manuscript. W.D. was a recipient of a Leukemia were crossed to rbf /FM7 females. Once eggs were laid, the vials were Society Fellowship, and currently is a scholar of the Kimmel Foundation 11 14 heat-shocked for1hat 37°C per day. rbf /rbf ,P[w; hsp70-RBF] for Cancer Research. This work was supported by NIH grant females represented ~20% of the adults. Theoretically, 25% of the R01GM53203. 924 Role of RBF in G regulation in Drosophila embryogenesis References down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell, 77, 107–120. Asano,M., Nevins,J.R. and Wharton,R.P. 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(1994) Cyclin E controls S-phase progression and its http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis

Du, Wei; Dyson, Nicholas
The EMBO Journal , Volume 18 (4) – Feb 15, 1999
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Copyright © European Molecular Biology Organization 1999
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Abstract

The EMBO Journal Vol.18 No.4 pp.916–925, 1999 The role of RBF in the introduction of G regulation during Drosophila embryogenesis 1,2,3 1 progression through the cell cycle and changes in cell Wei Du and Nicholas Dyson cycle composition occur in precise temporal and spatial MGH Cancer Center, Building 149, 13th Street, Charlestown, patterns that have been described in great detail (reviewed MA 02129 and Ben May Institute for Cancer Research and Center for in Foe et al., 1993). The synchrony and reproducibility Molecular Oncology, University of Chicago, JFK R314, 924 E. 57th of this process has facilitated investigations into the Street, Chicago, IL 60637, USA regulatory mechanisms responsible for these transitions. Corresponding author How are different types of cell cycle regulation e-mail: [email protected] imposed? The best understood transition is the introduction of G regulation. The appearance of G in cell cycle 14 The first appearance of G during Drosophila embryo- 2 2 results from the degradation of maternally supplied string, genesis, at cell cycle 17, is accompanied by the down- and a subsequent requirement for de novo string synthesis regulation of E2F-dependent transcription. Mutant (Edgar and O’Farrell, 1989, 1990; Edgar and Datar, 1996). alleles of rbf were generated and analyzed to determine The string-encoded phosphatase promotes M-phase entry the role of RBF in this process. Embryos lacking both by activating Cdc2-containing kinases. String synthesis is maternal and zygotic RBF products show constitutive regulated developmentally in a complex pattern, and cycles expression of PCNA and RNR2, two E2F-regulated 14, 15 and 16 vary considerably in length between genes, indicating that RBF is required for their tran- cell types. scriptional repression. Despite the ubiquitous expres- The mechanisms responsible for the imposition of G sion of E2F target genes, most epidermal cells enter 1 regulation are less well understood, and it is unclear G normally. Rather than pausing in G until the 1 1 how many factors are required for this process. Two appropriate time for cell cycle progression, many of different mutants have been described in which cells these cells enter an ectopic S-phase. These results fail to arrest in G at the appropriate time: dacapo indicate that the repression of E2F target genes by (dap) (de Nooij et al., 1996; Lane et al., 1996) and RBF is necessary for the maintenance but not the fizzy-related (fzr) (Sigrist and Lehner, 1997). In wild- initiation of a G phase. The phenotype of RBF- type embryos, cells of the epidermis leave mitosis of deficient embryos suggests that rbf has a function that cell cycle 16 relatively synchronously and enter a is complementary to the roles of dacapo and fizzy- sustained period of quiescence, the G phase of cell related in the introduction of G during Drosophila cycle 17 [G (17)]. These cells do not normally enter embryogenesis. S-phase until the embryo has hatched and the larva has Keywords: cell cycle/embryogenesis/Drosophila/RBF/ begun to feed. In dap mutant embryos, epidermal cells transcription factor E2F do not arrest following mitosis of cell cycle 16 but continue through an additional cycle (cell cycle 17) before arresting in G of cell cycle 18 (de Nooij et al., Introduction 1996; Lane et al., 1996). dap encodes a cyclin-dependent kinase (cdk) inhibitor with homology to human p21 In mice and humans, most somatic cells progress through and p27 cdk inhibitors. It has been proposed that the a cell cycle in which DNA synthesis (S-phase) and mitosis additional cycle results from a failure to inactivate the (M-phase) are separated by two gap phases (G and G ). 1 2 cyclin E–Cdc2c kinase. Several lines of evidence support However, cycles that differ from the G /S/G /M cycle also 1 2 the idea that inactivation of cyclin E is important for occur in many species, including mammals. Polyploid G to be established. Cyclin E is broadly expressed in cells, for example, have been observed in a wide variety the early embryo but is down-regulated as cells reach of plants, animals and ciliates (for examples, see Nagl, G (Richardson et al., 1993; Knoblich et al., 1994). 