Download Functional monopolar spindles caused by

Functional monopolar spindles caused by mutation in mgr, a cell
division gene of Drosophila melanogaster
CAYETANO GONZALEZ*, JOSE CASAL and PEDRO RIPOLL
Cenlm cle Biolo/fia Molecular (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Madrid, Spain
•Present address, for correspondence: Department of Biochemistry, Imperial College of Science and Technology, London SW7 2AZ,
England
Summary
Mutation in the gene merry-go-round (mgr) of
Drosophila causes a variety of phenotypic traits
in somatic and germinal tissues, such as polyploid cells, metaphasic arrest, postmeiotic cysts
with 16 nuclei, and spermatids with four times the
normal chromosome content. The most characteristic phenotype is the appearance of mitotic
and meiotic figures where all chromosomes are
arranged in a circle. Treatment with anti-mitotic
drugs and the phenotype of double mutants mgr
asp (asp being a mutation altering the spindle)
Introduction
The poleward movement of chromosomes during anaphase has attracted the interest of cell biologists since it
was beautifully described over one hundred years ago
(Flemming, 1878). Though a great amount of work has
been devoted to cytological and biochemical aspects of
this process, our knowledge of it is still rather unsatisfactory. Genetics provides a powerful tool for identifying the structural or regulatory components involved in
chromosome movement and their function during cell
division. Genetic analysis has been successful in dissecting the cell cycle in yeast and other fungi (Pringle &
Hartwell, 1981; Nurse, 1985), and it is yielding interesting results in mammalian cell cultures (Simchen,
1978; Marcus et al. 1985) and mouse (Magnuson &
Epstein, 1984). Recently, several groups have concentrated on the genetic dissection of cell division in
Drosophila melanogaster, where a fairly large collection
of mutants is already available (Gatti et al. 1983; Ripoll
et al. 1987).
The spindle is the structure responsible for the
accurate segregation of chromatids (or chromosomes)
Journal of Cell Science 89, 39-47 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
show that these circular figures need a functional
spindle for their formation. These abnormal figures are caused by monopolar spindles similar to
those observed after different treatments in several organisms. All mutant traits indicate that
mgr performs a function necessary for the correct
behaviour of centrosomes, thus opening this organelle to genetic analysis.
Key words: monopolar spindles, mgr, cell division,
Divsophila.
to daughter cells during mitosis and meiosis, as well as
for the equitable partition of other subcellular organelles. Morphologically, the spindle is formed by the
centrosomes, which are composed of centrioles and
pericentriolar material, and the fibres joining the
centrosomes to each other or to the kinetochores.
Compared with what we have learned about spindle
fibres (Dustin, 1984), centrosomes have managed to
escape biochemical and genetical approaches (Fulton,
1971; Mazia, 1984). In this report we present and
discuss the phenotype caused in Drosophila melanogaster by mutation in the gene merry-go-round (mgr),
which we interpret as resulting in abnormal centrosomc
behaviour.
Materials and methods
Isolation and location of mgr
Meny-go-round was recovered among a collection of 30 late
(larval and pupal) lethals induced with X-rays in the third
chromosome of an isogenic red strain (for description of
mutations see Lindsley & Grell, 1968). Zygotic lethality,
mitotic phenotypes, and testicular phenotypes, co-mapped at
39
51'3cM (centiMorgans), based on 28 recombinant chromosomes between scarlet (44-0cM) and red (S3-6cM). The
gene was cytologically localized in region 86E3-6; 86E12-20,
based on its inclusion in Df(JR)TE61 (86E3-6;87A6-10) but
not in Df(3R)TE41 (86E12-20;87Cl-2) (we have redefined
the breakpoints of these deficiencies and they do not agree
with previous cytological description given by Lindsley &
Zimm, 1987). All mutant traits are fully recessive and no
alteration has been found in salivary gland chromosomes of
either homozygous or heterozygous larvae. Mutant strains
were balanced over TM6 B,Hu e Tb ca, and all cultures
reared on standard medium at 29, 25 or 17°C.
