Developmental regulation of the cell cycle Robert Saint and Peter L. Wigley University of Adelaide, Adelaide, Australia The control of metazoan cell proliferation, a problem long the domain of cell culture studies, is now being examined in developing animals. Surprisingly, developmental regulation is mediated at a variety of cell-cycle stages. Highly conserved cell-cycle control mechanisms provide a focus for studying the regulatory processes involved.
Current Opinion in Genetics and Development 1992, 2:614-620
Introduction This review examines the emerging relationship between the molecular regulation of development and the control of the eukaryotic cell cycle. This relationship bears directly on the problem of developmental growth regulation, and indirectly on the problem of uncontrolled proliferation in neoplastic tissues. Interesting examples of cell-cycle control during development continue to be identified, but the underlying regulatory processes are, as yet, poorly understood. In this review, we focus particularly on the following questions: at what cell-cycle stages, and by virtue of the activity of which cell-cycle control genes, is developmental regulation of cell proliferation mediated? We will not discuss, for the most part, the vast body of data that has come from studies of cultured cells (reviewed in [1]). The molecular control of cell-cycle progression has been a fertile field of research for some time (reviewed in [2,3]). Here, we describe only those features relevant to our discussion. The cell cycle can be divided into four sequential phases: mitosis, or M-phase; a gap or Gl-phase that follows mitosis; S-phase during which DNA replication occurs; and a second gap or G2-phase prior to entry into mitosis. Central to the control of cell-cycle progression is a serine/threonine kinase known as p34 cdc2. For activity, this protein requires association with one of a family of proteins termed cyclins. The activi W of the cyclins varies during the cell cycle, with different cyclins being active in different cell-cycle phases. The phosphorylation state of p34 cdc2 also regulates its activity. Dephosphorylation of p34 cdc2 by a phosphatase, encoded in Schizosaccharomyces pombe by cdc25, is required for its activation at the G2-M phase boundary (reviewed in [4]). Proteolytic breakdown of cyclin is required for inactivation of the p34 cdc2 kinase during metaphase and, therefore, for exit from mitosis.
On the basis of sequence homologies and complementation of yeast cell-cycle mutants with metazoan homologues, these eukaryotic cell-cycle controls appear to be universal. The control mechanisms are much more complex than suggested in this outline. For exanlple, metazoans have additional cyclin-dependent kinases that are also implicated in cell-cycle control (reviewed in [5] ). The role of these homologues in the developmental regulation of the cell cycle is yet to be studied.
Developmental regulation during the Gl-phase Cyclins and G1 arrest during yeast mating Tissue culture studies have long pointed to the Gl-phase as the stage during which key cell-cycle control mechanisms operate. It is no surprise, therefore, that we find examples in which cell proliferation during development is controlled during this phase. One of the best characterized examples of developmental regulation during the Gl-phase is found in the budding yeast, Saccharomyces ceret,isiae. In response to mating pheromone, haploid S. cerevisiae cells fuse to form diploid cells. Mating pheromone induces arrest in the Gl-stage prior to mating. Normal progression from G1- into S-phase in this organism requires the activity of a functionally redundant group of G1 cyclins, CLN1, CLN2 and CLN3 (reviewed in [3]). The Gl-phase arrest induced by mating pheromone, is mediated by a signal transduction cascade that ultimately results in functional inactivation of all three G1 cyclins (reviewed in [6]). Two genes required for this inactivation have been identified by mutant analysis. Firstly, the FARI gene was identified because far1 mutant cells fail to arrest in response to mating pheromone [7]. Using far1 cln double mutants, FAR1 was shown to be specifically required for the in-
Abbreviations CSF--cytostatic factor; CSF-l--colony-stimulating factor-I; DER--Drosophila EGF-receptorhomologue; MPF---maturation (or M-phase) promoting factor.
614
(~ Current Biology Ltd ISSN 0959-437X
Developmental regulation of the cell cycle Saint, Wigley hibition of CLN2, since only far1 cln2 double mutants arrest normally in the presence of pheromone. Unfortunately, the sequence of FAR1 provides no hints to its molecular action. Secondly, the product of the FUS3gene was also shown to be involved in the inhibition of the G1 cycllns [8"]. FUS3 encodes a protein kinase required for at least two responses to mating pheromone, one being G1 arrest [9]. Mutant analysis demonstrated that FUS3 mediates inhibition of CLNI and CLN2 by decreasing their mRNA levels. FUS3 is also required for CLN3 inhibition, since fus3 cln3 double mutants arrest normally, but this inhibition must be post-transcriptional, as the level of CLN3 mRNA is unaffected in a fus3 mutant. The CLN3 repression may, in part, explain the effect on the other CLN genes, as CLN3 stimulates transcription of CLIV1 and CLIV2 [10,11 ]. Even in this, the best characterized example of developmental cell-cycle regulation, we are only beginning to understand the pathways that transduce the pheromone signal into CLN inactivation, and consequently, cell-cycle arrest.
One such example of tissue-specific cyclin expression has come from our studies of a Drosophila cyclin-E homologue (HE Richardson, LV O'Keefe, SI Reed, R Saint, unpublished data). Drosophila cyclin-E transcripts are present initially as uniformly distributed maternal mRNA that persists during the first 13 synchronous syncytial embryonic cycles. During cycle 14, immediately prior to the onset of the asynchronous mitoses that characterize organogenesis, the level of transcripts decreases dramatically. Expression is then observed specifically in proliferating neuroblasts of the central and peripheral nervous systems, but not in proliferating epidermal cells. Developmental regulation of cyclin gene expression may, therefore, turn out to be a widespread mechanism of tissue-specific cell-cycle control. Not all G1 cyclins are transcriptionally regulated. The mRNA products of the cyclin-C homologue of Drosophila appear to be uniformly distributed in developing embryos [14,15]. This cyclin may, however, play a role in developmental regulation of proliferation as posttranscriptional regulation of cyclins is very common. A more detailed understanding of the role of these G1 cyclins in metazoan development awaits the analysis of the proteins themselves and of cyclin mutant phenotypes.
Cyclins and G1 regulation in metazoans G1 cyclins are also likely candidates for the control of the G1-S-phase transition in metazoans. Recently, several candidate metazoan G1 cyclins have been identified by their ability to rescue yeast strains deficient in the three yeast G1 cycllns (reviewed in [3]). These cyclins have been assigned the names cyclin-C, cyclin-D (which has several related members) and cyclin-E. Two preliminary lines of evidence suggest a role for these cyclins in the developmental regulation of the cell cycle. The first comes from the study of mouse macrophage cultured cells in which expression of a mouse cycllnD homologue, termed CYL1, was shown to respond to a growth factor that stimulates proliferation [12-]. In these cells, colony-stimulating factor-1 (CSF-1) is required throughout G1 for both cell survival and commitment to S-phase. Withdrawal of CSF-1 results in arrest in the Gl-phase, while the subsequent addition of CSF-1 stimulates re-entry into the cell cycle. CYL1 mRNA and protein levels decline abruptly following CSF-1 with. drawal, while re-stimulation with the growth factor very quickly induces C"YL1expression [12--]. Interestingly, the level of CYLI mRNA in the re-stimulated cells is maintained throughout the cycle and into the next Gl-phase, so that CYL1 mRNA levels appear to respond to growthfactor activity and not to the phase of the cell cycle. In the same study, two related murine cyclin-D homologues, termed CYL2 and CYL3, were identified [12-.]. Northern-blot analysis demonstrated that two of the three cyclin-D genes were expressed in a tissue-specific manner. Furthermore, a candidate bcl-1 oncogene, which is a member of the human cyclin-D family, also exhibits lineage-specific expression in a variety of human cell lines [13o]. This raises the provocative possibility of developmental regulation of cell proliferation mediated by stage- and tissue-specific cyclin expression.
Developmental regulation in the Gl-phase during Drosophila development Development of the compound eye of Drosophila melanogaster is well characterized (reviewed in [16,17]). The eye derives from the eye imaginal disc, a single cell layer epithelium that is generated by proliferation of a group of founder cells during the first two larval instars. Differentiation occurs as a wave that sweeps across the epithelium in eye imaginal discs of third instar larvae and early pupae. The first event in this progressive process of differentiation is the arrest of cells in the G1phase. While arrested, groups of five cells form a 'precluster' of differentiating photoreceptor cells, each precluster corresponding to the primordium of a single ommatidium. At a later time, all cells other than the differentiating precluster cells enter S-phase /18-], and many of them subsequently enter mitosis. These cells either differentiate to form the remaining photoreceptor cells, cone cells, pigment cells and bristle cells of the mature ommatidium, or undergo a programmed cell death. In dominant Ellipse mutants of the Drosophila EGFreceptor homologue (DER), only a small number of ommatidia form [19]. Most of the cells that would normally differentiate to form the precluster cells rather enter S-phase along with the other undifferentiated cells [20.,21.]. As such, the over-activity of the DER gene either drives these cells into S-phase or causes the cells to enter an alternative developmental pathway that involves a programmed entry into S-phase. Developmental regulation of the G2-M-phase transition is -also implicated in the developing eye. Mitoses in the region of differentiation in Ellipse eye discs were found clustered near the few ommatidia that formed. The remainder of the cells appeared to undergo a programmed cell death. Based on these observations it has been pos-
615
616
Patternformation and developmental mechanisms tulated that an inductive signal from the five precluster cells may drive adjacent G2-phase cells into mitosis, and that in the absence of this signal, the remaining G2-phase cells undergo a programmed cell death [21o]. Another interesting exanlple of developmental regulation during the Gl-phase is found in the developing brain of Drosophila larvae. The lamina neurons that receive direct synaptic input from photoreceptor axons derive from lamina precursor cells which undergo a regulated set of cell movements and cell cycles prior to innervation. In sine occulis mutants, which lack eyes and therefore the incoming photoreceptor cell axons, progression of the lamina precursor cells from G1- into S-phase of the final cell cycle does not occur [22-]. Presumably the incoming photoreceptor axons stimulate entry of the lanaina cells into their final cell cycle.