1978; Brodsky and Uryvaeva, 1984; MacAuley et al., Moreover, the ectopic expression of cyclin E drives 1998). In Drosophila, non-G /S/G /M cell cycles are 1 2 cells from G (17) into S-phase (Knoblich et al., 1994; widespread throughout development. The first 13 cell Richardson et al., 1995). cycles, that occur synchronously and rapidly in the embryo, In fzr mutant embryos, like dap mutant embryos, consist only of alternating S-phases and M-phases without epidermal cells progress through an additional cell cycle any significant gap phases. G phase first appears in cell following mitosis of cell cycle 16 (Sigrist and Lehner, cycle 14, but G regulation is not apparent until the 1997). Fizzy (fzy) and fzr promote the destruction of completion of mitosis of cell cycle 16 (Foe and Alberts, A- and B-type cyclins (Dawson et al., 1995; Sigrist 1983; Foe, 1989; Edgar and O’Farrell, 1990; Smith and et al., 1995). Studies of mutant embryos lacking Fzy Orr-Weaver, 1991). Endoreduplication cycles, consisting of alternating gap and S-phases, have been observed in and/or Fzr indicate that Fzr is required specifically for many, if not most, larval and adult tissues (Spradling and the down-regulation of cyclins A, B and B3 in G Orr-Weaver, 1987). During Drosophila embryogenesis, when epidermal cells cease to proliferate, or in G 916 © European Molecular Biology Organization Role of RBF in G regulation in Drosophila embryogenesis preceding salivary gland endoreduplication (Sigrist and carrying deletions of the 1B–2A region were analyzed Lehner, 1997). and two deficiencies were identified that delete rbf In both dap and fzr mutant embryos, epidermal cells [Df(1)AD11 and Df(1)su(s)83]. Consistent with the complete only one additional cycle before entering G , mapping data, deficiencies that extend from 1E to 2B suggesting that other regulatory mechanisms can over- [Df(1)A94 and Df(1)S39] leave rbf intact. Previously, ride the proliferative stimulus to these cells. The identity we have shown that the overexpression of dE2F and of the other regulators is unclear. One potential target dDP in the developing eye generates a rough eye of this regulation is the E2F transcription factor. dE2F phenotype that is suppressed by the co-expression of and dDP, two components of E2F, are broadly expressed RBF (Du et al., 1996a). We found that the two in Drosophila embryos (Duronio et al., 1995; Hao deficiencies that delete rbf caused a moderate enhance- et al., 1995). However, the expression of RNR2 and ment of the GMRdE2FdDP eye phenotype (Figure 1). PCNA, two genes whose transcription requires dE2F While individual ommatidia are relatively normal in the and dDP, is down-regulated in wild-type embryos as eyes of GMRdE2FdDP flies (Figure 1C and D), the cells enter G (Duronio and O’Farrell, 1994; Duronio 1 introduction of either Df(1)AD11 or Df(1)su(s)83, that et al., 1995). It is unclear whether this decline in E2F remove one copy of rbf, resulted in abnormal, variably activity is important in establishing G control, or shaped ommatidia and, in places, additional bristles simply a consequence. Although ectopic expression of (Figure 1E and F, and data not shown). Expression of dE2F and dDP can drive cells from G (17) into S- human p21 in the eye (GMRp21) blocks the second phase (Duronio and O’Farrell, 1995; Duronio et al., mitotic wave during eye development, resulting in 1996), the analysis of dDP and dE2F mutant embryos abnormal eyes that are characterized by missing cone shows that regulated S-phase entry can occur in the cells, pigment cells and bristles (de Nooij and Hariharan, absence of measurable expression of E2F target genes 1995). Interestingly, these deficiencies that delete the (Royzman et al., 1997). rbf gene strongly suppressed the eye phenotypes caused The abundance and activity of E2F complexes are by the expression of human p21 (Figure 1K and L). subject to multiple levels of control. Studies of E2F in These genetic interactions suggest that this region mammalian cells have illustrated how E2F activity is contains an important negative regulator of eye cell altered by changes in E2F gene expression, subcellular proliferation. Although both Df(1)AD11 and Df(1)su(s)83 location, phosphorylation, ubiquitination and by protein are large deletions and are likely to remove many association (reviewed in Dyson, 1998). In particular, genes, rbf represented the most likely candidate for the pRB family proteins act to repress E2F-dependent critical gene. We sought mutants within this region that transcription and are thought to provide an important specifically affect rbf using these observations. level of regulation. Mammalian cells contain at least three pRB family members, and the analysis of cells Generation of mutant alleles of RBF lacking these proteins has been complicated by evidence Pre-existing mutants that had been mapped to the 1B– that there is extensive functional overlap and/or functional 1DE interval were obtained, but none enhanced the compensation between family members in knockout GMRdE2FdDP phenotype. Overlapping cosmids of the cells (Mulligan and Jacks, 1998). cytological region 1B–1DE were obtained from the RBF, a Drosophila protein with homology to the European Genome Mapping project and used to charac- pRB family of proteins, has a sequence and structural terize the rbf genomic locus further. Colony hybridization organization that is intermediate between that of human identified two overlapping cosmids (158H9 and 26B3) pRB, p107 and p130, raising the possibility that RBF that map to cytological region 1C and contain rbf might represent the archetypal family member (Du sequences (data not shown; see Figure 6 for a diagram). et al., 1996a). RBF associates with dE2F/dDP and Lines carrying P-elements inserted in the 1C region inhibits the effects of dE2F/dDP overexpression (Du were obtained from the Bloomington Stock Center and et al., 1996b). Here, we have generated mutant alleles the