Cytology
Homozygous mutant larvae or pharate adults were recognized by the red coloration of their Malpighian tubules, that
are yellowish in heterozygous individuals, and the absence of
the shortened body shape produced by the dominant mutation Tubby (Tb). Larval brains were stained with orcein
following Gatti el al. (1974), and testes following Lifshytz &
Hareven (1977). Live cells during spermatogenesis were
observed with phase-contrast optics after dissection of adult
testes in saline (Hardy et al. 1981).
To quantify mitotic phenotypes we have referred each type
of mitotic figure to a 'field', which is defined as the area seen
under the microscope with the following constants: Zeiss
Universal Microscope, plan-neofluar 63x/l-25 oil objective,
kpl-W lOx/18 ocular, Optovar in position 1-2SX. Under
these conditions an average wild-type brain yields some 200
fields. To study a larger population and thus minimize the
effect of quantitative variations among individuals, only 15
fields per brain were scored. Determination of the nuclear
size of onion stage-spermatids was performed by measuring
the diameter of nuclei in micrographs taken under standard
conditions with a 6CT2 Nikon Profile Projector.
Results
Mitotic defects
Homozygosity for mgr results in lethality late in
development, a majority of the mutant animals dying
just before emergence from the pupal case. Many of
these pharate adults have slight cuticular abnormalities
typical of viable mitotic mutants, such as rough eyes.
Since lethality is meiotically inseparable from the
mitotic abnormalities seen in larval brains, most probably the failure to emerge from the pupal case is due to
general muscular or neurological defects. Larval brains
homozygous for mgr show a variety of alterations both
in frequency and phenotype of mitotic figures (Figs 1,
2). As shown in Fig. 2 all these mutant traits are
stronger when larvae are grown at 17 °C than at 29 °C;
culture at 25 °C results in intermediate values (data not
presented). The most conspicuous mitotic abnormalities are: (1) an increase in the mitotic index (Fig. 2A),
accompanied by a reduction of the relative number of
anaphases (Fig. 2B). (2) 50-60% of the cells have Xshaped over-condensed chromosomes (Fig. 1C). This
40
C. Gonzalez et al.
phenotype is indicative of arrest during prometaphase
or metaphase; for simplicity, we will always refer to
these figures as metaphases. (3) Around 15 % of the
mitotic figures seen at 17 °C ( 3 % at 29°C) have
numbers of chromosomes exceeding the normal diploid
complement of eight (Fig. 2B). Roughly a quarter of
these cells seem to be true polyploids, the number of
chromosomes being an exact multiple of the haploid
complement; the remaining cells are aneuploid, with
intermediate numbers of chromosomes (Fig. IB).
Unless specifically stated, we will call both true polyploid and aneuploid cells 'polyploid'. (4) The most
conspicuous cellular phenotype is the appearance of
circular mitotic figures (CMFs; Fig. IE); in these
figures the major autosomes and sex chromosomes are
arranged in a circle with the centromeres pointing
towards the centre and the chromatids pointing
towards the periphery. The small fourth chromosomes
are always located in the centre. This regular configuration is somewhat disturbed when the circular figures
are polyploid (Fig. 1G). CMFs represent 25-35% of
the mitotic figures in mutant larvae while, even with
very permissive criteria, they never exceed 1 % in wild
type (Fig. 2D). (5) Occasionally, abnormal anaphases
are found in which at least one of the poles appears as a
circle of chromatids similar to that described for
CMFs; the majority of these anaphases are asymmetric, with a circular figure in one pole and a normal
anaphasic plate in the other. Fig. IF shows one of the
rare symmetric circular anaphases found in mutant
brains. Larval brains hemizygous for mgr (mgr/
Df(3R)TE61) show a more dramatic phenotype, indicative of the hypomorphic nature of the mutant allele and
of the locus-specific nature of the mutant phenotype.
The fraction of normal mitotic figures is decreased 10fold relative to homozygous mutant brains, the relative
numbers of CMFs and polyploid cells increase twice,
normal anaphases practically disappear, and CMFs are
more frequently polyploid.