Developmental regulation during the G2-phase The mos gene and oocyte maturation in Xenopus It is a c o m m o n
belief that cell-cycle regulation occurs
during the Gl-phase. It is therefore particularly significant that developmental regulation of the cell cycle has also been found to occur during the G2-phase. One example where G2-phase regulation is implied is in the developing Drosophila eye, as discussed above. A much better characterized exan~ple concerns maturation of Xenopus oocytes (reviewed in [23]). As for most vertebrates, Xenopus oocytes are arrested in the G2-phase of the first meiotic division. Progesterone, released by follicle cells, induces maturation of the oocytes. Maturation involves progression through the cell cycle until arrest again occurs at metaphase of the second meiotic division. This arrest is released following fertilization. The crucial event in oocyte maturation is the activation of maturation (or M-phase) promoting factor (MPF), which drives entry into mitosis. MPF corresponds to the regulatory p34Ca'C2-cyclin complex. It is now clear that induction of MPF activity, and thus of maturation, requires the production of the pp39 'n°s protein kinase, encoded by the c-mos proto-oncogene (reviewed in [24]). In the normal situation, hormonal stimulation induces translation of maternal c-mos mRNA and, thereby, maturation. This effect is mimicked by injection of mos RNA into oocytes [25,26]. Injection of an engineered mos fusion protein caused the onset of maturation in the absence of protein synthesis [27.°], showing that the mos-encoded product is the only protein required to be synthesized for maturation induction. Direct evidence confirms that new cyclin translation is not necessary for this induction; sufficient stores already exist in the arrested oocyte [28-30]. Induction of maturation is greatly enhanced when, in addition to mos-encoded protein, progesterone is added to oocytes [27°°]. This suggests either that the hormone potentiates mos action, or that it removes an inhibitor. Furthermore, oocytes induced to mature do not progress to the normal end-point of maturation, namely, metaphase
arrest in meiosis II. New protein synthesis is required for completion of maturation, and the evidence suggests that cyclin synthesis is not sufficient to account for this requirement - - another, as yet unknown, protein must be synthesized. The metaphase arrest of Xenopus eggs (and eggs of other vertebrates) is mediated by an activity long known as cytostatic factor (CSF) (not to be confused with CSF-1). Microinjection and immunodepletion experiments have now shown that the c-mos-encoded protein kinase is a component of CSF [31]. Furthermore, proteolysis of the pp39 mos protein following fertilization is necessary to allow cell-cycle progression [32]. Recent evidence shows, however, that cyclin degradation, and therefore MPF inactivation, is mediated by a CSF: mos-independent pathway, probably in response to the calcium-transient induced by fertilization [33",34"]•
• The~s~ing gene and mitotic domains during Drosophila organogenesis Another exm~ple of developmental regulation at the G2stage is found in embryos of D. melanogaster. Following fertilization, the Drosophila embryo undergoes 13 rapid, synchronous and syncytial mitoses before arresting in the G2-phase of cycle 14. At this time, membranes extend from the periphery of the embryo to enclose the nuclei. The subsequent mitoses are dramatically different to the earlier cleavage mitoses, not only because the embryo is now cellular, but also because the mitoses are asynchronous. The cycle-14 mitoses occur synchronously in groups of cells termed mitotic domains [35], but each domain undergoes mitosis at a different, developmentally regulated time. A striking observation is that some, and perhaps all, of the mitotic domains correspond to organ or tissue-type primordia, and therefore represent one of the earliest signs of organogenesis. Several lines of evidence indicate that expression of string regulates the appearance of the mitotic domains [36,37]: string mutants arrest in the G2-phase of cycle 14; accumulation of string mRNA precisely anticipates the pattern of mitotic domains; and ectopic string expression induces synchronous mitoses throughout the embryo, indicating that string is the only limiting factor for cycle-14 divisions. Finally, string is a homologue of the S. pombe mitotic activator gene, cdc25. Regulation of string expression occurs at the transcriptional level (PL Wigley, B Patterson, LV O'Keefe, R Saint, unpublished data)[38]. Moreover, it is predicted that the genes required for pattern formation during embryogenesis, many of which encode transcriptional regulators, directly activate string expression to effect the mitotic domain pattern [36,38]. This prediction is supported by observations that the patterns of both cycle-14 mitoses and string expression are altered in dorsal-ventral patterning gene mutants (cited in [38,39,40"]). Exactly how the patchwork of regulatory gene expression generates the complex string transcription pattern during cycle 14 constitutes a fascinating problem that we are currently addressing.
Developmental regulation of the cell cycle Saint, Wigley One surprising observation from our studies is that the control of string expression also appears to be complex at later stages of embryonic development (during neurogenesis), and in proliferating imaginal tissues (PL Wigley, P Kylsten, B Patterson, R Saint, unpublished data). For example, string transcriptional enhancers have been identified that are specific for particular imaginal tissues, and even for subsets of cells within a single tissue. This indicates either that evolution has opportunistically utilized many different regulators to achieve the end result of string expression in all dividing cells, or that the G2-M-phase transition is developmentally regulated in many more circumstances than previously suspected (other examples of G2 regulation in Drosophila are known, for instance in leg and wing imaginal discs [41,42]).
Developmentally specialized cell cycles
Cleavage cycles The metazoan cell cycle can be substantially modified in particular developmental circumstances. It can, for example, be dramatically accelerated, as occurs in cleavage divisions following fertilization. The rapid cleavage divisions of Xenopus and Drosophila are simple biphasic cycles lacking observable G1- and G2-phases. This is a remarkable modification of the more common complex cell cycle and is very likely to require regulatory specializations. Evidence for specialization in the regulation of these cleavage cycles has come from the study of maternaleffect mutations in Drosophila. For example, nuclei in eggs from gnu mutant females aberran@ enter S-phase prior to fertilization [43]. Eggs derived from pan gu and plutonium mutant females exhibit similar phenotypes to those of gnu [44-], thus all three genes may regulate the same process. Also, hypomorphic mutations in the pan gu gene suggest a role for this gene in the regulation of entry into S-phase during the later cleavage divisions. The Drosophila j~(1)Ya mutation defines another gene required for the cleavage mitoses [45"]. Thefi(1)Ya gene encodes a cell-cycle-dependent.nuclear envelope component that is required for mitosis during the cleavage divisions, including the first mitosis following fertilization. Mutations in fs(1)Ya, gnu, pan gu and plutonium appear to be strictly maternal-effect; homozygous mutant individuals develop normally from heterozygous mothers, showing that the gene products are required only for the early mitoses.
Syncytial versus cellular divisions Syncytial divisions, which are nuclear divisions without cytokinesis, are a common variation on the normal cell cycle. Little is known about the developmental regulation of cytokinesis, but the analysis of the Drosophila pebble gene may provide some insights into this problem. Mutant pebble embryos fail to undergo cytokinesis, at the fourteenth and subsequent cycles of embryonic devel-
opment [46"]. These cycles are the first cellular cycles during embryogenesis, following the thirteen syncytial cleavage mitoses. The requirement for zygotic pebble expression is likely to be a consequence of this change in the nature of the cell cycles following cellularization. Indeed, pebble expression is likely to be an example of a type of developmental cell-cycle control that must act in tissues that convert from syncytial to cellular divisions or
vice versa.
Polytenization and the endo-cell-cycles Another example of developmental modification of the cell cycle is the generation of polyteny. In Drosophila, polytene chromosomes are generated by 'endo-cell-cydes': S-phases, separated by a gap phase, that proceed without cell division [47".]. Several features of the endocell-cycles are intriguing. Firstly, the S-phases occur in a developmentally regulated fashion, in domains of cells that correspond to particular organs or cell types. Secondly, entry into the first S-phase appears to proceed either from G1 or G2 depending on the tissue. Thirdly, regulation of the endo-cell-cycles appears to be dramatically different to the normal cycles. None of the cell-cycle regulatory genes, the cyclins A, B, C and E, and the two p34 cac2 homologues, appear to be expressed in polytenizing tissues (HE Richardson, LV O'Keefe, SI Reed, R Saint, unpublished data)[48-51] and mutant analysis has shown that string is not required [47-.].
Germline-specific cell-cycle genes Germline tissues use specialized cell-cycle genes. The twine gene of Drosophila, a homologue of string, is expressed in premeiotic spermatocytes, and in nurse ceils that provide maternal mRNA to the oocyte, but not in other tissues [52]. Mutations in the gene cause both male and female sterility, confirming a requirement for twine in sperm formation and in oogenesis. Several other genes have been identified that appear to be required specifically for aspects of cell-cycle progression during Drosophila spermatogenesis (M Fuller, personal communication).