Berkeley Drosophila Genome Project. To identify of rbf and used these to investigate the role of RBF P-elements inserted in the vicinity of the rbf gene, in the embryonic cell cycles. The phenotype of mutant probes corresponding to the genomic sequences flanking embryos lacking both maternal and zygotic RBF products the P-element insertion sites were generated by an reveals that RBF is dispensable for the early cell cycles inverse PCR approach (Dalby et al., 1995) and screened but plays an essential role in the introduction of G for hybridization to the 158H9 and 26B3 cosmids. By control during development. The cell cycle defects wd1 this method, one P-element line, P[w]cx31A.2 (see observed in RBF-deficient embryos are strikingly differ- Materials and methods), generated probes that hybridized ent from those described previously in dap and fzr with the cosmid 26B3 but not with 158H9 (data not mutant embryos, and suggest that Dacapo, Fizzy-related shown; see Figure 6 for a diagram of the rbf genomic and RBF provide distinct functions that are required locus). This P-element insertion is not lethal and fails for the timely cessation of cell cycle progression. to enhance the GMRdE2FdDP phenotype. Western blot analysis showed that the P-element does not alter the Results level of RBF protein (data not shown), and Southern Chromosomal deletions in the 1B–2A region blot analysis indicated that the P-element lies at least modify eye phenotypes that result from altered 20 kb 3 to the RBF coding sequences (data not shown). cell proliferation A local hopping strategy was used to generate P- The rbf gene was mapped by in situ polytene element insertions in the rbf locus. The P-element in wd1 hybridization to the cytological region 1CD. Stocks P[w]cx31A.2 was mobilized by the introduction of 917 W.Du and N.Dyson Fig. 1. rbf mutants enhance the phenotypes of GMRdE2FdDP and suppress the phenotypes of GMRp21. Scanning electron micrographs of adult eyes. (A), (C), (E), (G), (I) and (K–N) were at the same magnification, the white bar in (A) corresponding to 100 μm. (B), (D), (F), (H) and (J) were at the same magnification, the white bar in (B) corresponding to 10 μm. Genotypes: (A and B) wild-type; (C and D) GMRdE2FdDP/; 120a 14 (E and F) Df(1)su(s)83/;GMRdE2FdDP/;(G and H) P[w]wd /Y; GMRdE2FdDP/;(I and J) rbf /;GMRdE2FdDP/;(K) GMRp21/; 120a 14 (L) Df(1)su(s)83/;GMRp21/;(M) P[w]wd /;GMRp21/; and (N) rbf /;GMRp21/. Note that the GMRdE2FdDP phenotype (in C and D) is enhanced in (E), (G) and (I), and in (F), (H) and (J), whereas the GMRp21 phenotype (in K) is suppressed in (L), (M) and (N). a Δ2-3 transposase, and two complementary screening lines were established and rbf was analyzed by Southern strategies were used to identify insertions in the vicinity blot analysis (see Figure 2C and D for an example). rbf , 14 16 of rbf as described in Materials and methods. A total of rbf and rbf are three alleles obtained by this method that 15a 38a three new P-element lines (P[w]wd , P[w]wd , contain complete deletions of the RBF coding sequence. 120a and P[w]wd ) were obtained. 15a 38a 120a Analysis of P[w]wd , P[w]wd and P[w]wd RBF mutations enhance a phenotype caused by revealed that in each case the P-element was inserted 5 the overexpression of dE2F/dDP and suppress a to the rbf open reading frame. This is illustrated by the phenotype caused by the overexpression of p21 PCR analysis shown in Figure 2. PCR reactions using a Mutant alleles of rbf were assessed for interactions with the primer from the P-element and a primer from the rbf 5- GMRdE2FdDP and GMRp21 phenotypes as predicted from untranslated region (Figure 2A) specifically amplified the interactions observed using the large deficiencies. As DNA fragments from DNA prepared from each of these shown in Figure 1, reducing the gene dosage of rbf by either lines (Figure 2B). The size of these fragments indicates 14 120a the null allele RBF or the viable weak allele P[w]wd that the P-elements lie ~700 bp from the translation start both strongly suppressed the GMRp21 phenotype (Figure codon. Flies homozygous for any one of these new 1K–N). In addition, reducing the gene dosage of rbf also insertions were viable, and Western blot analysis showed enhanced the GMRdE2FdDP phenotype (Figure 1C–J). that RBF levels varied between 50 and 100% of wild- 120a 15a These interactions indicate that P[w]wd indeed type (data not shown), indicating that P[w]wd , 38a 120a behaves like a viable weak allele of RBF, and showed that P[w]wd and P[w]wd are not null alleles of rbf. the activity of ectopic dE2F/dDP is limited by endogenous To generate null alleles of rbf, these new P-element RBF protein and that the p21-mediated cell cycle arrest lines were used to carry out imprecise excision. After crossing with flies carrying the transposase Δ2-3, new depends on RBF. These results strongly suggest that RBF 918 Role of RBF in G regulation in Drosophila embryogenesis Fig. 2. PCR and Southern blotting analysis illustrating P-element insertion in the rbf locus and its subsequent excision to generate mutant alleles of 120a rbf.