Whereas many of the mitotic phenotypes described
above are common to other mitotic mutants (Gatti et
al. 1983; Ripoll et al. 1985, 1987), CMFs and asymmetric anaphases are typical of mgr. Figures somewhat
similar to CMFs have recently been found in individuals mutant for polo (Sunkel & Glover, 1988). When
homo- or hemizygous mgr larval brains are treated with
colchicine (a drug resulting in depolymerization of
microtubules), CMFs as well as asymmetric anaphases
are no longer found. Incubating mutant brains in taxol
(a microtubule-stabilizing drug) does not have any
effect on the frequency of CMFs. Nevertheless, this
treatment leads to a change in the typical phenotype of
CMFs: although the centromeres are still arranged in a
circle with the small fourth chromosomes in its centre,
the chromatids are no longer seen pointing towards the
periphery. The effects observed after treatment with
VH %
• £**
Fig. 1. Mitotic phenotypes found in wild-type and mgr larval brains. A. Wild-type (red) female metaphase; note the
arrangement of homologous chromosomes paired along their length as a result of somatic pairing during interphase.
B. Aneuploid metaphase (1QA"; 10V; 56 major autosomes). C. Tetraploid male metaphase with overcondensed
chromosomes; the arrow points to a chromosome-like big ugly piece of dirt (after Postlethwait, 1978). D. Wild-type {red)
anaphase. E. Circular mitotic figure; note the small dot-like fourth chromosomes in the centre, and the two chromatids per
chromosome. F. Mutant anaphase showing two circular poles. G. Polyploid CMF. Aceto-orcein stained squash
preparations; phase-contrast optics. Only in B was the brain cultured in colchicine and given a hypotonic shock. Bar, 4/tm.
both antimitotic drugs indicate that the typical CMF
structure requires microtubules for its maintenance.
Since microtubules are the major component of the
mitotic spindle, these results suggest that a functional
spindle is needed for CMFs to appear. There is no
spindle-specific drug to test this hypothesis, but we can
nevertheless substitute drug treatment for the genetic
interaction between mgr and mutations known specifically to alter spindles. We have constructed individuals
simultaneously mutant for mgr and abnormal spindle
(asp, 3-85-2), a mutation thought to alter the spindle
but not other microtubule-dependent structures
(Ripoll el al. 1985; Wandosell el al., unpublished
data). Brains homozygous only for asp show a variety
of mitotic phenotypes such as high mitotic index,
polyploid cells arrested at metaphase, and absence of
anaphases. Since the original mutant allele is coldsensitive, for our present analysis we have used aspLJ, a
strong mutant allele with smaller temperature-dependent phenotypic variations (Fig. 2). While individuals
homozygous for asphj die as mature pupae or soon after
emergence, and homozygous mgr as pharate adults,
double mutant individuals die as third-instar larvae.
These larvae lack imaginal discs and their brains are
reduced in size (60 % of the size of single mutant
brains, measured as amount of total protein). Since
imaginal discs and larval brains are among the few
tissues mitotically active during larval development,
this decrease in size suggests a high cell mortality
associated with mitotic defects.
Quantification of the mitotic phenotypes shown by
larval brains doubly homozygous for mgr and asp is
presented in Fig. 2. As happens with whole individuals, most cellular phenotypes suggest synergistic
interactions, between both mutations. The reduction
in the mitotic index, which is the result of an increased
Monopolar spindles in Drosophila
41
361
12
I
3
SO
*
«"l
4
0J
D
45
I 30
u
o
V
o
O.151
o
112
I
CO
a° J
Fig. 2. Quantification of mitotic figures in brains from
larvae grown at different temperatures. Genotypes: w, wild
type (red) (500 mitotic figures scored at 29 °C, 500 at
17°C); m, red mgr (893 and 418); a, red asp1^ (621 and
832); ma, red mgr aspliJ (378 and 448). In genotype m
only those CMFs that fulfilled all the characteristics
described in the text were recorded as such, while in the
rest of the genotypes any figure resembling a circle was
considered a CMF. Asymmetric anaphases are rare (around
5 % of the cells in mitosis in genotype m) and they have
not been included in these figures. Vertical bars,
percentage confidence interval (90% significance level).
cell mortality, indicates that both mutations lead to cell
lethality through independent mechanisms. A similar
interpretation is derived from the increase in the
relative number of polyploid cells, so that the cause of
the failure in chromatid segregation is different in each
mutant strain. Only the frequency of anaphases is
indicative of an epistatic interaction between both
mutations: anaphases are practically absent in the
double mutant, as is the case in asp individuals. The
same happens with CMFs, even using very loose
criteria to define a figure as a circle in doubly mutant
brains. Since the effect of asp is thought to be spindlespecific, the absence of CMFs in mgr asp brains shows
that a functional spindle is needed for these figures to
be formed and maintained.
