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y. Cell Sci. Suppl. 12, 277-291 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 111 Mitosis in Drosophila development D . M. G L O V E R , L . A L P H E Y , J . M. A X T O N , A. C H E S H I R E , B. D A L B Y , M. F R E E M A N * , C. G I R D H A M , C. G O N Z A L E Z , R. E. K A R E S S f , M. H. L E I B O W I T Z , S. L L A M A Z A R E S , M. G. M A L D O N A D O - C O D I N A , J . W. R A F F , R. S A U N D E R S , C. E. S U N K E L J a n d W. G. F . W H I T F I E L D Cancer Research Campaign, Eukaryotic Molecular Genetics Research Group, Department o f Biochemistry, Imperial College of Science Technology and Medicine, London SW 7 2AZ, UK Summary Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. T he pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed. Introduction Drosophila melanogaster is an excellent organism in which to study mitosis. Like the yeasts it has the advantage of being genetically well characterised, but unlike the yeasts it has to face the problems of all multicellular organisms of co-ordinating cell proliferation with development. The mitotic divisions in early embryos of echinoderms, molluscs, amphibians and insects consist of rapid successions of M and S phases with no discernible Gi or G 2 phases as found at later stages of development. The Drosophila embryo is initially a syncytium in which thirteen rapid rounds of nuclear division occur at approximately 10 minute intervals. The first nine cycles occur within the embryo and then, at telophase of nuclear cycle nine, the majority of the nuclei migrate to the cortex. The nuclei that reach the posterior pole of the Present addresses: * Department of Biochemistry, University of California, Berkeley, California 94720, U SA . ■f Department of Biochemistry, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA. J Centro de Citologia Experimental, Instituto Nacional de Investigacao Cientifica, Rua do Campo Alegre 823 , 4100 Porto, Portugal. Key words: mitosis mutants, centrosome, chromosome condensation, cyclin. 278 ü . M. Glover et al. embryo undergo cellularisation ahead of the rest to form the pole cells that will develop into the germ-line. A small number of nuclei, the yolk nuclei, are left behind in the interior of the embryo. These cease dividing and lose their centrosomes, and eventually become polyploid. This represents the first example in Drosophila development of a switch from mitotic to polyploid cell cycles that later occurs in many tissues. Once at the surface, the majority of the nuclei undergo a further four division cycles before cellularisation occurs at interphase of cycle fourteen (Zalokar and Erk, 1976; Foe and Alberts, 1983). The organisation of the cytoskeleton during this period of rapid nuclear divisions has been carefully documented in both fixed and living embryos (K arr and Alberts, 1986; Warn et al. 1987; Kellogg et al. 1988). The cell cycle lengthens following cellularisation and there is a distinct interphase period enabling transcription to occur. The division cycles that follow cellularisation occur in complex ‘mitotic domains’ which develop following a specific temporal programme (Hartenstein and Campos-Ortega, 1985; Foe, personal communication). This is co-ordinated with a complex programme of gene expression in the morphogenesis of specific tissues within the embryo, which hatches as a larva after about 24 hours. Most of subsequent larval development involves cell growth with the endoreduplication of DNA in the absence of mitosis. Nevertheless the imaginai cells, destined to form the adult organism and not themselves necessary for the survival of the larva, continue to divide throughout larval development as do cells of the central nervous system. These imaginai tissues will develop into the adult organism during pupation. Uncoupling of mitotic cycles from DNA replication in the early embryo Some years ago, we described the embryonic phenotype of a mutation, gnu, a gene whose product is needed for nuclear division during early development. Females homozygous for gnu lay eggs (G N U eggs) which develop giant nuclei as a result of continued DNA replication in the absence of chromosome segregation and nuclear division (Freeman et al. 1986). Such an embryo can be seen in panel G of Fig. 1. Fertilisation of G N U eggs is not required for the development of giant nuclei, contrasting with wild-type eggs in which fertilisation is required before any DNA replication or mitotic events can take place (Freeman and Glover, 1987). It seems that somehow, the G N U cytoplasm lifts the repression of DNA synthesis that normally occurs following the completion of meiosis until the fusion of the male and female pronuclei has taken place. Thus, whether or not the G N U egg is fertilised, any of the four products of female meiosis, the three polar bodies or female pronucleus, can participate in DNA synthesis to give giant nuclei. We