(A) Diagram showing the relative position of the P-element insertion in P[w]wd and the position of primers used for PCR. P1, P2 and P3 are primers from the ends of the P-element; a and b indicate the primers from the RBF 5-untranslated region. Arrows indicate the direction of the primers from 5 to 3. ATG indicates the start of translation. (B) PCR products from the P-element insertion lines. DNA isolated from the P-element 120a 38a 15a lines P[w]wd , P[w]wd and P[w]wd are in lanes 1–6, 7–12 and 14–19, respectively. Lane 13 is a molecular weight marker. Primer pairs: (P3 and a) are used in lanes 1, 7 and 14; (P2 and a) in lanes 2, 8 and 15; (P1 and a) in lanes 3, 9 and 16; (P3 and b) in lanes 4, 10 and 17; (P2 and b) in lanes 5, 11 and 18; and (P1 and b) in lanes 6, 12 and 19. Note that PCR products were generated from each of the lines by the 120a combination of primers P1 and b. (C) Diagram showing the position of HindIII sites at the rbf locus in P[w]wd and FM6.In FM6 (as in wild- 120a type), the RBF cDNA hybridizes to two HindIII fragments (fragment II and III). The P-element insertion in P[w]wd (labeled 120a) introduces 120a a new HindIII site, resulting in the appearance of an additional short fragment I. Thus the two fragments that hybridize to RBF in P[w]wd are fragments I and II, and the two fragments that hybridize to RBF in FM6 are fragments II and III. (D) Southern blot analysis illustrating a P-element 14 16 insertion in the RBF locus and its subsequent excision in RBF and RBF . The genotypes of the DNA samples: lane 1, FM6; lane 2, 120a 14 16 P[w]wd /FM6; lane 3, RBF /FM6; and lane 4, RBF /FM6 as indicated. The full-length cDNA RBF was used as a probe. Note that the 120a 14 16 additional HindIII fragment (fragment I) introduced in P[w]wd is deleted in RBF and RBF . In addition, the intensity of fragment II is also reduced by 50% in lanes 3 and 4 compared with lane 2, whereas the fragment III that is derived solely from the FM6 chromosome is unchanged (compare lanes 2, 3 and 4). Lane 1 contains less DNA than lanes 2–4. normally acts as a negative regulator of cell proliferation RBF is an essential regulator of E2F-dependent during eye development. transcription in the embryo Previous work has demonstrated that the transcription of Expression of RBF cDNA rescues the lethality of PCNA and RNR2, two genes that are coordinately RBF mutants expressed as cells enter S-phase, requires both dE2F and Mutations resulting in the complete deletion of rbf were dDP and is induced by ectopic expression of dE2F/dDP lethal as homozygotes, hemizygotes or trans-heterozy- (Duronio and O’Farrell, 1994, 1995; Duronio et al., 1995, gotes. To demonstrate that the lethality of these rbf alleles 1996, 1998; Royzman et al., 1997). In wild-type embryos, is due to the lack of RBF activity, rescue experiments RNR2 is expressed uniformly during early stages but is were carried out by expressing the RBF cDNA under the down-regulated following cell cycle 16 in cells entering control of a heat shock promoter. The lethality of a trans- G , the first time that G regulation is apparent (Duronio 1 1 11 14 heterozygous combination of rbf and rbf , two null and O’Farrell, 1994). Periodic expression of RNR2 is seen alleles in which the RBF coding sequence is completely in the gut and peripheral nervous system (PNS) as cells deleted, was rescued efficiently by expression of the RBF enter S-phase. RNR2 expression persists in the CNS that cDNA from the heat shock-regulated transgene hsRBF contains actively dividing cells, but RNR2 is not re- (see Materials and methods). Similarly, the hsRBF trans- expressed in epidermis of the embryo following mitosis gene also allowed rescue of viable males carrying rbf of cell cycle 16 as these cells remain quiescent until the or rbf . For these experiments, vials were incubated at first larval instar. 11 14 37°C for 1 h/day throughout development. No viable rbf / While no rbf /rbf trans-heterozygous adult flies were 14 11 14 rbf females, or rbf or rbf male adult flies were found in the absence of the hsRBF transgene, egg counts 11 14 observed without heat shock treatment or by heat shock showed that most rbf /rbf trans-heterozygous embryos in the absence of the hsRBF transgene. Thus, rbf is the hatched (data not shown). Embryos homozygous for 14 11 only essential function missing in rbf and rbf . mutant alleles for rbf were assayed by in situ hybridization 919 W.Du and N.Dyson using a probe for RNR2 expression, but no abnormalities deficient embryos examined, BrdU incorporation was were apparent. In particular, RNR2 expression was observed in only a subset of the epidermal cells. These repressed after mitosis 16 in rbf mutant embryos in a observations suggest that epidermal cells initially enter manner that appeared identical to the wild-type embryos G following mitosis 16 but, in the absence of RBF, a (data not shown). These observations might suggest that significant proportion of the cells were unable to remain RBF is neither required for the repression of RNR2 in G and entered S-phase. In situ hybridization with a expression nor has an essential function during embryogen- probe for cyclin E showed that the level of cyclin E esis; alternatively, RBF may have essential functions that mRNA is significantly elevated in the epidermis of RBF- can be performed by maternally supplied products. To test deficient embryos (Figure 4G and H). In these embryos, this, we generated rbf mutant embryos that were derived cyclin E expression is only partially deregulated, consistent from germline clones and lacked both maternal and zygotic with previous evidence that dE2F and dDP provide only RBF. As described below, such RBF-deficient embryos one of several activities that regulate cyclin E expression displayed a variety of phenotypes that reveal essential (Duronio and O’Farrell, 1995; Royzman et al., 1997; roles for RBF in E2F regulation and cell cycle control Duronio et al., 1998). Interestingly, cyclin E expression during embryogenesis. was