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y. Cell Sci. Suppl. 12, 277-291 (1989)
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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.
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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
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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
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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
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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
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♦
B
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_
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•
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%
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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.
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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
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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
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F
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-
F
^ A
^
H
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-
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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.
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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
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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.
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