also showed that the paternal genome could replicate in fertilised G N U eggs, even if the mothers were also homozygous for the maternal haploid mutation, mh, a mutation that otherwise results in the failure of the paternal genome to replicate (Freeman and Glover, 1987). The gene therefore appears to play a role in the correct establishment of co-ordinated DNA replication and mitosis in zygotic development. One of the striking features of G N U embryos is that although nuclear division Mitosis in Drosophila development 279 does not take place, centrosomes continue to replicate. In the field of anaphase figures from wild-type embryos (Fig. 1, panels A and B), single centrosomes can be seen at spindle poles. This is in contrast to the two fields irom gnu embryos, one with a developing giant nucleus (panels C and D) and the other with no nuclei, where the centrosomes are completely dissociated from nuclei and do not function in the formation of mitotic spindles. They are, however, capable of nucleating asters of microtubules (Freeman et al. 1986). The increase in number and migration of centrosomes in the developing G N U embryo indicates the independence of the centrosome cycle from the nuclear division cycle. DNA synthesis is, however, an ongoing process in G N U embryos, as indicated by the increase in size and fluorescence of the Hoechst-labelled nuclei, and by molecular hybridisation exper iments (Freeman and Glover, 1987). There are no obvious cycles of chromosome condensation-decondensation, although the nuclear envelope may be undergoing cyclical changes by the criterion of staining with an anti-lamin antibody. The embryo depicted in Fig. 1 (panels G and H ), for example, has three giant nuclei, two of which are stained with the anti-lamin antibody, and one of which is not. In order to determine the degree of autonomy of mitotic cycles, we recently investigated the effects of microinjecting aphidicolin, an inhibitor of D NA polym erase, into syncytial wild-type Drosophila embryos (Raff and Glover, 1988). Not only were centrosome replication and nuclear division uncoupled, but also centro somes proceeded through multiple rounds of division in the absence of DNA replication. Cortical budding cycles (Foe and Alberts, 1983) also continue in aphidicolin-treated embryos, and as with untreated embryos, spread in waves from both poles. When the buds are present at the surface of aphidicolin-injected embryos, the nuclei have decondensed chromatin surrounded by nuclear membranes as judged by bright annular staining with an anti-lamin antibody. As the buds recede, the unreplicated chromatin condenses and lamin staining becomes weak, diffuse and cytoplasmic (Fig. 2). There seems therefore to be no absolute requirement for the correct completion of S phase in order for both nuclear and cytoplasmic events of M phase to take place. This is not to say that some critical aspect of S phase is not completed, and if indeed aphidicolin has its only primary effects on DNA polymerases a and <5, this could well be possible. Nevertheless, DNA synthesis is dramatically inhibited and chromosome replication, a major objective of the cell cycle, does not occur. In an extension of these experiments, we showed that if aphidicolin was injected into embryos before nuclear cycle 7 - 8 , the normal migration of nuclei to the embryo cortex is completely inhibited (Raff and Glover, 1989). Centrosomes can continue to migrate to the cortex, however, where they nucleate asters of microtubules, each of which is overlaid with an actin cap. This suggests that the co-ordinated movement of nuclei to the embryo cortex is normally mediated by forces acting on the centrosome rather than on the nucleus itself. Cellularisation, thought to be triggered by the nuclei:cytoplasmic ratio (Edgar et al. 1986), does not occur in these aphidicolintreated embryos except at the posterior pole. Remarkably, the centrosomes that migrate into the posterior pole plasm appear to initiate formation of pole 280 D. M. Glovei' et al. DNA Centrosomes Fig. 1. Immunostaining of G N U embryos. A field of anaphase figures from a wild-type embryo stained with Hoechst to reveal DNA (panel A) and the Bx63 antibody (panel B) that recognises an antigen associated with the centrosome (Frasch et al. 1985; Whitfield et a l. 1988) is shown for comparison with two fields from G N U embryos (panels C - F ) (Freeman et a l. 1986). Panels G and H show a whole GNU embryo stained with Hoechst and an anti-lamin antibody respectively. Note the lack of staining by the anti-lamin antibody of the nucleus at the pole. Panels I and J show a higher magnification of giant nuclei stained with Hoechst and the anti-lamin antibody (Freeman, 1987). Scale bars are 20 [im in panels A - F , 80jUm in panels G and H and 20,um in panels I and J. cells which lack nuclei (Fig. 3). It has long been known that determinants localised in the posterior pole plasm are required for pole