slightly higher in epidermal cells near the segment In situ hybridization experiments show that the expres- boundaries, in a pattern that resembled the pattern of sion of RNR2 is strongly deregulated in the RBF-deficient BrdU incorporation. It is possible that RBF is more embryos. In these embryos, RNR2 was expressed ubiquit- important for cyclin E regulation in these cells. Alternat- ously and uniformly in both early stages of embryogenesis ively, these cells may be more sensitive to deregulation and also in later stage embryos following germband of E2F, and the elevated level of cyclin E expression may retraction (Figure 3E–H, and data not shown). The expres- be caused by S-phase entry. sion of PCNA was altered similarly. In RBF-deficient To determine whether ectopic S-phases resulted in cell embryos, the level of PCNA expression in the epidermis proliferation, RBF-deficient embryos were stained with is equivalent to that seen in the PNS, where PCNA staining an antibody to phosphorylated histone H3 that detects is detected in wild-type embryos at this stage (Figure 3A– mitotic chromosomes (de Nooij et al., 1996). No extra D). Using RNR2 and PCNA expression as a measure of mitotic cells were found in the epidermis in RBF-deficient E2F activity, these results suggest that E2F-dependent embryos (data not shown), indicating that the epidermal transcription is constitutively elevated in the absence of cells that entered S-phase did not complete a mitotic cell RBF. Thus, RBF is required for the developmental down- cycle. The disparity between the S-phase and M-phase regulation of E2F that occurs when G regulation is markers prompted us to assess the level of apoptosis introduced during Drosophila embryogenesis. in these embryos. Wild-type embryos show a low but significant level of apoptosis in the epidermis that can be Defective G regulation in the absence of RBF detected by the TUNEL assay. In contrast, extensive Crosses between females bearing rbf germline clones apoptosis was found in the epidermis of stage 13 RBF- and wild-type males gave viable females, indicating that deficient embryos (Figure 4E and F). Although the majority expression of the wild-type rbf gene from the paternal X of cells incorporating BrdU were located near the segment chromosome was sufficient for viability. As the first 13 boundaries, TUNEL-positive cells were distributed cell cycles are driven exclusively by maternally encoded throughout the segment. This difference suggests that, in products, the paternal rescue indicates that RBF has no many cells, induction of apoptosis is unlinked to S-phase essential functions during these early stages. entry. Taken together, these observations indicate that Bromodeoxyuridine (BrdU) incorporation was used to epidermal cells initially stop in G following mitosis of monitor DNA synthesis in the RBF-deficient embryos. cell cycle 16, but that these cells are unable to maintain Aberrant cell cycle control was observed in cells of the this arrest effectively. With time, an increasing proportion midgut in stage 12–14 embryos. In wild-type embryos, of cells enter S-phase. In addition, many cells are elimin- pulses of BrdU incorporation are observed in the midgut ated by apoptosis. due to differential timing of G (17) in subsets of cells. In RBF-deficient embryos, the pulses of BrdU incorporation Discussion that normally distinguish the central and anterior midgut were weaker, and BrdU incorporation occasionally was RBF-deficient embryos provide a third example, in addi- seen throughout the midgut region (data not shown). tion to dap and fzr mutants (de Nooij et al., 1996; Lane However, the most dramatic changes in DNA synthesis et al., 1996; Sigrist and Lehner, 1997), where epidermal were seen in the epidermis. In wild-type embryos, cells cells are unable to stop cell cycle progression following of the epidermis complete cell cycle 16 in late stage 11; mitosis of cell cycle 16. Although the same cells enter an these cells enter G of cell cycle 17 and no longer ectopic S-phase in each case, the phenotypes of these incorporate BrdU (Figure 4A and C). DNA synthesis is embryos are quite different. First, in dap and fzr mutants, evident in mid-stage 12 in the PNS cells that lie just epidermal cells entering an ectopic S-phase complete a below the epidermis (Figure 4A). No defect was observed mitotic cell cycle; in RBF mutants, no additional mitotic in the pattern of S-phases of RBF-deficient embryos until cells were detected. A second distinction lies in the mid-stage 12 when the germband was partially retracted. persistence of ectopic S-phases. In dap and fzr mutants, In these embryos, ectopic S-phase cells were first observed ectopic S-phases were seen for a short time window; once in the dorsal epidermis. In stage 13 and stage 14 embryos, epidermal cells complete an additional cycle, they remain ectopic S-phases had become more abundant and extended arrested in G . In RBF-deficient embryos, ectopic S-phases to the ventral epidermis (Figure 4B and D). In all RBF- persisted in the epidermis and even increased as the 920 Role of RBF in G regulation in Drosophila embryogenesis Fig. 3. Deregulation of E2F activity in embryos lacking RBF. Endogenous E2F activities were detected by in situ hybridization with antisense probes to PCNA (A–D) or RNR2 (E–H); the same patterns of expression were detected for these two genes. Two different stage of embryos are shown. (A–D) Embryos at the beginning of germband retraction; (E–H) embryos with a completely retracted germband. RNR2 and PCNA are