Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis Karsten Sauer, 1,3 Jiirgen A. Knoblich, 1,3,4 Helena Richardson, 2 and Christian F. Lehner l's ~Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tfibingen, Germany; 2Department of Genetics, University of Adelaide, South Australia 5005, Australia Drosophila cyclin E (DmcycE) is required in embryos for S phase of mitotic and endoreduplication cycles. Here, we describe regulatory differences characteristic for these two cell cycle types. While DmcycE transcript levels decline in DmcycE mutant cells programmed for mitotic proliferation, they are maintained and no longer restricted to transient pulses in DmcycE mutant cells programmed for endoreduplication. Moreover, DmcycE expression in endoreduplicating cells is down-regulated by ectopic expression of a heat-inducible cyclin E transgene. DmcycE expression in endoreduplicating tissues, therefore, is restricted by a negative feedback to the transient pulse triggering entry into S-phase. Conversely, during mitotic cycles, where S phase entry is not only dependent on cyclin E but also on progression through M phase, cyclin E and associated Dmcdc2c kinase activity are present throughout the cell cycle. Reinitiation of DNA replication during the G 2 phase of the mitotic cell cycle, therefore, is prevented by cyclin E/Dmcdc2c kinase-independent regulation. Observations in cyclin A mutants implicate G 2 cyclins in this regulation. Our results suggest molecular explanations for the different rules governing S phase during mitotic and endoreduplication cycles. [Key Words: Cyclin A; cyclin E; Dmcdc2c kinase; S phase; endoreduplication; Drosophila] Received December 1, 1994; revised version accepted April 11, 1995. The essential steps of the mitotic cell cycle, S and M and vertebrate tissue culture cells results in a periodic phase, have to be strictly alternating to preserve genome and ordered accumulation of G1 and G2 cyclins and, ploidy. Recent progress in understanding the regulation therefore, contributes to the ordered progression through of entry into S and M phase provides a basis for the anal- the cell cycle. In budding yeast, this transcriptional con- ysis of the molecular mechanisms that maintain the cor- trol involves positive and negative feedback loops. The rect temporal order of these essential events. Entry into G1 cyclins Clnl and Cln2, as well as the G2 cyclin Clb2, both S and M phase is controlled by cyclin-dependent have been shown to stimulate the transcription of their kinases. Specialized cyclins and cdc2-1ike kinases con- own genes via positive feedback loops {Cross and Tink- trol either the G1/S or the Gz/M transition in higher elenberg 1991; Dirick and Nasmyth 1991; Amon et al. eukaryotes (Fang and Newport 1991; Hamaguchi et al. 1993). Importantly, the G2 cyclin Clb2 is also capable of 1992; Baldin et al. 1993; Knoblich and Lehner 1993; shutting off the transcription of the GI cyclins Clnl and Quelle et al. 1993; Stem et al. 1993; van den Heuwel et Cln2 (Amon et al. 1993). al. 1993; Knoblich et al. 1994; for review, see Sherr 1993; A number of observations indicate that post-transcrip- Solomon 1993). The G2/M transition is controlled by the tional mechanisms are also involved in ordering cell cy- cdc2 kinase in association with G2 cyclins (cyclins A and cle phases. Replacing the periodic transcription of either B). In contrast, progression through the G1 phase and G1 or G 2 cyclins by continuous transcription throughout entry into S phase is controlled by the cyclin-dependent the cell cycle is not incompatible with an ordered cell kinase 4/6 (cdk4/6) and cdk2 in conjunction with G~ cycle progression in budding yeast {see, e.g., Ghiara et al. cyclins (cyclins D and E). 1991; Amon et al. 1994}. Moreover, early embryonic cy- Transcriptional control of cyclin expression in yeast cles in many species, including Xenopus and Drosophila, rely exclusively on maternal stores deposited in the egg during oogenesis and proceed normally in the complete 3These authors contributed equally to this work. absence of transcription. A post-transcriptional mecha- 4Present address: Howard Hughes Medical Institute, University of Cal- ifornia, San Francisco, Cafifomia 94143-0724 USA. nism, which is of paramount importance for the order of SCorresponding author. cell cycle phases, is the control of G2 cyclin stability. GENES & DEVELOPMENT 9:1327-1339 91995 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/95 $5.00 1327 Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Saner et al. The abrupt degradation of these proteins during mitosis E-associated kinase activity. Our observations in cyclin results in the inactivation of the associated cdc2 kinase A mutant embryos, however, suggest that G2 cyclins are and allows exit from mitosis (Evans et al. 1983; Glotzer required for preventing inappropriate endoreduplication et al. 1991; Holloway et al. 1993; Surana