cell formation (Okada et al. 1974; Illmensee and Mahowald, 1974; Illmensee and Mahowald, 1976). Although the molecular nature of these determinants is unknown, structures called polar granules can be seen in the posterior pole plasm (Counce, 1963; Mahowald, 1962; Mahowald, Mitosis in Drosophila development DNA 281 Lamin 1968). Our finding raises the question of how an interaction between polar determinants and the centrosome (or the microtubules it nucleates) might direct the formation of pole cells. It is a demonstration of how centrosomes can direct a major re-organisation of the cortical cytoskeleton upon their arrival at the surface of the embryo. Cyclins It is clear from the studies described above that many aspects of embryonic mitoses can cycle independently of D N A synthesis. By its very nature, mitosis is the sum of a number of cyclical processes, and consequently it is difficult to know which events are key stages in regulating or coordinating the cycle. Studies on other organisms may be helpful in this respect. Several observations point towards a unique step in the yeast cell cycle that has been termed ‘start’, which has to be completed in order to 282 D. M. Glover et al. initiate DN A synthesis and subsequent mitotic events. T h e cdc2 gene of Saccharo myces pombe is required not only for ‘start’, but also for the G j to M transition. T h e cdc2 gene product has homologues in other eukaryotes (the subject of several articles in this volume), most strikingly a protein of similar molecular mass that is a component of mitosis-promoting factor (M P F ) purified from Xenopus (Gautier et al. 1988; Dunphy et al. 1988). One of the 5 . pombe cell cycle genes, cdcl3, that interacts with cdc2, encodes a protein homologous to the cyclins (Solomon et al. 1988; Goebl and Byers, 1988; Hagan et al. 1988), a family of proteins that undergo dramatic cycles of synthesis and degradation in the cell cycle. Cyclins, originally discovered in the eggs of marine invertebrates, are present in a wide variety of eukaryotes (Evans et al. 1987; Swenson et al. 1986; Standart et al. 1987). T h e Lamin DNA Centrosomes A t i * ft * ♦ B • _ ft * • • % m 1 ft $ % mm Fig. 2. Wild-type embryos that have been injected with aphidicolin and fixed for immunostaining after 90 minutes (Raff and Glover, 1988). Lamin and centrosomes have been revealed by immunostaining with antibodies that recognise Drosophila lamin and the centrosome-associated Bx63 antigen, respectively. DNA is stained with Hoechst. Note the increased ratio of centrosomes to nuclei in both fields. T he nuclei in field A have condensed chromatin and diffuse lamin staining, in contrast to field B, in which the chromatin is decondensed and the anti-lamin antibody stains the nuclear envelope. Scale bar, 10 fim. Mitosis in Drosophila development 283 numbers of eyclin genes vary in different organisms: clams, for example, have two cyclins termed A and B , whereas sea urchins have a single B-type cyclin. It is possible that the two cyclins play differing roles in the cell cycles of clams since their degradation occurs at different times relative to the metaphase-anaphase transition, although the significance of this is not understood. Lehner and O ’Farrell (1989) have DNA Centrosomes Fig. 3. The posterior poles of wild-type embryos injected with aphidicolin between nuclear cycles 7—8, approximately 0 -2 0 minutes before the nuclei at the posterior pole of the embryo would have reached the surface and initiated pole bud formation. The embryos were fixed for immunostaining after pole cell formation had occurred (Raff and Glover, 1989). A and B are from two different embryos in which the nuclei have remained within the interior of the embryo (Hoechst staining in the left-hand panels), whereas the centrosomes have migrated to the cortex (punctate staining with R bl88 antibody, Whitfield et a l. 1988, in the right hand panels) and initiated the formation of pole cells. 284 D. M. Glover et al. cloned the cyclin A gene from Drosophila, raised an antibody against its gene product expressed in E. coli, and shown that after cellularisation the protein undergoes cycling in the mitotic domains of the developing embryo. Embryos homozygous for mutations in the cyclin A gene utilise maternally-provided cyclin A to complete up to 15 rounds of division, after which no mitotic figures can be seen (Lehner and O ’Farrell, 1989). We also have cloned the gene for cyclin A together with the cyclin B gene of Drosophila (Whitfield et al. 1989). Both genes encode abundant maternal mRNAs, but whereas the cyclin A m RN A is relatively uniformly distributed prior to cellularisation, the cyclin B m RN A becomes localised to the developing pole cells, the precursors of the germ-line (Fig . 