expressed ubiquitously at high levels at each of the stages shown in RBF-deficient embryos. (A and B) A wild-type embryo at the beginning of germband retraction; the image was focused on the epidermis in (A) and on the midline in (B). (C and D) An RBF maternal and zygotic null embryo; the image was focused on the epidermis in (C) and on the midline in (D). (E and F) A germband-retracted wild-type embryo; the image was focused on the epidermis in (E) and on the midline in (F). Note that the anterior and posterior midgut staining in (F) is out of focus in (E). (G and H)A germband-retracted RBF maternal and zygotic null embryo; the image was focused on the epidermis in (G) and on the midline in (H). embryos aged. Third, in RBF-deficient embryos, most of E2F-dependent transcription (Duronio and O’Farrell, epidermal cells initially enter G (17). Because epidermal 1994). As the overexpression of dE2F and dDP is able to cells of dap mutant embryos enter S-phase synchronously drive cells from G into S-phase, it appeared likely that and rapidly following mitosis of cell cycle 16, it has been this inhibition of E2F activity would be essential for the unclear whether these cells enter G (17) or whether they appearance of G . The analysis of RBF-deficient embryos 1 1 progress directly from mitosis to S-phase (de Nooij et al., argues against this conclusion. RNR2 and PCNA, the two 1996; Lane et al., 1996). In mid-stage 12 RBF-deficient genes that have been used most widely in previous studies embryos, only a subset of cells incorporate BrdU, indicat- to provide a measure of endogenous E2F activity, are ing that the majority of cells initially remained in G (17). constitutively expressed in RBF-deficient embryos. Never- Cells of the dorsal epidermis were already post-mitotic theless, the majority of epidermal cells are able to enter by this stage in control embryos. G , and it is only as the embryo ages that large numbers We infer from these observations that RBF plays an of these cells enter ectopic S-phases. Thus, most epidermal important role in the imposition of G regulation during cells require neither RBF nor the repression of E2F- Drosophila development. It is well established that the dependent transcription to enter G . Instead RBF’s role appearance of G (17) correlates with the down-regulation appears to lie in the maintenance of the G phase. 1 1 921 W.Du and N.Dyson Fig. 4. Ectopic S-phases and increased apoptosis in RBF-deficient embryos. BrdU incorporation of wild-type embryos (A and C) or RBF maternal and zygotic null embryos (B and D). (A and B) Germband partially retracted stage 12 embryos. Cells in the epidermis were not labeled with BrdU in wild-type embryos (A); the cells that are labeled with BrdU in the middle of each segment are PNS cells just below the epidermis. In contrast, cells in the epidermis in RBF-deficient embryos were labeled with BrdU (B); note that the cells that are labeled are on the dorsal epidermis and are adjacent to the segment boundary. (C and D) Germband completely retracted embryos. Cells in the epidermis were not labeled with BrdU in wild- type embryos (C); the dark shadow in the middle of the embryo is due to BrdU staining of midgut and hindgut. CNS cells at the ventral side of the embryo are labeled strongly; these cells are still proliferating at this stage. Cells in the epidermis in RBF-deficient embryos were labeled with BrdU (D); note that cells in the ventral epidermis also incorporate BrdU. CNS cells are below this focal plane. (E and F) TUNEL staining of a wild-type embryo shown in (E) and an RBF-deficient embryos shown in (F). Note that the number of TUNEL-stained cells is significantly increased; however, these cells do not appear to have the same pattern of BrdU-labeled cells (compare B, D and F). (G and H) Cyclin E expression was detected by whole-mount in situ hybridization. A germband-retracted wild-type embryo is shown in (G); cyclin E transcripts were not detected in the epidermis. A similar staged RBF-deficient embryo is shown in (H); a significant level of cyclin E RNA was detected in the epidermis. Note that cyclin E expression is elevated in cells adjacent to the segment boundary, resembling the pattern of BrdU incorporation in these embryos. The properties of dap mutant embryos and RBF- exit the cell cycle (de Nooij et al., 1996; Lane et al., deficient embryos are highly consistent with a model in 1996). Our results suggest that the repression of cyclin E which the complementary roles of RBF and Dacapo in and other E2F-regulated genes by RBF is not important G control are due to their complementary roles in the at this stage, but becomes essential later, as the expression regulation of cyclin E activity (Figure 5). In this model, of Dacapo declines. In RBF-deficient embryos, the elev- G is triggered by the inhibition of the cyclin E–Cdc2c ated expression of cyclin E in cells that lack Dacapo will kinase. Dacapo is required for this process (de Nooij et al., lead to the accumulation of an active kinase, and eventually 1996; Lane et al., 1996), although it is not certain that it to S-phase entry. In dap mutants, however, RBF repression is the only mechanism involved. Previous studies have of cyclin E expression would limit its ability to drive shown that Dacapo is only transiently expressed as cells epidermal cells through multiple cycles. 