The molecular genetic basis of growth regulation In Drosophila, mutations in a number of genes result in overgrowth of imaginal tissues (reviewed in [53] ). During the past year, analysis of two such genes have been reported. Mutations in disc~large result in neoplasia, while mutations in fat result in hyperplasia. The disc~large gene encodes a protein that is located at septate junctions in epithelial tissues and contains a domain highly homologous to a yeast guanylate kinase [54"], while the fatgene is a cadherin homologue [55"]. The genes are therefore implicated in processes of cell adhesion and/or intercellular signalling. The relationship between such
617
618
Patternformation and developmental mechanisms genes and the activity of cell-cycle control genes has yet to be investigated.
8. •
It is tempting to speculate that all genes defined by tissue overgrowth mutant phenotypes, such as recessive oncogenes, are involved in growth regulation during development. A simple relationship between oncogenesis and developmental growth control is, however, challenged by the fact that mice homozygous for a null mutation in the p53 recessive oncogene do not have specific developmental growth defects [56•].
9.
EUON E, GmSAFI PL FINK GR: FUS3 Encodes a cdc2+/CDC28-related Kinase Required for the Transition from Mitosis into Conjugation. Cell 1990, 60:649-664.
10.
CROSSFR, TINKELENBERGAH: A Potential Positive Feedback Loop Controlling CLNI and CLN2 Gene Expression at the Start of the Yeast Cell Cycle. Cell 1991, 65:875-883.
11.
DIRICKL NASM~q'HK: Positive Feedback in the Activation of G1 Cyclins in Yeast. Nature 1991, 351:754-757.
Conclusion We are only nov,, beginning to appreciate the complexi W of cell-cycle regulation during development. Regulation is mediated at a number of points in the cell cycle, and regulatory genes specialized for particular developmental situations have been found. The identification of specific instances of cell-cycle regulation during development, and a growing understanding of the molecular basis of pattern formation and of cell-cycle control, provide a solid foundation for understanding the control of cell proliferation during development.
Acknowledgements We wish to thank the other members of our laborato~, for many helpful discussions. Work of the authors is supported by grants from the National Heahh and Medical Res~lrch Council of Australia, and the Australian Research Council. PL Wigley is supported by an Australian Research Council Postdoctoral Fellowship.
ELION EA, BRILLJA, FINK GR: FUS3 Represses CLNI and CLN2 and in Concert with KSS1 Promotes Signal Transduction. Proc Natl Acad Sci USA 1991, 88:9392-9396. Mutant analysis of the FUS3 gene of S. cerevisiae. Demonstrates that FUS3 mediates cell-cycle arrest in response to mating pheromone through transcriptional repression of two G1 cyclins, CLN1 and CLN2, and through post-transcriptional inhibition of a third G1 cyclin, CLN3.
12. ..
MATSUSHIblEH, ROUSSEI. MF, ASHMUN RA, SHERR CJ: Colonystimulating Factor 1 Regulates Novel Cyclins during the G1 Phase of the Cell Cycle. Cell 1991, 65:701-713. Reports the isolation of three routine cyclin-D genes, CYL1, C-7}72and C3"L3. Using a mouse macrophage cell line, dependent on CSF-1, the authors show that C3Z-1 expression is induced or repressed in response to CSF-1 addition or withdrawal, respectively. This suggests that the CYL genes may function during S-phase commitment. It is "also shown that two of the three genes are expressed in a cell-type-specific manner. 13.
WITHERS DA, HARVEY RC, FAUST JB, MEI2~'K O, CAREY K, MEEKERTC: Characterization of a Candidate bcl. 1 Gene. Mol Cell Biol 1991, ! 1:4846-4853. A human cydin-D homologue is shown to be expressed in a cell-typespecific manner in a range of human cultured cell lines. •
14.
L',HUEEE, SMITH AV, ORR-WEAVERTL: A Novel Cyclin Gene from Drosophila C o m p l e m e n t s CLN Function in Yeast. Genes Dev 1991, 5:2166-2175.
15.
LEOPOLDP, O'F,~RELL PH: An Evolutionarlly Conserved Cyclin Homolog from Drosophila Rescues Yeast Deficient in G1 Cyclins. Cell 1991, 66:1207-1216.
16.
TOMLINSON A: Cellular Interactions in the Developing Drosophila Eye. Developmen! 1988, 104:183-189.
17.
READYD: A Multifaceted Approach to Neural Development. Trends Nettrosci 1989, 12:102-110.
18. •
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of OULStanding interest I.
PARDEEAB: GI Events and Regulation of Cell Proliferation. Science 1989, 246:603-608.
2.
NURSE P: Universal Control Mechanism Regulating Onset of M-phase. Nature 1990, 344:503-508.
3.
REED SI: A Proliferation of Cyclins. Trends Cell Biol 1992, 2:77-81.
4.
MILLM~JBA, RUSSELL P: The cde25 M-phase Inducer - - an Unconventional Protein Phosphatase. Cell 1992, 68:407-410.
5.
PINES J, HUiX~FERT: Cyclin-dependent Kinases: a New Cell Cycle Motif?. Trends Cell Biol 1991, 1:117-121.
6.
SPRAGUEGF: Signal Transduction in Yeast Mating: Receptors, Transcription Factors, and the Kinase Connection. Trends Genet 1991, 7:393-398.
7.
CHANG F, HERSKOWI17. l: Identification of a Gene Necessary for Cell Cycle Arrest by a Negative Growth Factor of Yeast: FAR1 is an Inhibitor of a G1 Cyclin, CLN2. Cell 1990, 63:999-1011.
WOLFFT, RE*'.D¥ DF: The Beginning of Pattern Formation in the Drosophila C o m p o u n d eye: The Morphogenetic Furrow and the Second Mitotic Wave. Development 1991, 113:841-850. Reports a det:tiled examination of the distribution of S-phase cells in the developing compound eye of D. melanogaster It concludes that all cells, other than the differentiating precluster cells, enter S-phase following the initiation of cellular differentiation. 19.
BAKERNE, RUBIN GM: Effect on Eye Development of Dominant Mutations in Drosophila Homologue of EGF-receptor. Nature 1989, 340:150-153.
ZAK NB, SHILO B-Z: Localization of DER and the Pattern of Cell Divisions in Wild-type and Ellipse Eye Imaginal Discs. Dev Biol 1992, 149:448-456. This, together with [21 •], reports a study of developing compound eyes of dominant Ellipse mutants in O. melanogastet: Ellipse mutant cells are shown to aberrantly enter S-phase instead of differentiating into photoreceptor cells. 20. •
BAKERNE, ROBIN GM: Ellipse Mutations in the Drosophila Homologue of the EGF Receptor Affect Pattern Formation, Cell Division and Cell Death in Eye Imaginal Discs. Dev Biol 1992, 150:381-396. This, together with [20•], reports a study of S-phases in developing compound eyes of dominant Ellipse mutants in D. melanogastet: Cells that would nomlally differentiate to fore1 the precluster are rather shown to aberrantly enter S-phase. In this paper, mitoses are shown to cluster near the few ommatidia that form in Ellipse mutants, suggesting that a signal from the differentiating ommatidia induces G2-phase cells to enter mitosis. 21. •
Developmental regulation of the cell cycle Saint, Wigley SELLECKSB, GONZALEZC, GLOVERDM, WHITE K: Regulation of the G1-S Transition in Postembryonic Neuronal Precursors by Axon Ingrowth. Nature 1992, 355:253-255. Cell-cycle phases of lamina precursor cells are shown to occur in specific locations within the developing Drosopbila brain. Lamina precursor cells in sine occulis mutant brains not innewated by photoreceptor cell axons, remain arrested in Gl-phase, instead of undergoing a further cycle. Innervation appears to generate a signal that induces the final cell cycle in the lamina precursor cells. 22. •,
23.
MURRAYAW, KIRSCHNER MW: Dominoes and Clocks: the Union of Two Views of the Cell Cycle. Science 1989, 246:614--621.
c-mos.
HUNT T: Cell Cycle Arrest and 355:587-588.
25.
SAGATAN, OSKAP~ChSONM, COPELAND T, BRUMBAUGHJ, VANDE WOUDE GF: Function of c-mos Proto.oncogene Product in Meiotic Maturation in Xenopus Oocytes. Nature 1988, 335:519-525.
26.
SAGATAN, DAARI, OSKARSSONM, SHOWAI.TERSD, VtM'qDEWOUDE GF: The Product of the mos Proto-oncogene as Candidate 'Initiator' for Oocyte Maturation. Science 1989, 245:643-646.
27. YEW N, MELUNI ML, VANDE WOLIDE GF: Meiotic Initiation by •• the mos Protein in Xenopus. Nature 1992, 355:649-652. Addresses the role of mos-encoded protein in the maturation of Xeno pus oocytes. Shows that a bacterially expressed mos fusion protein can induce maturation initiation, in the absence of protein s3,'nthesis, indicating that pp39 m°s is the only protein required to be made for the induction of maturation. Protein sTnthesis is, however, reqt, ired for progression to the end-point of maturation: metaphase arrest in meiosis I1. Also demonstrates that post-translational changes, induced by progesterone, are required in addition to mosaction, for fully efiqcient initiation of maturation. 28.