4 ). As the somatic nuclei complete their syncytial mitotic cycles, cyclin B transcripts show a clear association with the cortex in the vicinity of nuclei, and then disappear abruptly during cellularisation of somatic nuclei. Transcripts either persist or continue to be produced in the pole cells as they move dorsally and anteriorly in germ-band elongation. Although some zygotic cyclin B transcription appears to ensue in the soma in later embryos, the predominant labelling is still in pole cells. Microinjection of actinomycin indicates that the initial localisation of cyclin B transcripts to the pole cells does not represent de novo Fig. 4. Localisation of cyclin RNAs in sections of Drosophila embryos by in situ hybridisation (Whitfield et a l. 1989). The two embryos shown are both around nuclear cycle 8, at which time nuclei are migrating towards the cortex. The upper embryo (A) is hybridised with cyclin A probe, the lower (B) with cyclin B. The anterior of the embryos are to the left, and the left and right panels show the corresponding bright field and dark field images respectively. Mitosis in Drosophila development 285 transcription but redistribution of maternal m RN A. The two genes are differentially expressed in mitotically-dividing tissues in larval development. Cyclin A, but not cyclin B , transcripts are readily detectable over proliferating cells in the periphery of the brain lobes, and, at a lower level, in imaginal discs of third instar larvae. Cyclin B transcripts, on the other hand, predominate in the larval testes (Whitfield et al. 1989). At present, we can only speculate that there is a special requirement for cyclins in an aspect of germ cell proliferation that differs from somatic cell division. In the imaginal discs, for example, there appears to be a target cell number that may be controlled by achieving cell-cell contacts in a desired pattern. By contrast, cell division continues in the stem cells of the testes and ovary throughout the life of the organism to produce daughter stem cells and primary gonial cells. In addition, the premeiotic mitoses of the gonial cells in both male and female are accompanied by incomplete cytokinesis to produce groups of cells that share cytoplasm connected by canals. It is possible that there is a need for a specific cyclin either to maintain stem cell proliferation, to provide for the peculiarities of what are essentially syncytial divisions in the gonial cysts, or to mediate the ultimate transition between mitotic and meiotic divisions. Mutations that affect mitosis About 70 genes essential for mitosis are known in Drosophila (see Ripoll et al. 1987, and Glover, 1989, for reviews). In this article, we will concentrate upon briefly describing the ongoing efforts in our own laboratory to characterise some of these mutations. These are all recessive lethal mutations that show mitotic phenotypes at various developmental stages. As all of the gene products required for mitotic cycles of the syncytial embryo are supplied maternally, one class of mutations shows maternal effect lethal phenotypes. A female homozygous for such a mutation produces embryos in which the early divisions are not completed successfully. In many cases such maternal effect mitotic mutations also show an additional zygotic effect upon cell proliferation in the diploid imaginal and neuroblast cells of the larvae, and in such cases cytological examination of the neuroblasts of homozygous larvae often reveals mitotic abnormalities. The consequence to the animal depends upon the severity of the lesion and the developmental regulation of the gene in question. Some homozygous mutant larvae survive to adulthood, and lethality is only seen as a maternal effect. However, many mitotic mutations are zygotic lethals. The wild-type gene product supplied by heterozygous mothers is sufficient for the syncytial divisions and can persist throughout part or all of subsequent embryo genesis. The cyclin A gene (see above) and string (Edgar and O’Farrell, 1989) are examples of genes whose products are needed to complete the remaining three or four rounds of cell division that occur following cellularisation. Many homozygous mitotic mutants can survive utilising maternally-supplied proteins until late larval development. In such cases, the imaginal cells of the homozygous mutant larvae cannot proliferate, and consequently death ensues during the 286 D. M. Glover et al. larval or early pupal stages, a phenotype first recognised by Baker, Gatti and co workers (see, for example, Baker et al. 1982; Gatti and Baker, 1989). In some cases, the mitotic mutation also affects meiosis, and leads to chromosome non-disjunction if not sterility. Mutations at the polo locus show many of these phenotypic aspects (Sunkel and Glover, 1988). Females homozygous for polo survive to adulthood to lay eggs that die during embryogenesis, but nevertheless show some mitotic abnormalities in neuro blast cells during the larval stages of their development. Immunocytological studies on PO LO embryos reveal highly branched mitotic spindles with broad irregular poles that do not have distinct