Defects during spermatogenesis
Mitosis and meiosis are different types of cell division
sharing many steps and differing in others. The
common steps are likely to be under the control of the
same set of genes, and mutations altering both processes have been described (e.g. see Baker et al. 1978;
Ripoll et al. 1985). Mutant testes were examined to
ascertain if mgr was needed during spermatogenesis.
42
C. Gonzalez et al.
Observation of homo- and hemizygous testes under
phase-contrast optics reveals a series of defects. (1) Absence of recognizable meiotic spindles. In a sample of
30 testes no structure resembling a spindle could be
found, while between one and two cysts with clear
spindles per testis were observed in wild type. Therefore, meiotic spindles, if they exist, must be highly
abnormal. (2) The size of post-meiotic nuclei in early
spermatids (onion stage) is noticeably larger than in
wild type (Fig. 3A,B). At this stage the large majority
of cysts in which all nuclei could be counted had 16
nuclei instead of the normal 64. (3) The mitochondrial
derivatives (Nebenkern) are very abnormal: they lack
both their typical circular shape and uniform appearance, they are disaggregated, and they are hardly ever
found associated with the nuclei (Fig. 3B). Mitochondrial disorganization is more obvious in testes obtained
from pharate adults than from mature larvae. The
reason for this age-dependent phenotypic variation is
unclear, although it could be related to a decrease in the
ability of persisting maternal products to perform the
wild-type function partially. (4) Spermatids degenerate
during early stages of elongation. During this stage,
flagellar basal bodies, which are easily identifiable in
wild type, are either absent or unrecognizable in
mutant individuals. This phenotypic heterogeneity
does not necessarily mean that mgr is needed during
different stages of spermiogenesis, since defects in an
early stage can result in a cascade of effects producing
abnormalities later on (Hardy et al. 1981). All the
defects observed in mgr testes can be traced back to
abnormal behaviour of components of the meiotic
spindle. Failures in spermatid elongation could be due
to non-functionality of the mitochondrial aggregates;
the abnormalities observed in these aggregates could
result either from the absence or functional failure of
the spindle (the equitable partition of mitochondria
during both meiotic divisions is achieved through their
association with this structure) or from defective
elongation of the axoneme, a derivative of the centriole
(Tokuyasu, 1974).
Onion-stage spermatid cysts are formed by 16 large
nuclei, the same number with which meiosis is initiated
in wild type. Variations in the nuclear size of early
spermatids have been taken to indicate variations in
their chromosome content due to abnormal chromosome partition during meiosis (Lifschytz & Hareven,
1977; Hardy et al. 1981; Kemphues et al. 1982; Ripoll
et al. 1985; Fuller et al. 1987). We have found
(unpublished data) that during the onion stage there is
a geometric relationship between the amount of DNA
contained in a nucleus and its diameter, so that nuclear
volume is roughly proportional to chromosome content. A quantification of the amount of DNA contained
in mgr postmeiotic nuclei is shown in Fig. 3C. A total
of 96% of the nuclei have a chromosome content
Wild type
y
mgr
B
4A ' " -
r.
r*
e 8N
|4N
e
2N
^l
IN/2
SN/4-,
4-4
5-4
6-4
7-4
Nuclear diameter (pm)
8-4
9-4
Fig. 3. Onion stage spermatids in wild-type (A) and mgr
testes (B), and quantification of the DNA content in
mutant nuclei (C). A larval testis has been chosen for B, so
that the mitochondrial derivatives can still be easily
recognized. A,B. Live cells; phase-contrast optics;
spermatid nuclei are seen as white circles (arrows), and the
mitochondrial derivatives as black circles (arrowheads).
Bar, lOjum. In C, the frequency distribution of spermatid
nuclear diameters in red mgr testes (104 nuclei measured)
is compared with the variations in nuclear diameter as a
function of the chromosome content in C(2L),dp;
C(2R),px\ C(3L),h; C(3R) males (Gonzalez et al.,
unpublished data). N, chromosome complement equivalent
to a wild-type gamete.
around four times that of wild-type onion-stage nuclei,
while the remaining 4 % are similar to wild type.