KOBAYASHII-I, MINSHULLJ, FORD C, GOLSTEYNR, POON R, HUNT T: On the Synthesis and Destruction of A- and B-type Cyclins During Oogenesis and Meiotic Maturation in Xenopus laevis. J Cell Biol 1991, 114:755-765.
29.
MINSHULLJ, MURRAYA, COla~,b\NA, HUNT T: Xenopus Oocyte Maturation does not Require New Cyclin Synthesis..I Cell Biol 1991, 114:767-772.
30.
GAUTIERJ, MALt.ERJL: Cyclin B in Xenopus Oocytes: Implications for the Mechanism of pre-MPF Activation. ~I,IBO J 1991, 10:177-182.
31.
SAGATAN, WKFANABEN, VANDE \'{tOUDE GF, IKAWAY: T h e cmos Proto-oncogene Product is a Cytostatic Factor Responsible for Meiotic Arrest in Vertebrate Eggs. Nature 1989, 342:512-518. WATANABEN, VANDE WOUDE GF, IKAWAY, SAGATA N: Specific Proteolysis of the c-mos Proto-oncogene Product by Calpain on Fertilization of Xenopus Eggs. Nature 1989, 342:505-511.
33.
LORCAT, GALASS, FESQUET D, DEVAULTA, CAVADOREJ, DOREE Degradation of the Proto-oncogene Product p 3 9 m o s is not Necessary for Cyclin Proteolysis and Exit from Meiotic Metaphase: Requirement for a Ca 2+-calmodulin D e p e n d e n t Event. FMBOJ 1991, 10:2087-2093. Extracts prepared from metaphase-arrested Xenopus eggs are used to analyze cyclin degradation in response to increased calcium levels. At physiological calcium concentrations, proteolysis of cyclin but not of mos:encoded protein occurs, suggesting that pp39ma*destmction is not required for MPF inactivation following fertilization. •
34. ,
FOE VE: Mitotic Domains Reveal Early C o m m i t m e n t of Cells in Drosophila Embryos. Development 1989, 107:1-22.
36.
EDGARBA, O'FARRELL PH: Genetic Control of Cell Division Patterns in the Drosophila Embryo. Cell 1989, 57:177-187.
37.
EDGARBA, O'FARREI± PH: T h e Three Postblastoderm Cell Cycles of Drosophila Embryogenesis are Regulated in G2 by string. Cell 1990, 62:469-480.
38.
O'FARRELLPH, EDGARBA, LAKICHD, LEHNERCF: Directing Cell Division during Development. Science 1989, 246:635--640.
39.
FOE VE, ODELL GM: Mitotic Domains Partition Fly Embryos, Reflecting Early Cell Biological C o n s e q u e n c e s of Determination in Progress. Am Zool 1989, 29:617-652.
Nature 1992,
24.
32.
35.
M:
WATANABEN, HUNT T, IKAW^ Y, SAGATAN: I n d e p e n d e n t Inactivation of MPF and Cytostatic Factor (Mos) u p o n Fertilization of Xenopus Eggs. Nature 1991, 352:247-248. Xenopus eggs and egg extracts were used to show that during meiotic release, cyclin subunits of MPF are degraded before mos proteolysis, and accordingly, that functional MPF is inactivated prior to inactivation of cytostatic factor (CSF). Supports the idea that CSF inactivation allows cell-cycle progression following fertilization, but that there is a CSF-independent pathway for MPF inactivation.
40. ,
ARORAK, NUSSLEIN-VOIMARDC: Altered Mitotic Domains Reveal Fate Map Changes in Drosophila Embryos Mutant for Zygotic Dorsoventral Patterning Genes. Development 1992, 114;1003-1024. The patterns of Drosophila cycle-14 mitotic domains, known to be under the direct regulation of string expression, are shown to be altered in a number of dorsoventral patterning mutants. 41.
GRAVESBJ, SCHUBIGERG: Cell Cycle Changes during Growth and Differentiation of Imaginal Leg Discs in Drosophila melanogaste~ Det, Biol 1982, 93:104-110.
42.
FAIN MJ, STEVENS B: Alterations in the Cell Cycle of Drosophila Imaginal Disc Cells Precede Metamorphosis. Det, Biol 1982, 92:247-258.
43.
FREEMANM, GLOVEI DM: The gnu Mutation of Drosophila Causes Inappropriate DNA Synthesis in Unfertilized and Fertilized Eggs. Genes Dev 1987, 1:924-930.
SHAMANSKIFL, ORR-WEAVERTI2 The Drosophila plutonium and pan gu Genes Regulate Entry into S Phase at Fertilization. Cell 1991, 66:1289-1300. Two genes, in addition to a third previously described, are shown to be required for file inhibition of DNA synthesis prior to fertilization in mature eggs. The mutations are shown to be stricdy maternal-effect mutations, suggesting that the genes are not required for cell-cycle regulation later in development. 44. •
[.IN H, WOLFNER ME: The Drosophila Maternal-effect Gene f s ( 1 ) Y a Encodes a Cell Cycle-dependent Nuclear Envelope C o m p o n e n t Required for Embryonic Mitoses. Cell 1991, 64:49-62. Describes the isolation and characterization of a Drosophila gene, mutations in which are strict maternal-effect mutations that show a requirement for the gene in the first and subsequent mitoses following fertilization.
45. •
HIME G, SAINT R: Zygotic Expression of the pebble Locus is Required for Cytokinesis during the Post-blastoderm Mitoses of Drosophila. Development 1992, 114:165-171. The D. melanogaster pebble gene is shown to be required for cytokinesis in the cellular post-blastodeml mitoses that follow the s3'ncytial cleavage mitoses. 46. •
47. **
SMITHAV, ORR-WEAVERTL: The Regulation of the Cell Cycle during Drosophila Embryogenesis: the Transition to Polyteny. Development 1991, 112:997-1008. Reports a study of the cycles of endo-replication, termed endo-cell-cy. cles, that lead to polyteny in Drosophila tissues. Several interesting features are noted: the cycles are comprised of S-phases separated by gap phases without cell division; the S.phases occur in developmentally regulated domains that correspond to organ or tissue types; each S-phase replicates most, but not all, of the DNA once; S-phases appear to be entered from G1- or G2-phase; none of the currently characterized cell-cycle control genes appear to be active in regulating these S-phases. 48.
WHrITIELDW, GONZALEZC, MALDONADO-CODINAG, GLOVER D: The A- and B-type Cyclins of Drosophila are Accumulated and Destroyed in Temporally Distinct Events that Define Separable Phases of the G2-M Transition. EMBO J 1990, 9:2563-2572.
49.
LEHNERCF, O'FARRELL PH: Drosophila cdc2 Homologs: a Functional Homolog is Coexpressed with a Cognate Variant. EMBO J 1990, 9:3573-3581.
619
620
Pattern formation and developmental mechanisms 50.
LEHNER CF, O'FARRELL PH: Expression and Function of Drosophila Cyclin A during Embryonic Cell Cycle Progression. Cell 1989, 56:957-968.
51.
JIMENEZ J, AI.PHE'," I~ NURSE P, GLOVER DM: Complementation of Fission Yeast cdc2ts and cdc25ts Mutants Identifies Two Cell Cycle Genes from Drosophila:. a cdc2 Homologue and string. EMBO J 1990, 9:3565-3571.
52.
ALPHEY L, JIMENEZ J, WHITE-COOPER H, DA\X~ON I, NURSE P, GI.OVER DM: twine, a cdc25 Homologue that Functions in the Male and Female Germ-line of Drosophila. Cell 1992, 69:977-988.
53.
GATEFF EA, MECHLER BM: Tumor-suppressor Genes of Drosophila melanogaster CRC Crit Rev Oncogen 1989, 1:221-245.
WOODSDF, BR'~'ANTPJ: The discs-large T u m o r Suppressor Gene of Drosophila Encodes a Guanylate Kinase Homolog Localized at Septate Junctions. Cell 1991, 66:451-464. The sequence and sub-cellular location of the discs.large gene of Drosophila are described. Mutations in the gene cause disc overgrowdl.
Tile sequence of the gene and the sub-cellular location of the gene product suggest a role in signal transduction. 55. ..
MAHONEYPA, WEBER U, ONOFRECHUKP, BIESS~bkNN H, BR','ANT PJ, GOODMAN CS: The f a t T u m o r Suppressor Gene i n Drosophila Encodes a Novel Member of the Cadherin Gene Superfamily. Cell 1991, 67:853-868. Mutations in the fat gene result in hyperplasia of the imaginal discs. This paper reports that fat is a member of the cadherin family of cell adhesion molecules.
56.
DONEHOWER LA, HARVEY M, SLAGLE BL, MCARTHU8 MJ, MONTGOMERYCA, BUTEL JS, BRADI.E't' A: Mice Deficient for p53 are Developmentally Normal but Susceptible to Spontaneous Tumours. Nature 1992, 356:215-221. Mice homozygous for a mutation in tile p53 gene are shown to develop nomlally, but are more susceptible to neoplastic diseases. This result challenges the idea that recessive oncogenes are genes normally im,oh,ed in the developmental regulation of growth. •
54. •.