centrosomes. The centrosome-associated antigen, Bx63, (see below) is present as particulate matter which gradually coalesces throughout the abnormal development of the embryo. Neuroblast cells in larvae homozygous for the mutation show abnormal polyploid circular mitotic figures, and anaphase figures in which chromosomes appear to be randomly oriented with respect to at least one of the spindle poles. One explanation that has been proposed for such circular mitotic figures, also seen in three other mitotic mutations under study in our laboratory (see below), is that they are monopolar spindles (Gonzalez et al. 1988; Sunkel and Glover, 1988). polo also shows aberrant meiotic divisions in which the spindles in testes are often multi-polar. This results in chromosome non-disjunction that can be seen genetically and cytologically by the diverse sizes of the spermatid nuclei (Sunkel and Glover, 1988). Thus certain mutations at polo manifest their phenotypes at a number of developmental stages. Other more extreme alleles have strong zygotic phenotypes. One allele, generated in a P —M dysgenic cross, for example, is a larval lethal. We are confident that this P-element-tagged mutation will lead to the cloning of the polo gene. Although mitotic mutations can affect a variety of developmental stages, most show phenotypes in larval neuroblasts. In the remaining part of this article we will exemplify the variety of phenotypes that can be observed referring to mutations under study in our laboratory. Fig. 5 (panels B - I ) shows micrographs of neuroblast cells from larvae bearing these mutations, together with the diploid chromosomal complement of a wild-type cell (panel A) for comparison. Mutations affecting chromosome condensation Of the many genes that affect chromosome condensation in Drosophila, we have chosen to study mus 101, originally isolated as a mutagen-sensitive mutation, but subsequently discovered by Gatti and co-workers to have a striking effect upon the condensation of heterochromatin but not euchromatin (Gatti et al. 1983). An example of this phenotype, in a mus 101 allele identified in our laboratory, is shown in panel B of Fig. 5 (Axton et al. unpublished). The availability of a temperature sensitive mutant allele of the locus allowed Gatti and his colleagues to follow the onset of abnormal chromosome condensation after cells are shifted to the restrictive temperature. There appears to be no gross effect of mus 101 upon the replication of DNA in heterochromatin as judged by an autoradiographic study of [3H]thymidine incorporation, and it has been suggested that the effects of the mutation upon Mitosis in Drosophila development 287 mutagen sensitivity and DN A repair are secondary consequences of the primary effect on condensation of heterochromatin. Nevertheless, there are instances in which mutant alleles of this locus do affect DN A replication. One allele, K 451, prevents the extra rounds of DN A replication that occur at the X and 3rd chromosome clusters of chorion genes in follicle cells at a developmentally specific phase of oogenesis (Orr et al. 1984; Snyder et al. 1986). In addition to these female sterile mutants affecting chorion biosynthesis, we have found that other female sterile m uslO l alleles have mitotic defects in the syncytial embryo. We are investigating whether one temperature-sensitive allele can be used to give a better indication of the phenocritical phase of the cell cycle at which the gene product is required. W hilst it is possible that effects upon the organisation of chromatin could have secondary consequences upon DN A replication, it is probably prudent to await further molecular characterisation of this locus before drawing any conclusions about the mode of action of the gene. Towards this end, we have localised m us 101 to a small interval on the X-chrom osom e, microdissected this region from polytene salivary gland chromosomes, cloned its DN A into a bacteriophage insertion vector following limit digestion with E coR l and have used these clones as multiple starting points in a chromosome walk to isolate overlapping clones of DN A in the cytological interval (Axton et al. unpublished). Abnorm al spindle abnormal spindle (asp) is a well characterised mitotic locus that was first described by Ripoll et al. (1985). Larvae homozygous for asp show an elevated mitotic index, a reduced frequency of anaphases, and aneuploid cells (Fig. 5, panel D ). If asp is A A 4-j * ^ F • c • % V* * * & - F ^ A ^ H \ - •i Fig. 5. Mitotic chromosomes in squashes of Drosophila larval neuroblasts. A, wild-type; B, muslOl-, C, l(3)snap\ D , asp ; E, polo\ F , aurora', G , merry-go-round ; H, rough-deal; I, lodestar. 