The number and size of onion-stage nuclei in mutant
testes can be explained if spermatocytes can differentiate as spermatids without going through meiosis or,
alternatively, if both meiotic divisions take place in the
Fig. 4. Cytology of meiosis in red (wild type) and mutant
(mgr) males. A,B. Prometaphase I; C,D, metaphase I;
E, anaphase I; F, abnormal meiotic figure that we believe
to be a mutant anaphase I; in every case observed all 16
members of the cyst show the same configuration.
G,H. Metaphase II; note that in H the chromosomes are
not arranged in a plate, and that the number of
chromosomes is twice that of G. Aceto-orcein stained
squash preparations; phase-contrast optics. Bar, 10/um.
absence of karyo- and cytokinesis. Cytological observation of orcein-stained mutant testes reveals that both
meiotic divisions actually take place (Fig. 4), ruling out
the first possibility. The first meiotic division starts
normally in cysts of 16 primary spermatocytes, with a
euploid number of perfectly paired bivalents that end
up arranged in nearly normal plates during the first
metaphase (Fig. 4B,C). Disarranged metaphase II
figures are also found, with their chromosomes showing the typical configuration of this meiotic stage
(Fig. 4H); these metaphases differ from wild type
(Fig. 4G) in that they remain diploid, indicating that
the reductional segregation failed during anaphase I.
We have never observed normal anaphases in mutant
Monopolar spindles in Drosophila
43
testes. Instead, cysts where all 16 cells show the
phenotype presented in Fig. 4F are found: there is a
ring of heavily stained chromatin that, most probably,
is formed by all bivalents arranged in a circle. We
interpret these circular figures as mutant first meiotic
anaphases. Failure of the whole chromosome complement to segregate during the first meiotic division
explains the diploid configuration of second division
metaphases (Fig. 4H). If a similar failure in segregation also occurs during anaphase II, both the number
and the tetraploid size of postmeiotic nuclei are readily
explained. The same result could be obtained if anaphase 11 is simply absent instead of abnormal. Since we
have not found any mutant cyst showing what could be
considered as a second division anaphase, whether
anaphase II is abnormal or absent remains an open
question. In any case, the tetraploid size of the
spermatid nuclei correlates with the cytological observations and indicates that both meiotic divisions have
proceeded in the absence of karyo- and cytokinesis. A
similar phenotype is observed in mutants lacking /32tubulin, the testis-specific tubulin isotype, where spermatocytes progress through meiosis in the absence of
spindles (Kemphues et al. 1982).
Discussion
CMFs are caused by monopolar spindles
All the mutant traits observed during mitosis in mgr
cells can be traced back to structural or functional
defects in the mitotic apparatus: aneuploid cells can
result from abnormal chromatid segregation, true polyploid cells from total failure of chromatid segregation
followed by DNA replication, and arrested cells from
absence or lack of function of the spindle. Although
less obvious, the presence of CMFs could also be
attributable to defects in spindle components. The
study of mutant brains incubated with anti-mitotic
drugs, as well as the analysis of brains from mgr asp
doubly mutant larvae, has shown that CMFs need a
spindle to be formed and maintained. CMFs could
therefore be either metaphases or anaphases, the stages
of mitosis or meiosis that require a spindle for their
maintenance. While metaphase is a stage of dynamic
equilibrium dependent on forces exerted from opposite
poles, anaphase is characterized by the synchronous
movement of sister chromatids to the poles. Anaphase
can therefore be defined either by the separation of
sister chromatids or by the initiation of poleward
migration, two inseparable phenomena in Drosophila
wild-type divisions. CMFs are formed by chromosomes, not chromatids as in normal anaphases. Similar
chromosome behaviour is observed whenever functional monopolar spindles are formed, either spontaneously (Bajer, 1982), due to mutation (Wang et al.