R Saint, PL Wigley, Department of Biochemistry, University of Adelaide, GPO Box 498, Adelaide, Sot, th Australia 5001, Australia.
Current Opinion in Genetics and Development 1992, 2:614-620
Introduction This review examines the emerging relationship between the molecular regulation of development and the control of the eukaryotic cell cycle. This relationship bears directly on the problem of developmental growth regulation, and indirectly on the problem of uncontrolled proliferation in neoplastic tissues. Interesting examples of cell-cycle control during development continue to be identified, but the underlying regulatory processes are, as yet, poorly understood. In this review, we focus particularly on the following questions: at what cell-cycle stages, and by virtue of the activity of which cell-cycle control genes, is developmental regulation of cell proliferation mediated? We will not discuss, for the most part, the vast body of data that has come from studies of cultured cells (reviewed in [1]). The molecular control of cell-cycle progression has been a fertile field of research for some time (reviewed in [2,3]). Here, we describe only those features relevant to our discussion. The cell cycle can be divided into four sequential phases: mitosis, or M-phase; a gap or Gl-phase that follows mitosis; S-phase during which DNA replication occurs; and a second gap or G2-phase prior to entry into mitosis. Central to the control of cell-cycle progression is a serine/threonine kinase known as p34 cdc2. For activity, this protein requires association with one of a family of proteins termed cyclins. The activi W of the cyclins varies during the cell cycle, with different cyclins being active in different cell-cycle phases. The phosphorylation state of p34 cdc2 also regulates its activity. Dephosphorylation of p34 cdc2 by a phosphatase, encoded in Schizosaccharomyces pombe by cdc25, is required for its activation at the G2-M phase boundary (reviewed in [4]). Proteolytic breakdown of cyclin is required for inactivation of the p34 cdc2 kinase during metaphase and, therefore, for exit from mitosis.
On the basis of sequence homologies and complementation of yeast cell-cycle mutants with metazoan homologues, these eukaryotic cell-cycle controls appear to be universal. The control mechanisms are much more complex than suggested in this outline. For exanlple, metazoans have additional cyclin-dependent kinases that are also implicated in cell-cycle control (reviewed in [5] ). The role of these homologues in the developmental regulation of the cell cycle is yet to be studied.
Developmental regulation during the Gl-phase Cyclins and G1 arrest during yeast mating Tissue culture studies have long pointed to the Gl-phase as the stage during which key cell-cycle control mechanisms operate. It is no surprise, therefore, that we find examples in which cell proliferation during development is controlled during this phase. One of the best characterized examples of developmental regulation during the Gl-phase is found in the budding yeast, Saccharomyces ceret,isiae. In response to mating pheromone, haploid S. cerevisiae cells fuse to form diploid cells. Mating pheromone induces arrest in the Gl-stage prior to mating. Normal progression from G1- into S-phase in this organism requires the activity of a functionally redundant group of G1 cyclins, CLN1, CLN2 and CLN3 (reviewed in [3]). The Gl-phase arrest induced by mating pheromone, is mediated by a signal transduction cascade that ultimately results in functional inactivation of all three G1 cyclins (reviewed in [6]). Two genes required for this inactivation have been identified by mutant analysis. Firstly, the FARI gene was identified because far1 mutant cells fail to arrest in response to mating pheromone [7]. Using far1 cln double mutants, FAR1 was shown to be specifically required for the in-
Abbreviations CSF--cytostatic factor; CSF-l--colony-stimulating factor-I; DER--Drosophila EGF-receptorhomologue; MPF---maturation (or M-phase) promoting factor.
614
(~ Current Biology Ltd ISSN 0959-437X
Developmental regulation of the cell cycle Saint, Wigley hibition of CLN2, since only far1 cln2 double mutants arrest normally in the presence of pheromone. Unfortunately, the sequence of FAR1 provides no hints to its molecular action. Secondly, the product of the FUS3gene was also shown to be involved in the inhibition of the G1 cycllns [8"]. FUS3 encodes a protein kinase required for at least two responses to mating pheromone, one being G1 arrest [9]. Mutant analysis demonstrated that FUS3 mediates inhibition of CLNI and CLN2 by decreasing their mRNA levels. FUS3 is also required for CLN3 inhibition, since fus3 cln3 double mutants arrest normally, but this inhibition must be post-transcriptional, as the level of CLN3 mRNA is unaffected in a fus3 mutant. The CLN3 repression may, in part, explain the effect on the other CLN genes, as CLN3 stimulates transcription of CLIV1 and CLIV2 [10,11 ]. Even in this, the best characterized example of developmental cell-cycle regulation, we are only beginning to understand the pathways that transduce the pheromone signal into CLN inactivation, and consequently, cell-cycle arrest.
One such example of tissue-specific cyclin expression has come from our studies of a Drosophila cyclin-E homologue (HE Richardson, LV O'Keefe, SI Reed, R Saint, unpublished data). Drosophila cyclin-E transcripts are present initially as uniformly distributed maternal mRNA that persists during the first 13 synchronous syncytial embryonic cycles. During cycle 14, immediately prior to the onset of the asynchronous mitoses that characterize organogenesis, the level of transcripts decreases dramatically. Expression is then observed specifically in proliferating neuroblasts of the central and peripheral nervous systems, but not in proliferating epidermal cells. Developmental regulation of cyclin gene expression may, therefore, turn out to be a widespread mechanism of tissue-specific cell-cycle control. Not all G1 cyclins are transcriptionally regulated. The mRNA products of the cyclin-C homologue of Drosophila appear to be uniformly distributed in developing embryos [14,15]. This cyclin may, however, play a role in developmental regulation of proliferation as posttranscriptional regulation of cyclins is very common. A more detailed understanding of the role of these G1 cyclins in metazoan development awaits the analysis of the proteins themselves and of cyclin mutant phenotypes.
Cyclins and G1 regulation in metazoans G1 cyclins are also likely candidates for the control of the G1-S-phase transition in metazoans. Recently, several candidate metazoan G1 cyclins have been identified by their ability to rescue yeast strains deficient in the three yeast G1 cycllns (reviewed in [3]). These cyclins have been assigned the names cyclin-C, cyclin-D (which has several related members) and cyclin-E. Two preliminary lines of evidence suggest a role for these cyclins in the developmental regulation of the cell cycle. The first comes from the study of mouse macrophage cultured cells in which expression of a mouse cycllnD homologue, termed CYL1, was shown to respond to a growth factor that stimulates proliferation [12-]. In these cells, colony-stimulating factor-1 (CSF-1) is required throughout G1 for both cell survival and commitment to S-phase. Withdrawal of CSF-1 results in arrest in the Gl-phase, while the subsequent addition of CSF-1 stimulates re-entry into the cell cycle. CYL1 mRNA and protein levels decline abruptly following CSF-1 with. drawal, while re-stimulation with the growth factor very quickly induces C"YL1expression [12--]. Interestingly, the level of CYLI mRNA in the re-stimulated cells is maintained throughout the cycle and into the next Gl-phase, so that CYL1 mRNA levels appear to respond to growthfactor activity and not to the phase of the cell cycle. In the same study, two related murine cyclin-D homologues, termed CYL2 and CYL3, were identified [12-.]. Northern-blot analysis demonstrated that two of the three cyclin-D genes were expressed in a tissue-specific manner. Furthermore, a candidate bcl-1 oncogene, which is a member of the human cyclin-D family, also exhibits lineage-specific expression in a variety of human cell lines [13o]. This raises the provocative possibility of developmental regulation of cell proliferation mediated by stage- and tissue-specific cyclin expression.
Developmental regulation in the Gl-phase during Drosophila development Development of the compound eye of Drosophila melanogaster is well characterized (reviewed in [16,17]). The eye derives from the eye imaginal disc, a single cell layer epithelium that is generated by proliferation of a group of founder cells during the first two larval instars. Differentiation occurs as a wave that sweeps across the epithelium in eye imaginal discs of third instar larvae and early pupae. The first event in this progressive process of differentiation is the arrest of cells in the G1phase. While arrested, groups of five cells form a 'precluster' of differentiating photoreceptor cells, each precluster corresponding to the primordium of a single ommatidium. At a later time, all cells other than the differentiating precluster cells enter S-phase /18-], and many of them subsequently enter mitosis. These cells either differentiate to form the remaining photoreceptor cells, cone cells, pigment cells and bristle cells of the mature ommatidium, or undergo a programmed cell death. In dominant Ellipse mutants of the Drosophila EGFreceptor homologue (DER), only a small number of ommatidia form [19]. Most of the cells that would normally differentiate to form the precluster cells rather enter S-phase along with the other undifferentiated cells [20.,21.]. As such, the over-activity of the DER gene either drives these cells into S-phase or causes the cells to enter an alternative developmental pathway that involves a programmed entry into S-phase. Developmental regulation of the G2-M-phase transition is -also implicated in the developing eye. Mitoses in the region of differentiation in Ellipse eye discs were found clustered near the few ommatidia that formed. The remainder of the cells appeared to undergo a programmed cell death. Based on these observations it has been pos-
615
616
Patternformation and developmental mechanisms tulated that an inductive signal from the five precluster cells may drive adjacent G2-phase cells into mitosis, and that in the absence of this signal, the remaining G2-phase cells undergo a programmed cell death [21o]. Another interesting exanlple of developmental regulation during the Gl-phase is found in the developing brain of Drosophila larvae. The lamina neurons that receive direct synaptic input from photoreceptor axons derive from lamina precursor cells which undergo a regulated set of cell movements and cell cycles prior to innervation. In sine occulis mutants, which lack eyes and therefore the incoming photoreceptor cell axons, progression of the lamina precursor cells from G1- into S-phase of the final cell cycle does not occur [22-]. Presumably the incoming photoreceptor axons stimulate entry of the lanaina cells into their final cell cycle.