288 D. M. Glover et al. made heterozygous with a deficiency, the resulting larvae show an increased mitotic index with some overcondensation of chromosomes as compared with wild-type larvae, but all the cells are diploid, as if abruptly arrested in metaphase, asp is thought to affect the mitotic spindle, and biochemical studies have shown that microtubules are more stable in mutant than in wild-type cell extracts (Ripoll et al. 1985). A polypeptide has been identified by 2D electrophoretic analysis which varies in concentration as a function of gene dosage of the region containing the asp locus (Wandosell et al. 1989). It is proposed that this protein acts to modify a second protein involved in spindle dynamics. Genetic and cytological analyses both indicate incorrect chromosome segregation in male meiosis (Ripoll et al. 1985). In collabor ation with Ripoll’s group, we have examined the maternal effect phenotype in ASP embryos, the larval neuroblast phenotype, and the male meiotic phenotype by immunostaining. All of these developmental stages show unusually long arrays of microtubules, consistent with the biochemical indications of their increased stability (unpublished data). More work needs to be done to confirm the model of increased microtubule stability, and it will be helped by a molecular analysis. Towards this end, we have microcloned the chromosomal region 96A21-25 and 96B1-10 in which asp lies (Gonzalez, 1986). Approaches to the analysis of the Drosophila centrosome We routinely stain centrosomes either with a monoclonal antibody, Bx63 (Frasch et al. 1986), or a polyclonal serum raised against the Bx63 antigen synthesised as a fusion protein in Escherichia coli (Whitfield et al. 1988). The cloning of the gene for the Bx63 antigen and its mapping by in situ hybridisation to the salivary gland chromosome region 88E mean that it is now possible to attempt to generate mutations at this locus, and thereby examine the mutant phenotype. In addition we are following a complementary approach by studying three mutations that appear to affect the centrosome. Each mutation leads to the formation of circular chromosomal figures in larval neuroblasts. We mentioned above that the larval neuroblast phenotype associated with mutations at the polo locus (Fig. 5 panel E) might be indicative of a lesion affecting the centrosome. Similar phenotypes have also been seen with alleles of merry-go-round (mgr ; Gonzalez et al. 1988, Fig. 5 panel G ), and aurora (Leibowitz and Glover, unpublished, Fig. 5 panel F ). aurora only shows this phenotype when uncovered by a deficiency for the region. Mutation in mgr causes polyploid cells, metaphase arrest, circular mitotic and meiotic figures, post-meiotic cysts with 16 rather than 64 cells and spermatids with four times the normal chromosome content (Gonzalez et al. 1988). The integrity of microtubules appears to be required for the circular figures to form, as they are no longer seen if the cells are treated with colchicine. This is supported by observations on the phenotype of larvae homozygous for both mgr and asp , which do not show circular figures. The mgr gene maps at 51.3 cM and has been localised cytologically to 86E3-10 (Gonzalez et al. 1989), a region which we have now microcloned. aurora, like polo, was originally identified by maternal effect lethal mutations. Embryos from homozygous aurora females have mitotic spindles in which there is a Mitosis in Drosophila development 289 characteristic change in the pattern of centrosome staining in the progression from anaphase to telophase. In anaphase there are well defined ‘dot-like’ centrosomes that nucleate the spindle poles. These spindles develop into broad, telophase-like structures apparently nucleated from points around the nuclear envelope and showing weak, indistinct centrosome staining. We have cloned the DNA correspond ing to the cytological interval to which we have mapped aurora , and have used the cloned D N A to probe Northern blots of RNA from different developmental stages, and so identify transcripts across the region. It would seem from the sizes and developmental profiles of the RNAs that there are either six or seven transcription units in the interval. The ultimate proof that one of these is the aurora gene will come from P-element-mediated transformation experiments to see whether or not a particular DNA fragment is capable of rescuing the mutation. Whilst the phenotypes of these mutations do suggest lesions affecting the centrosome, the primary effect might well be upon other components of the mitotic apparatus that have to interact with the centrosome. This should become clear with studies using antibodies raised against protein expressed from the cloned genes. Chromosome segregation Finally, we are studying two loci, rough deal and lodestar, representative of those affecting mitotic segregation at anaphase (Fig. 5 panels H and I; Karess et al. unpublished; Girdham et al. unpublished). These have similar phenotypes in which the mitotic figures in neuroblasts of homozygous larvae show aneuploidy, and anaphases with lagging chromatids and sometimes with chromatin bridges and broken chromosomes, lodestar also displays a