922 Role of RBF in G regulation in Drosophila embryogenesis Previous studies have shown that PCNA and RNR2 are not expressed in dE2F or dDP mutant embryos (Duronio et al., 1995, 1998; Royzman et al., 1997). These results suggested that the induction of gene expression that occurs in wild-type embryos as cells enter S-phase was due primarily to transcriptional activation by a dE2F–dDP complex. However, the finding that these genes are con- stitutively expressed in RBF-deficient embryos adds a level of complexity. This result indicates that RBF actively represses the expression of PCNA and RNR2 in G phase cells and suggests that the pulses of gene expression seen as cells enter S-phase could be due largely to the release of repression. If RBF, dE2F and dDP are common compon- ents of a repressor complex, one wonders why PCNA and Fig. 5. RBF and Dacapo cooperate to regulate cyclin E kinase activity RNR2 are not constitutively expressed in dE2F and dDP and to establish G . The cyclin E-associated kinase activity after mutant embryos? One possible explanation is that dE2F mitosis 16 in wild-type, dap or RBF-deficient embryos are drawn in black, blue and red, respectively. Lines on top show the time at which and dDP are not only co-repressors with RBF but are also RBF and Dacapo are important. In this model, we suggest that the required to activate the RNR2 and PCNA promoters. An functions of rbf and dap converge on the regulation of cyclin E, alternative possibility is that RBF repression of PCNA Dacapo acting to inhibit the residual cyclin E kinase activity following and RNR2 expression is not mediated by dE2F/dDP, but mitosis 16, and RBF acting to repress cyclin E expression. In dap by a different RBF-binding protein. The recent discovery mutants, residual cyclin E-associated kinase activity is sufficient to drive cells into S-phase following mitosis 16. Only one additional of dE2F2, that associates with dDP and RBF in Drosophila cycle occurs because cyclin E-associated kinase activity drops below embryos (D.Huen, W.Du, Y.Chen, and N.Dyson, unpub- the threshold due to the repression of cyclin E expression by RBF. In lished observations), opens up a variety of potential RBF-deficient embryos, the expression of Dacapo inhibits the cyclin explanations. It is also evident that RNR2/PCNA and E-associated kinase, initially causing a G arrest. However, in the absence of RBF, cyclin E is mis-expressed and cells accumulate cyclin cyclin E represent two different types of E2F target genes. E kinase activity as Dacapo levels decline and the cyclin E level Although expression of all three genes can be induced by increases. Once the cyclin E kinase activity passes the threshold for ectopic expression of dE2F and dDP, RNR2 and PCNA S phase, these cells will initiate DNA replication. were completely derepressed by the absence of RBF, whereas cyclin E was deregulated in a more subtle manner. The level of cyclin E expression observed in the epidermis Why do the ectopic S-phase cells in RBF-deficient of RBF-deficient embryos remains considerably lower mutants fail to progress to mitosis? We suggest that this than the level of cyclin E expressed in the central nervous may occur for several reasons. The levels of cyclins A system and brain. In some cell types, expression of cyclin and B drop rapidly once cells enter G (Lehner and E is independent of both dE2F and dDP (Duronio and O’Farrell, 1989, 1990). In dap mutant embryos, where O’Farrell, 1995; Royzman et al., 1997; Duronio et al., cells enter S-phase rapidly after mitosis of cell cycle 16, 1998), and it seems likely that the expression of cyclin E, there may still be sufficient A- and B-type cyclins to like that of string and other critical cell cycle regulators, allow these cells to progress to mitosis 17. Given the is under the control of multiple enhancer elements. delay before ectopic S-phase cells appear in RBF-deficient The phenotype of RBF-deficient embryos has several embryos, the levels of mitotic cyclins may be insufficient –/– features in common with the phenotype of Rb mice for cell cycle progression. In wild-type animals, epidermal (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992, cells will leave G (17) to enter an endoreduplication S- 1994). Both mutants are characterized by ectopic S-phases, phase. By the time that ectopic S-phase cells appear the expression of E2F-regulated genes and high levels of in RBF-deficient embryos, these cells may already be apoptosis. Such similarities emphasize that these homolog- committed to an endoreduplication cycle. In addition, ous proteins serve analogous functions. Interpretation of there is evidence that the down-regulation of E2F activity the phenotypes of pRB, p107 and p130 knockout mice is is important for cells to exit S-phase (Krek et al., 1995). complicated by the fact that these proteins share overlap- Potentially, the failure to down-regulate E2F or, alternat- ping functions. Even in studies of double knockout mice, ively cyclin E expression (Follet et al., 1998; Weiss et al., these animals progress through many stages of mouse 1998), may arrest cells either in S-phase or post-S-phase, development before cell cycle defects become apparent in RBF-deficient embryos. Finally, the overexpression of (Cobrinik et al., 1996; Lee et al., 1996; Mulligan and E2F genes induces apoptosis in a variety of experimental Jacks, 1998). It has been unclear whether pRB family systems (Qin et al., 1994; Shan and Lee, 1994; Wu and proteins are unimportant for many cell cycles or whether Levine, 1994; Kowalik et al., 1995; Asano et al., 1996; they have a redundant but critical function. The phenotype Du et al., 1996b). Although our results suggest that S- of RBF-deficient embryos reveals that RBF is important phase entry is not a prerequisite for apoptosis in the RBF- at the very first cell cycle in Drosophila development deficient embryos, cells that enter S-phase inappropriately where G regulation is introduced. Interestingly, RBF is may be especially sensitive to the effects of elevated E2F required for the maintenance of G , but not for its activity and may not survive to complete the cell cycle. initial appearance. By analogy, one of the initial roles of The changes in expression of E2F-regulated genes caused by the absence of RBF provide new insights into mammalian pRB family members may be to allow rapidly the regulation of E2F activity in the Drosophila embryo. proliferating cells to pause in G . 