Developmental regulation during the G2-phase The mos gene and oocyte maturation in Xenopus It is a c o m m o n
belief that cell-cycle regulation occurs
during the Gl-phase. It is therefore particularly significant that developmental regulation of the cell cycle has also been found to occur during the G2-phase. One example where G2-phase regulation is implied is in the developing Drosophila eye, as discussed above. A much better characterized exan~ple concerns maturation of Xenopus oocytes (reviewed in [23]). As for most vertebrates, Xenopus oocytes are arrested in the G2-phase of the first meiotic division. Progesterone, released by follicle cells, induces maturation of the oocytes. Maturation involves progression through the cell cycle until arrest again occurs at metaphase of the second meiotic division. This arrest is released following fertilization. The crucial event in oocyte maturation is the activation of maturation (or M-phase) promoting factor (MPF), which drives entry into mitosis. MPF corresponds to the regulatory p34Ca'C2-cyclin complex. It is now clear that induction of MPF activity, and thus of maturation, requires the production of the pp39 'n°s protein kinase, encoded by the c-mos proto-oncogene (reviewed in [24]). In the normal situation, hormonal stimulation induces translation of maternal c-mos mRNA and, thereby, maturation. This effect is mimicked by injection of mos RNA into oocytes [25,26]. Injection of an engineered mos fusion protein caused the onset of maturation in the absence of protein synthesis [27.°], showing that the mos-encoded product is the only protein required to be synthesized for maturation induction. Direct evidence confirms that new cyclin translation is not necessary for this induction; sufficient stores already exist in the arrested oocyte [28-30]. Induction of maturation is greatly enhanced when, in addition to mos-encoded protein, progesterone is added to oocytes [27°°]. This suggests either that the hormone potentiates mos action, or that it removes an inhibitor. Furthermore, oocytes induced to mature do not progress to the normal end-point of maturation, namely, metaphase
arrest in meiosis II. New protein synthesis is required for completion of maturation, and the evidence suggests that cyclin synthesis is not sufficient to account for this requirement - - another, as yet unknown, protein must be synthesized. The metaphase arrest of Xenopus eggs (and eggs of other vertebrates) is mediated by an activity long known as cytostatic factor (CSF) (not to be confused with CSF-1). Microinjection and immunodepletion experiments have now shown that the c-mos-encoded protein kinase is a component of CSF [31]. Furthermore, proteolysis of the pp39 mos protein following fertilization is necessary to allow cell-cycle progression [32]. Recent evidence shows, however, that cyclin degradation, and therefore MPF inactivation, is mediated by a CSF: mos-independent pathway, probably in response to the calcium-transient induced by fertilization [33",34"]•
• The~s~ing gene and mitotic domains during Drosophila organogenesis Another exm~ple of developmental regulation at the G2stage is found in embryos of D. melanogaster. Following fertilization, the Drosophila embryo undergoes 13 rapid, synchronous and syncytial mitoses before arresting in the G2-phase of cycle 14. At this time, membranes extend from the periphery of the embryo to enclose the nuclei. The subsequent mitoses are dramatically different to the earlier cleavage mitoses, not only because the embryo is now cellular, but also because the mitoses are asynchronous. The cycle-14 mitoses occur synchronously in groups of cells termed mitotic domains [35], but each domain undergoes mitosis at a different, developmentally regulated time. A striking observation is that some, and perhaps all, of the mitotic domains correspond to organ or tissue-type primordia, and therefore represent one of the earliest signs of organogenesis. Several lines of evidence indicate that expression of string regulates the appearance of the mitotic domains [36,37]: string mutants arrest in the G2-phase of cycle 14; accumulation of string mRNA precisely anticipates the pattern of mitotic domains; and ectopic string expression induces synchronous mitoses throughout the embryo, indicating that string is the only limiting factor for cycle-14 divisions. Finally, string is a homologue of the S. pombe mitotic activator gene, cdc25. Regulation of string expression occurs at the transcriptional level (PL Wigley, B Patterson, LV O'Keefe, R Saint, unpublished data)[38]. Moreover, it is predicted that the genes required for pattern formation during embryogenesis, many of which encode transcriptional regulators, directly activate string expression to effect the mitotic domain pattern [36,38]. This prediction is supported by observations that the patterns of both cycle-14 mitoses and string expression are altered in dorsal-ventral patterning gene mutants (cited in [38,39,40"]). Exactly how the patchwork of regulatory gene expression generates the complex string transcription pattern during cycle 14 constitutes a fascinating problem that we are currently addressing.
Developmental regulation of the cell cycle Saint, Wigley One surprising observation from our studies is that the control of string expression also appears to be complex at later stages of embryonic development (during neurogenesis), and in proliferating imaginal tissues (PL Wigley, P Kylsten, B Patterson, R Saint, unpublished data). For example, string transcriptional enhancers have been identified that are specific for particular imaginal tissues, and even for subsets of cells within a single tissue. This indicates either that evolution has opportunistically utilized many different regulators to achieve the end result of string expression in all dividing cells, or that the G2-M-phase transition is developmentally regulated in many more circumstances than previously suspected (other examples of G2 regulation in Drosophila are known, for instance in leg and wing imaginal discs [41,42]).
Developmentally specialized cell cycles
Cleavage cycles The metazoan cell cycle can be substantially modified in particular developmental circumstances. It can, for example, be dramatically accelerated, as occurs in cleavage divisions following fertilization. The rapid cleavage divisions of Xenopus and Drosophila are simple biphasic cycles lacking observable G1- and G2-phases. This is a remarkable modification of the more common complex cell cycle and is very likely to require regulatory specializations. Evidence for specialization in the regulation of these cleavage cycles has come from the study of maternaleffect mutations in Drosophila. For example, nuclei in eggs from gnu mutant females aberran@ enter S-phase prior to fertilization [43]. Eggs derived from pan gu and plutonium mutant females exhibit similar phenotypes to those of gnu [44-], thus all three genes may regulate the same process. Also, hypomorphic mutations in the pan gu gene suggest a role for this gene in the regulation of entry into S-phase during the later cleavage divisions. The Drosophila j~(1)Ya mutation defines another gene required for the cleavage mitoses [45"]. Thefi(1)Ya gene encodes a cell-cycle-dependent.nuclear envelope component that is required for mitosis during the cleavage divisions, including the first mitosis following fertilization. Mutations in fs(1)Ya, gnu, pan gu and plutonium appear to be strictly maternal-effect; homozygous mutant individuals develop normally from heterozygous mothers, showing that the gene products are required only for the early mitoses.
Syncytial versus cellular divisions Syncytial divisions, which are nuclear divisions without cytokinesis, are a common variation on the normal cell cycle. Little is known about the developmental regulation of cytokinesis, but the analysis of the Drosophila pebble gene may provide some insights into this problem. Mutant pebble embryos fail to undergo cytokinesis, at the fourteenth and subsequent cycles of embryonic devel-
opment [46"]. These cycles are the first cellular cycles during embryogenesis, following the thirteen syncytial cleavage mitoses. The requirement for zygotic pebble expression is likely to be a consequence of this change in the nature of the cell cycles following cellularization. Indeed, pebble expression is likely to be an example of a type of developmental cell-cycle control that must act in tissues that convert from syncytial to cellular divisions or
vice versa.
Polytenization and the endo-cell-cycles Another example of developmental modification of the cell cycle is the generation of polyteny. In Drosophila, polytene chromosomes are generated by 'endo-cell-cydes': S-phases, separated by a gap phase, that proceed without cell division [47".]. Several features of the endocell-cycles are intriguing. Firstly, the S-phases occur in a developmentally regulated fashion, in domains of cells that correspond to particular organs or cell types. Secondly, entry into the first S-phase appears to proceed either from G1 or G2 depending on the tissue. Thirdly, regulation of the endo-cell-cycles appears to be dramatically different to the normal cycles. None of the cell-cycle regulatory genes, the cyclins A, B, C and E, and the two p34 cac2 homologues, appear to be expressed in polytenizing tissues (HE Richardson, LV O'Keefe, SI Reed, R Saint, unpublished data)[48-51] and mutant analysis has shown that string is not required [47-.].
Germline-specific cell-cycle genes Germline tissues use specialized cell-cycle genes. The twine gene of Drosophila, a homologue of string, is expressed in premeiotic spermatocytes, and in nurse ceils that provide maternal mRNA to the oocyte, but not in other tissues [52]. Mutations in the gene cause both male and female sterility, confirming a requirement for twine in sperm formation and in oogenesis. Several other genes have been identified that appear to be required specifically for aspects of cell-cycle progression during Drosophila spermatogenesis (M Fuller, personal communication).