maternal effect in which the embryos of homozygous lodestar females have anaphases with lagging chromatids or chromatin bridges. We are currently carrying out germ-line transformation exper iments with several chromosomal DNA segments containing transcription units from the region on chromosome 3 to which lodestar maps cytogenetically. Concluding remarks Mitosis involves many concurrent cyclical processes. Cyclical changes occur to chromosomes, the nuclear envelope, centrosomes, microtubules, and many other structures within the cell. Control of this complex process is required at several levels. Each of the cyclical processes involves a sequential chain of events, in which there are probably several critical steps that cannot be surmounted unless the preceding stages have been completed. Moreover, there have to be mesh-points at which concurrent cycles interact in order that the whole process is coordinately regulated. Above this is another level of control that regulates the proliferation of cells within the developing organism. Cell-cell interactions undoubtedly play a major role at this level, but these result in intracellular signals that must impinge upon mitotic control. Since the primary objective of the mitotic process is to segregate the replicated genome into daughter cells, we believe it is important to study aspects of chromosomal and cytoskeletal architecture essential for this 290 D. M. Glover et al. function. This will lead to an understanding of molecular interactions that control two of the major cyclical mitotic pathways. A genetic approach will be invaluable in this respect, but it has limitations that can be overcome only with the concerted application of molecular studies. It is only a matter of time before many of the genes we have described are cloned and sequenced. Antibodies raised against the product of the cloned gene expressed in Escherichia coli will be powerful tools in analysing the functions of the proteins in Drosophila cells. This will bring an understanding of a sub-set of proteins from which one may, by using both biochemical and genetic approaches, take steps to study the proteins with which they interact. Together with functional studies, this strategy should lead to a greater understanding of mitotic events. We are grateful to the Cancer Research Campaign, the Medical Research Council and the Science and Engineering Research Council for their support. References B a k e r , B . S ., Sm ith, D . A. a n d G a t t i , M. (1982). Region specific effects on chromosome integrity of mutations at essential loci in Drosophila melanogaster. Proc. natn. Acad. Sei. U .S A . 79, 1205-1209. C o u n c e , S. J . (1963). Developmental morphology of polar granules in Drosophila including observation on pole cell behavior and distribution during embryogenesis. J . Morph. 112, 129-145. D unphy, W. G ., B r i z u e la , L ., B e a c h , D . a n d N ew p o rt, J. (1988). T h e Xenopus cdc2 protein is a component of M PF, a cytoplasmic regulator of mitosis. Cell 54, 423-431. E d g a r, B. A ., K ie h le , C. P. a n d S ch u b ig e r, G. (1986). Cell cycle control by the nucleocytoplasmic ratio in early Drosophila development. Cell 44, 365-372. E d g a r , B. A. a n d O ’F a r r e l l , P. (1989). Genetic control of cell division patterns in the Drosophila embryo. Cell 57, 177-187. E v a n s, T ., R o s e n t h a l, E . T ., Y o u n g b lo o m , J . , D i s t e l , D . a n d H u n t, T . (1987). Cyclin-. a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cell division. Cell. 33, 389-396. F o e , V. a n d A lb e r ts , B. M. (1983). Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J'. Cell Sei. 61, 31 -7 0 . F r a s c h , M ., G lo v e r , D . M. a n d Saum w eber, H. (1986). Nuclear antigens follow different pathways into daughter nuclei during mitosis in Drosophila embryos. J'. Cell Sei. 82, 115-172. F re em a n , M. (1987). gnu, a nuclear replication mutant of Drosophila. PhD thesis, University of London, U K . F re em a n , M. a n d G lo v e r , D. M. (1987). T h t gnu mutation of Drosophila causes inappropriate D N A synthesis in unfertilised and fertilised eggs. Genes and Development 1, 924-930. F re em a n , M ., N u s s le in -V o lh a r d , C. a n d G lo v e r , D . M. (1986). T he dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila. Cell 46, 457-468. G a t t i , M. a n d B a k e r , B . S. (1989). Genes controlling essential cell cycle functions in Drosophila melanogaster. Genes and Development 3, 438-453. G a t t i , M ., Sm ith, D . A. a n d B a k e r , B . S . (1983). A gene controlling the condensation of heterochrom atin in Drosophila melanogaster. Science 221, 83-85. G a u t ie r , J ., N o rb u ry , C ., L o h k a , M ., N u rs e , P. a n d M a l l e r , J . (1988). Purified maturation promoting factor contains the product of a Xenopus homologue of the fission yeast cell cycle control gene cdc2+ . Cell 54, 433-439. G lo v e r , D . M. (1989). Mitosis in Drosophila. J. Cell Sei. 92, 137-146. G o e b l, M. a n d B y e rs , B . (1988). Cyclin in fission yeast. Cell 54, 739-740. G o n z a le z , C. (1986). Analisis genetico de la segregation cromosomica en Drosophila melanogas ter. Doctoral thesis, Universidad Autonoma de Madrid, Italy. G o n z a le z , C ., C a s a l, J . a n d R ip o ll, P. (1988). Functional monopolar spindles caused by mutation in mgr, a cell division gene of Drosophila melanogaster. J . Cell Sei. 89, 39-47. Mitosis in Drosophila development 291 H agan , I., H a y l e s , J. and N u r s e , P. (1988). Cloning and sequencing the cyclin related cdcl3 gene and a cytological study of its role in fission yeast mitosis. J . Cell Sei. 91, 587-596. H a r ten stein , V . and C am pos -O rteg a , J . A. (1985). F ate mapping in wild-type Drosophila melanogaster. I . T h e pattern of the em bryonic cell divisions. Wilhelm Roux Arch. D evi Biol. 194, 181-195. I l l m e n se e , K . and M a h o w a ld , A. P. (1974). Transplantation of posterior pole plasm: Induction of germ cells at the anterior pole of the egg. Proc. natn. Acad. Sei. U .S A . 71, 1016-1020. I llm e n s e e , K . a n d M a h o w a ld , A. P. (1976). The autonomous function of germ plasm in a somatic region of the Drosophila egg. E xpl Cell Res. 97, 127-140. K a r r , T . L . and A l b e r t s , B . M. (1986). Organisation of the cytoskeleton in early Drosophila embryos, jf. Cell Biol. 98, 156-162. K ello g g , D . R ., M itch iso n , T . J . and A l b e r t s , B. M . (1988). Behaviour of microtubules and actin filaments in living Drosophila embryos. Development 103, 675-686. L eh n er , C. and O ’F a r r e l l , P. (1989). Expression and function of Drosophila cyclin A during embryonic cell cycle progression. Cell 5 6, 957-968. M a h o w a ld , A. P. (1962). Fine structure of pole cells and polar granules in Drosophila melanogaster. J . exp. Zool. 151, 201-205. M a h o w a ld , A. P. (1968). Polar granules of Drosophila . I I . Ultrastructural changes during early embryogenesis. J . exp. Zool. 167, 237-262. O ka d a , M ., K lein m a n , I. A. and S ch n eid erm a n , H. A. (1974). Restoration of fertility in sterilized Drosophila eggs by the transplantation of polar cytoplasm. D evi Biol. 37, 4 3 -5 4 . O r r , W ., K om itopoulou , K . and K afatos , F . (1984). Mutants suppressing in trans chorion gene amplification in Drosophila. Proc. natn. Acad. Sei. U.S.A. 8 1 , 3773-3777. R a f f , J. W. and G lo ver , D . M . (1988). Nuclear and cytoplasmic mitoticcycles continue in Drosophila embryos in which DNA synthesis is inhibited with aphidicolin. J. Cell Biol. 107, 2009-2019. R a f f , J. W. a n d G lo v e r , D . M . (1989). Centrosomes, and not nuclei, initiate pole cell formation in Drosophila embryos. Cell 57, 611-619. R ip o l l , P ., C a sa l , J . and G o n z a l e z , C. (1987). Towards the genetic dissection of mitosis in Drosophila. BioEssays 7, 204-210. R ip o l l , P ., P im pin e l l i , S ., V a ld iv ia , M. M. and A v il a , J . (1985). A cell division mutant of Drosophila with a functionally abnormal spindle. Cell 4 1, 907-912. S n y d er , P. B ., G alan opoulos , V. K . and K afatos , F . C. (1986). Trans acting amplification mutants and other eggshell mutants of the third chromosome of Drosophila melanogaster. Proc. natn. Acad. Sei. U.S.A. 8 3, 3341-3345. S olomon , M ., B o o h er , R ., K ir sc h n er , M. and B ea ch , D . (1988). Cyclin in fission yeast. Cell 54, 738-739. S tandart , N ., M in sh u l l , J . , P in e s , J . N. and H u n t , T . (1987). Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte. D evi Biol. 124, 248-258. S u n k e l, C. E. a n d G lo v e r , D . M. (1988). polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J . Cell Sei. 89, 2 5 -3 8 . S w en so n , K . I ., F a r r e l l , K . M. and R u d erm a n , J . R . (1986). The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes. Cell 47, 861-870. W a n d o se ll , F ., G o n z a l e z , C ., R ip o l l , P. and A v ila , J . (1989). Identification of the gene products of abnormal spindle, a modifier of a new Drosophila MAP. (in preparation). W a rn , R . M ., F le g g , L . and W a rn , A. (1987). An investigation of microtubule organisation and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha tubulin. .7- Cell Biol. 105, 1721-1730. W h it f ie l d , W . G . F ., G o n z a l e z , C ., S an ch ez -H err er o , E . and G lo ver , D . M . (1989). Transcripts of one of two Drosophila cyclin genes become localised in pole cells during embryogenesis. Nature, Lond. 338, 337-340. W h it f ie l d , W . G . F ., M il l a r , S . E ., S a u m w eb er , H ., F ra sc h , M . and G lo ver , D . M . (1988). Cloning of a gene encoding an antigen associated with the centrosome in Drosophila. J . Cell Sei. 89, 4 6 7 -4 8 0 . Z a lo k a r , M . AND E r k , I. (1976). Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J. Microbiol. Cell 25, 97-106.