et al. 1993). during the G2 phase of the mitotic cycle. Recent observations in Drosophila and budding yeast in- dicate that the reaccumulation of G2 cyclins after mito- sis is dependent on the presence of G1 cyclins (Amon et Results al. 1994; Knoblich et al. 1994). The G2 cyclin degradation The control of cyclin E transcription system, which is activated during mitosis, appears to in endoreduplicating and mitotically proliferating remain active in the following interphase until it is in- tissues is distinct activated by G1 cyclins. Thus, G2 cyclin accumulation after mitosis cannot occur prior to G~ cyclin accumula- To determine whether cyclin E can regulate its own ex- tion. This control, therefore, presumably prevents entry pression, we expressed cyclin E from a heat-inducible into a second mitosis before entry into S phase has oc- transgene (Hs-cyclm E) and analyzed the effect on the curred, and additional checkpoint mechanisms assure expression of the endogenous cyclin E gene [DmcycE). that entry into mitosis occurs only after completion of S The results of these experiments are illustrated in Figure phase (Weinert and Hartwell 1988; Enoch et al. 1992; 1. A 30-min heat pulse resulted in abundant expression Kelly et al. 1993; Saka and Yanagida 1993; Weinert et al. of Hs-cyclin E as visualized by in situ hybridization with 1994). an antisense RNA probe derived from the cyclin box re- Here, we address the controls that prevent entry into a gion of DmcycE. As expected, transcripts were detected second S phase before mitosis has occurred. An attrac- in all cells of the Hs-cyclin E embryos immediately after tive model for this regulation invokes a licensing factor a heat pulse at stage 13, and these transcripts were found that has been postulated based on elegant experiments to disappear slowly within 60 min of recovery at 25~ with Xenopus egg extracts but not yet identified molec- (Fig. 1G-I). Expression from the endogenous cyclin E ularily (Blow and Laskey 1988; Blow 1993; Coverley et gene was analyzed with a probe derived from the 3'-un- al. 1993). According to this model, entry into S phase is translated region of DmcycE that is not included in the dependent on the presence of licensing factor in the nu- Hs-cyclm E transgene. The transcripts from the endog- cleus. Licensing factor is postulated to accumulate in the enous DmcycE gene were not distributed throughout the cytoplasm during interphase. Access to the nucleus is embryo. DmcycE transcripts were found predominantly only possible after nuclear envelope breakdown during in cells of the developing central nervous system [CNS mitosis. Moreover, nuclear licensing factor is thought to [Fig. 1D-F)], where cells are known to proliferate mitot- be consumed during S phase. ically at this stage. Only very weak signals were present Drosophila allows a comparison of S-phase regulation in intemal tissues that are known to progress through during mitotic cell cycles and endoreduplication cycles. endoreduplication cycles at this stage (Fig. IF, arrow- Most postmitotic cells in Drosophila development even- heads). Interestingly, signals were much stronger in tually enter endoreduplication cycles where they prog- these internal tissues when the same probe was used for ress through several rounds of S phase without interven- in situ hybridization with control embryos that did not ing mitoses (Smith and Orr-Weaver 1991). The depen- carry the Hs-c.yclin E transgene but had also been ex- dency of S phase on progression through M phase must posed to a 30-min heat pulse (Fig. 1C, arrowheads). The be absent or bypassed in these endoreduplication cycles. same transcript distribution and levels as in the heat- We have demonstrated recently that Drosophila cyclin shocked controls that did not carry the Hs--cyclin E E {DmcycE) is required for entry into S phase during mi- transgene were also obtained with Hs-cyclin E or wild- totic and endoreduplication cycles (Knoblich et al. 1994). type embryos that had not been exposed to a heat pulse Moreover, ectopic expression of cyclin E after the final (Knoblich et al. 1994; data not shown). These observa- mitotic division is sufficient to force the postmitotic tions indicate that ectopic Hs-cyclin E expression inhib- cells into S phase, and the normal pattern of DmcycE its the endogenous DmcycE transcription only in en- expression determines the spatial and temporal program doreduplicating tissues and not in mitotically proliferat- of endoreduplication during embryogenesis (Knoblich et ing cells of the CNS. The inhibition was maximally al. 1994). We show here that the brief pulses of DrncycE apparent after 60 min of recovery (Fig. 1, cf. C and F). expression that anticipate endoreduplication rely on a The opposite effect, induction by ectopic Hs-cyclin E negative feedback loop whereby cyclin E down-regulates expression, has been reported in the case of S-phase-spe- its own expression.