The molecular genetic basis of growth regulation In Drosophila, mutations in a number of genes result in overgrowth of imaginal tissues (reviewed in [53] ). During the past year, analysis of two such genes have been reported. Mutations in disc~large result in neoplasia, while mutations in fat result in hyperplasia. The disc~large gene encodes a protein that is located at septate junctions in epithelial tissues and contains a domain highly homologous to a yeast guanylate kinase [54"], while the fatgene is a cadherin homologue [55"]. The genes are therefore implicated in processes of cell adhesion and/or intercellular signalling. The relationship between such
617
618
Patternformation and developmental mechanisms genes and the activity of cell-cycle control genes has yet to be investigated.
8. •
It is tempting to speculate that all genes defined by tissue overgrowth mutant phenotypes, such as recessive oncogenes, are involved in growth regulation during development. A simple relationship between oncogenesis and developmental growth control is, however, challenged by the fact that mice homozygous for a null mutation in the p53 recessive oncogene do not have specific developmental growth defects [56•].
9.
EUON E, GmSAFI PL FINK GR: FUS3 Encodes a cdc2+/CDC28-related Kinase Required for the Transition from Mitosis into Conjugation. Cell 1990, 60:649-664.
10.
CROSSFR, TINKELENBERGAH: A Potential Positive Feedback Loop Controlling CLNI and CLN2 Gene Expression at the Start of the Yeast Cell Cycle. Cell 1991, 65:875-883.
11.
DIRICKL NASM~q'HK: Positive Feedback in the Activation of G1 Cyclins in Yeast. Nature 1991, 351:754-757.
Conclusion We are only nov,, beginning to appreciate the complexi W of cell-cycle regulation during development. Regulation is mediated at a number of points in the cell cycle, and regulatory genes specialized for particular developmental situations have been found. The identification of specific instances of cell-cycle regulation during development, and a growing understanding of the molecular basis of pattern formation and of cell-cycle control, provide a solid foundation for understanding the control of cell proliferation during development.
Acknowledgements We wish to thank the other members of our laborato~, for many helpful discussions. Work of the authors is supported by grants from the National Heahh and Medical Res~lrch Council of Australia, and the Australian Research Council. PL Wigley is supported by an Australian Research Council Postdoctoral Fellowship.
ELION EA, BRILLJA, FINK GR: FUS3 Represses CLNI and CLN2 and in Concert with KSS1 Promotes Signal Transduction. Proc Natl Acad Sci USA 1991, 88:9392-9396. Mutant analysis of the FUS3 gene of S. cerevisiae. Demonstrates that FUS3 mediates cell-cycle arrest in response to mating pheromone through transcriptional repression of two G1 cyclins, CLN1 and CLN2, and through post-transcriptional inhibition of a third G1 cyclin, CLN3.
12. ..
MATSUSHIblEH, ROUSSEI. MF, ASHMUN RA, SHERR CJ: Colonystimulating Factor 1 Regulates Novel Cyclins during the G1 Phase of the Cell Cycle. Cell 1991, 65:701-713. Reports the isolation of three routine cyclin-D genes, CYL1, C-7}72and C3"L3. Using a mouse macrophage cell line, dependent on CSF-1, the authors show that C3Z-1 expression is induced or repressed in response to CSF-1 addition or withdrawal, respectively. This suggests that the CYL genes may function during S-phase commitment. It is "also shown that two of the three genes are expressed in a cell-type-specific manner. 13.
WITHERS DA, HARVEY RC, FAUST JB, MEI2~'K O, CAREY K, MEEKERTC: Characterization of a Candidate bcl. 1 Gene. Mol Cell Biol 1991, ! 1:4846-4853. A human cydin-D homologue is shown to be expressed in a cell-typespecific manner in a range of human cultured cell lines. •
14.
L',HUEEE, SMITH AV, ORR-WEAVERTL: A Novel Cyclin Gene from Drosophila C o m p l e m e n t s CLN Function in Yeast. Genes Dev 1991, 5:2166-2175.
15.
LEOPOLDP, O'F,~RELL PH: An Evolutionarlly Conserved Cyclin Homolog from Drosophila Rescues Yeast Deficient in G1 Cyclins. Cell 1991, 66:1207-1216.
16.
TOMLINSON A: Cellular Interactions in the Developing Drosophila Eye. Developmen! 1988, 104:183-189.
17.
READYD: A Multifaceted Approach to Neural Development. Trends Nettrosci 1989, 12:102-110.
18. •
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of OULStanding interest I.
PARDEEAB: GI Events and Regulation of Cell Proliferation. Science 1989, 246:603-608.
2.
NURSE P: Universal Control Mechanism Regulating Onset of M-phase. Nature 1990, 344:503-508.
3.
REED SI: A Proliferation of Cyclins. Trends Cell Biol 1992, 2:77-81.
4.
MILLM~JBA, RUSSELL P: The cde25 M-phase Inducer - - an Unconventional Protein Phosphatase. Cell 1992, 68:407-410.
5.
PINES J, HUiX~FERT: Cyclin-dependent Kinases: a New Cell Cycle Motif?. Trends Cell Biol 1991, 1:117-121.
6.
SPRAGUEGF: Signal Transduction in Yeast Mating: Receptors, Transcription Factors, and the Kinase Connection. Trends Genet 1991, 7:393-398.
7.
CHANG F, HERSKOWI17. l: Identification of a Gene Necessary for Cell Cycle Arrest by a Negative Growth Factor of Yeast: FAR1 is an Inhibitor of a G1 Cyclin, CLN2. Cell 1990, 63:999-1011.
WOLFFT, RE*'.D¥ DF: The Beginning of Pattern Formation in the Drosophila C o m p o u n d eye: The Morphogenetic Furrow and the Second Mitotic Wave. Development 1991, 113:841-850. Reports a det:tiled examination of the distribution of S-phase cells in the developing compound eye of D. melanogaster It concludes that all cells, other than the differentiating precluster cells, enter S-phase following the initiation of cellular differentiation. 19.
BAKERNE, RUBIN GM: Effect on Eye Development of Dominant Mutations in Drosophila Homologue of EGF-receptor. Nature 1989, 340:150-153.
ZAK NB, SHILO B-Z: Localization of DER and the Pattern of Cell Divisions in Wild-type and Ellipse Eye Imaginal Discs. Dev Biol 1992, 149:448-456. This, together with [21 •], reports a study of developing compound eyes of dominant Ellipse mutants in O. melanogastet: Ellipse mutant cells are shown to aberrantly enter S-phase instead of differentiating into photoreceptor cells. 20. •
BAKERNE, ROBIN GM: Ellipse Mutations in the Drosophila Homologue of the EGF Receptor Affect Pattern Formation, Cell Division and Cell Death in Eye Imaginal Discs. Dev Biol 1992, 150:381-396. This, together with [20•], reports a study of S-phases in developing compound eyes of dominant Ellipse mutants in D. melanogastet: Cells that would nomlally differentiate to fore1 the precluster are rather shown to aberrantly enter S-phase. In this paper, mitoses are shown to cluster near the few ommatidia that form in Ellipse mutants, suggesting that a signal from the differentiating ommatidia induces G2-phase cells to enter mitosis. 21. •
Developmental regulation of the cell cycle Saint, Wigley SELLECKSB, GONZALEZC, GLOVERDM, WHITE K: Regulation of the G1-S Transition in Postembryonic Neuronal Precursors by Axon Ingrowth. Nature 1992, 355:253-255. Cell-cycle phases of lamina precursor cells are shown to occur in specific locations within the developing Drosopbila brain. Lamina precursor cells in sine occulis mutant brains not innewated by photoreceptor cell axons, remain arrested in Gl-phase, instead of undergoing a further cycle. Innervation appears to generate a signal that induces the final cell cycle in the lamina precursor cells. 22. •,
23.
MURRAYAW, KIRSCHNER MW: Dominoes and Clocks: the Union of Two Views of the Cell Cycle. Science 1989, 246:614--621.
c-mos.
HUNT T: Cell Cycle Arrest and 355:587-588.
25.
SAGATAN, OSKAP~ChSONM, COPELAND T, BRUMBAUGHJ, VANDE WOUDE GF: Function of c-mos Proto.oncogene Product in Meiotic Maturation in Xenopus Oocytes. Nature 1988, 335:519-525.
26.
SAGATAN, DAARI, OSKARSSONM, SHOWAI.TERSD, VtM'qDEWOUDE GF: The Product of the mos Proto-oncogene as Candidate 'Initiator' for Oocyte Maturation. Science 1989, 245:643-646.
27. YEW N, MELUNI ML, VANDE WOLIDE GF: Meiotic Initiation by •• the mos Protein in Xenopus. Nature 1992, 355:649-652. Addresses the role of mos-encoded protein in the maturation of Xeno pus oocytes. Shows that a bacterially expressed mos fusion protein can induce maturation initiation, in the absence of protein s3,'nthesis, indicating that pp39 m°s is the only protein required to be made for the induction of maturation. Protein sTnthesis is, however, reqt, ired for progression to the end-point of maturation: metaphase arrest in meiosis I1. Also demonstrates that post-translational changes, induced by progesterone, are required in addition to mosaction, for fully efiqcient initiation of maturation. 28.