923 W.Du and N.Dyson Materials and methods Fly stocks 1 1 1 1 Deficiency lines Df(1)AD11/FM7 and Df(1)su(s)83, y cho ras v / 2 1 1 Dp(1;Y)y sc/C(1)DX, y f , the P-element stock P1318 (P[w] cx31A.2) and the FLP, FRT lines for germline clonal analysis were obtained from the Bloomington Stock Center. P[w; hsp70-RBF] was generated by subcloning of the RBF full-length cDNA (Du et al., 1996a) into pCaSpeR-hs vector. A P[w; hsp-RBF] on the X chromosome was used for the rescue experiments. Characterization of the RBF chromosomal region Cosmid contigs between the 1B and 1DE polytene region were obtained from the European Genome Mapping Project, and were hybridized using the RBF cDNA as a probe. Two overlapping cosmids, 158H9 and 26B3, contain RBF sequences and were used to identify P-elements in the vicinity of rbf. P-element lines were obtained from the Bloomington Stock Center and the Berkeley Drosophila Genome Project. An inverse PCR method was used to make probes from the genomic sequences adjacent to the P-element insertions. These probes were hybridized to the two cosmids that contained RBF sequences. Stock number P1318 (P[w]cx31A.2) gave probes that hybridize to the cosmid 26B3 but not to 158H9. Since this stock contains two P-element insertions on the X chromosome, this stock was crossed with w , and the resulting two different classes of lighter eye color flies were established. One such wd1 line, P[w]cx31A.2 , was shown to retain the P-element in the vicinity of rbf and was used in subsequent experiments. Embryo analysis In situ hybridization. Digoxigenin-labeled antisense RNA probes were prepared by in vitro transcription reaction. Properly aged embryos were collected and fixed. Hybridization was carried out at 70°C as described (Duronio and O’Farrell, 1994). BrdU staining. Properly aged embryos were collected, dechorionated in 50% Chlorox, permeabilized in octane, and incubated in 1 mg/ml BrdU in Schneider’s medium for 20 min. Embryos were fixed in an equal volume of 4% formaldehyde and heptane. BrdU was detected using a mouse anti-BrdU antibody (Becton Dickenson, 1:100). TUNEL assay. Collected and fixed embryos were treated with proteinase K (10 μg/ml) for 5 min, washed twice with PBT and post-fixed with 4% formaldehyde for 20 min. These embryos were processed further using the Apotag kit from ONCOR (Gaithersberg, MD) according to the instructions provided. Generation of RBF mutants The scheme used for hopping is shown in Figure 6. Females from 20 different lines (one female each line) were pooled for inverse PCR to Fig. 6. The scheme for hopping. Two different crosses were used to generate probes. These probes were hybridized to the 26B3 and 158H9 generate rbf mutants. A transposase Δ2-3 was crossed to the cosmid DNA digested with NotI. A total of 1600 lines were established wd1 P[w]cx31A.2 line. Resulting males were either crossed to a first and tested using cross A (Figure 6); one line was found to have a chromosome balancer FM6 to establish lines in (A) or crossed to new P-element insertion in cosmid 158H9. Four hundred lines were GMRdE2FdDP to identified flies with slightly enhanced eye established using cross B (Figure 6) and two lines were found to have phenotypes in (B). new P-element insertions in cosmid 158H9. Southern and PCR analysis showed that all three P-elements were inserted in the 5-untranslated region of the RBF cDNA, ~700 bp from the start codon ATG. To generate null alleles of RBF, these three P-element lines isolated 15a 38a 120a (P[w]wd , P[w]wd and P[w]wd ) were crossed to the transposase Δ2-3 and new lines were established. The deletions of the progeny from this cross are expected to be the mutant class; however, rbf coding sequences were determined by Southern analysis. All lines the FM7 male is usually under-represented and, taking this into account, with deletions of the rbf coding sequences were found to be lethal. complete rescue of the mutant class could provide up to 30% of the adults. Generation of germline clones rbf mutants were recombined onto an X chromosome carrying an FRT Acknowledgements site inserted at 14AB. Germline clone females were generated as described (Soto et al., 1995). Virgin females with rbf germline clones We thank the Bloomington Stock Center and the Berkeley Drosophila were collected and were crossed with males carrying Eve-lacZ on the X Genome Project for the fly stocks used in this study, and the European chromosome. Embryos with the wild-type rbf gene were identified by Genome Mapping Project for the cosmid contigs. We are grateful to anti-β-gal staining. 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Journal

The EMBO Journal – Springer Journals

Published: Feb 15, 1999

Keywords: cell cycle; embryogenesis; Drosophila; RBF; transcription factor E2F

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