KOBAYASHII-I, MINSHULLJ, FORD C, GOLSTEYNR, POON R, HUNT T: On the Synthesis and Destruction of A- and B-type Cyclins During Oogenesis and Meiotic Maturation in Xenopus laevis. J Cell Biol 1991, 114:755-765.
29.
MINSHULLJ, MURRAYA, COla~,b\NA, HUNT T: Xenopus Oocyte Maturation does not Require New Cyclin Synthesis..I Cell Biol 1991, 114:767-772.
30.
GAUTIERJ, MALt.ERJL: Cyclin B in Xenopus Oocytes: Implications for the Mechanism of pre-MPF Activation. ~I,IBO J 1991, 10:177-182.
31.
SAGATAN, WKFANABEN, VANDE \'{tOUDE GF, IKAWAY: T h e cmos Proto-oncogene Product is a Cytostatic Factor Responsible for Meiotic Arrest in Vertebrate Eggs. Nature 1989, 342:512-518. WATANABEN, VANDE WOUDE GF, IKAWAY, SAGATA N: Specific Proteolysis of the c-mos Proto-oncogene Product by Calpain on Fertilization of Xenopus Eggs. Nature 1989, 342:505-511.
33.
LORCAT, GALASS, FESQUET D, DEVAULTA, CAVADOREJ, DOREE Degradation of the Proto-oncogene Product p 3 9 m o s is not Necessary for Cyclin Proteolysis and Exit from Meiotic Metaphase: Requirement for a Ca 2+-calmodulin D e p e n d e n t Event. FMBOJ 1991, 10:2087-2093. Extracts prepared from metaphase-arrested Xenopus eggs are used to analyze cyclin degradation in response to increased calcium levels. At physiological calcium concentrations, proteolysis of cyclin but not of mos:encoded protein occurs, suggesting that pp39ma*destmction is not required for MPF inactivation following fertilization. •
34. ,
FOE VE: Mitotic Domains Reveal Early C o m m i t m e n t of Cells in Drosophila Embryos. Development 1989, 107:1-22.
36.
EDGARBA, O'FARRELL PH: Genetic Control of Cell Division Patterns in the Drosophila Embryo. Cell 1989, 57:177-187.
37.
EDGARBA, O'FARREI± PH: T h e Three Postblastoderm Cell Cycles of Drosophila Embryogenesis are Regulated in G2 by string. Cell 1990, 62:469-480.
38.
O'FARRELLPH, EDGARBA, LAKICHD, LEHNERCF: Directing Cell Division during Development. Science 1989, 246:635--640.
39.
FOE VE, ODELL GM: Mitotic Domains Partition Fly Embryos, Reflecting Early Cell Biological C o n s e q u e n c e s of Determination in Progress. Am Zool 1989, 29:617-652.
Nature 1992,
24.
32.
35.
M:
WATANABEN, HUNT T, IKAW^ Y, SAGATAN: I n d e p e n d e n t Inactivation of MPF and Cytostatic Factor (Mos) u p o n Fertilization of Xenopus Eggs. Nature 1991, 352:247-248. Xenopus eggs and egg extracts were used to show that during meiotic release, cyclin subunits of MPF are degraded before mos proteolysis, and accordingly, that functional MPF is inactivated prior to inactivation of cytostatic factor (CSF). Supports the idea that CSF inactivation allows cell-cycle progression following fertilization, but that there is a CSF-independent pathway for MPF inactivation.
40. ,
ARORAK, NUSSLEIN-VOIMARDC: Altered Mitotic Domains Reveal Fate Map Changes in Drosophila Embryos Mutant for Zygotic Dorsoventral Patterning Genes. Development 1992, 114;1003-1024. The patterns of Drosophila cycle-14 mitotic domains, known to be under the direct regulation of string expression, are shown to be altered in a number of dorsoventral patterning mutants. 41.
GRAVESBJ, SCHUBIGERG: Cell Cycle Changes during Growth and Differentiation of Imaginal Leg Discs in Drosophila melanogaste~ Det, Biol 1982, 93:104-110.
42.
FAIN MJ, STEVENS B: Alterations in the Cell Cycle of Drosophila Imaginal Disc Cells Precede Metamorphosis. Det, Biol 1982, 92:247-258.
43.
FREEMANM, GLOVEI DM: The gnu Mutation of Drosophila Causes Inappropriate DNA Synthesis in Unfertilized and Fertilized Eggs. Genes Dev 1987, 1:924-930.
SHAMANSKIFL, ORR-WEAVERTI2 The Drosophila plutonium and pan gu Genes Regulate Entry into S Phase at Fertilization. Cell 1991, 66:1289-1300. Two genes, in addition to a third previously described, are shown to be required for file inhibition of DNA synthesis prior to fertilization in mature eggs. The mutations are shown to be stricdy maternal-effect mutations, suggesting that the genes are not required for cell-cycle regulation later in development. 44. •
[.IN H, WOLFNER ME: The Drosophila Maternal-effect Gene f s ( 1 ) Y a Encodes a Cell Cycle-dependent Nuclear Envelope C o m p o n e n t Required for Embryonic Mitoses. Cell 1991, 64:49-62. Describes the isolation and characterization of a Drosophila gene, mutations in which are strict maternal-effect mutations that show a requirement for the gene in the first and subsequent mitoses following fertilization.
45. •
HIME G, SAINT R: Zygotic Expression of the pebble Locus is Required for Cytokinesis during the Post-blastoderm Mitoses of Drosophila. Development 1992, 114:165-171. The D. melanogaster pebble gene is shown to be required for cytokinesis in the cellular post-blastodeml mitoses that follow the s3'ncytial cleavage mitoses. 46. •
47. **
SMITHAV, ORR-WEAVERTL: The Regulation of the Cell Cycle during Drosophila Embryogenesis: the Transition to Polyteny. Development 1991, 112:997-1008. Reports a study of the cycles of endo-replication, termed endo-cell-cy. cles, that lead to polyteny in Drosophila tissues. Several interesting features are noted: the cycles are comprised of S-phases separated by gap phases without cell division; the S.phases occur in developmentally regulated domains that correspond to organ or tissue types; each S-phase replicates most, but not all, of the DNA once; S-phases appear to be entered from G1- or G2-phase; none of the currently characterized cell-cycle control genes appear to be active in regulating these S-phases. 48.
WHrITIELDW, GONZALEZC, MALDONADO-CODINAG, GLOVER D: The A- and B-type Cyclins of Drosophila are Accumulated and Destroyed in Temporally Distinct Events that Define Separable Phases of the G2-M Transition. EMBO J 1990, 9:2563-2572.
49.
LEHNERCF, O'FARRELL PH: Drosophila cdc2 Homologs: a Functional Homolog is Coexpressed with a Cognate Variant. EMBO J 1990, 9:3573-3581.
619
620
Pattern formation and developmental mechanisms 50.
LEHNER CF, O'FARRELL PH: Expression and Function of Drosophila Cyclin A during Embryonic Cell Cycle Progression. Cell 1989, 56:957-968.
51.
JIMENEZ J, AI.PHE'," I~ NURSE P, GLOVER DM: Complementation of Fission Yeast cdc2ts and cdc25ts Mutants Identifies Two Cell Cycle Genes from Drosophila:. a cdc2 Homologue and string. EMBO J 1990, 9:3565-3571.
52.
ALPHEY L, JIMENEZ J, WHITE-COOPER H, DA\X~ON I, NURSE P, GI.OVER DM: twine, a cdc25 Homologue that Functions in the Male and Female Germ-line of Drosophila. Cell 1992, 69:977-988.
53.
GATEFF EA, MECHLER BM: Tumor-suppressor Genes of Drosophila melanogaster CRC Crit Rev Oncogen 1989, 1:221-245.
WOODSDF, BR'~'ANTPJ: The discs-large T u m o r Suppressor Gene of Drosophila Encodes a Guanylate Kinase Homolog Localized at Septate Junctions. Cell 1991, 66:451-464. The sequence and sub-cellular location of the discs.large gene of Drosophila are described. Mutations in the gene cause disc overgrowdl.
Tile sequence of the gene and the sub-cellular location of the gene product suggest a role in signal transduction. 55. ..
MAHONEYPA, WEBER U, ONOFRECHUKP, BIESS~bkNN H, BR','ANT PJ, GOODMAN CS: The f a t T u m o r Suppressor Gene i n Drosophila Encodes a Novel Member of the Cadherin Gene Superfamily. Cell 1991, 67:853-868. Mutations in the fat gene result in hyperplasia of the imaginal discs. This paper reports that fat is a member of the cadherin family of cell adhesion molecules.
56.
DONEHOWER LA, HARVEY M, SLAGLE BL, MCARTHU8 MJ, MONTGOMERYCA, BUTEL JS, BRADI.E't' A: Mice Deficient for p53 are Developmentally Normal but Susceptible to Spontaneous Tumours. Nature 1992, 356:215-221. Mice homozygous for a mutation in tile p53 gene are shown to develop nomlally, but are more susceptible to neoplastic diseases. This result challenges the idea that recessive oncogenes are genes normally im,oh,ed in the developmental regulation of growth. •
54. •.
R Saint, PL Wigley, Department of Biochemistry, University of Adelaide, GPO Box 498, Adelaide, Sot, th Australia 5001, Australia.