44
C. Gonzalez et al.
1983), or after experimental manipulation (Mazia,
1960). If the separation of sister chromatids is taken as
the peculiarity defining a stage as an anaphase, then the
CMFs should be regarded as metaphases. However, if
synchronous migration of kinetochores is taken as the
characteristic that defines a stage as an anaphase,
several observations lead us to consider CMFs not as
aborted metaphases but as a peculiar type of anaphase
configuration. (1) With the techniques we use, we have
never found a figure identical to a typical CMF in
wild type, where, even with very permissive criteria,
figures resembling a circle are rarely seen (Fig. 2D).
(2) Mitotic figures identical to CMFs, formed by
chromatids instead of chromosomes, are seen in asymmetric anaphases, where the stage of the cycle is
unmistakable. (3) There is an evident correlation
between the decrease in the number of normal anaphases and the increase in the number of CMFs in mgr
brains as the culture temperature is decreased
(Fig. 2B,D). (4) The orientation of the chromosome
arms in these circular figures (Fig. IE) suggests that
the chromosomes are being moved towards the centre.
In fact, when taxol is added to the culture medium this
'dynamic' aspect is lost. (5) The central position
attained by the small fourth chromosomes correlates
with their position closer to the poles that is usually
found in normal anaphases (Kaufmann, 1934).
The configuration shown by CMFs can be explained
if mitosis is started with a single pole. The forces
moving the chromosomes towards this single pole
could end up placing the centrosome and the centromeres in the same plane, resulting in a circle with the
chromatids pointing outwards. In all respects these
figures are identical to the star configuration spontaneously occurring in functional monocentric anaphases in primary cultures of lung epithelium of the
newt Taricha granulosa (Bajer, 1982). Functional
monopolar spindles are also found in ts-745, a mutant
line of Syrian hamster ovary cells (Wang et al. 1983),
where the chromosomes are arranged around the single
pole in a spherical configuration like the one found in
some round cells in newts (Bajer, 1982). Treatmentinduced monopolar spindles have been elegantly shown
to be functional by in toto observation after inactivation
of centrosome replication with /3-mercaptoethanol
(Mazia, 1960), and after microtubule breakdown (see
Mazia (1961) for a review). After these treatments
disorganized chromosomes and chromatids reorganize
again following partial reconstruction of the mitotic
apparatus. The end result is that chromosomes or
chromatids arrange in circles around each pole, a figure
that has been called a 'quasirosette'. These poles are
functional since chromosomes or chromatids move
towards them. These quasirosettes are similar to the
CMFs and asymmetric anaphases found in mgr cells.
We have obtained equivalent results after submitting
wild-type Drosophila larval brains to low-temperature
pulses, and after addition and removal from the culture
medium of MTC, a reversible analogue of colchicine
(Fitzgerald, 1976). The figures obtained after these
treatments are identical to the asymmetric or symmetric circular anaphases found in mgr brains. The
high frequency with which CMFs are found in mutant
brains contributes to a great extent to the elevated
mitotic index they present. This abundance indicates
either that many cells arrest their cycle at the stage
characteristic of CMFs, or that the duration of this
monopolar stage is longer than that of normal anaphases in wild-type cells. We believe the second interpretation to be correct since the presence of polyploid cells, the orientation of chromosome arms
pointing outwards, and the absence of chromatin overcondensation, all suggest that the spindle in CMFs is
functional. A considerable increase in the time spent
during the cell cycle seems to be a characteristic
common to all cells with monopolar spindles (Bajer,
1982; W a n g l e / . 1983).
Monopolar anaphases can also explain the rings of
chromatin observed during mutant meiosis (Fig. 3F).
These rings would be formed by all bivalents migrating
to a single pole during the first meiotic anaphase; as a
consequence, the following metaphase would have
a diploid chromosome complement (Fig. 3H). If
second-division anaphases during mgr meiosis are also
monopolar (or if they never take place) the number of
postmeiotic nuclei per mutant cyst corresponds with
the one expected, since the establishment of bipolarity
is a prerequisite for cytokinesis (discussed by Mazia,
1961).
Is mgr a centrosome-i elated function?
The existence of monopolar spindles points to defects
in the centrosome cycle as the primary cause of the
appearance of CMFs. These defects could consist of
the absence of centrosome replication (as happens after
treatment with /3-mercaptoethanol) or of failure in the
segregation of centrosomes (as is the case of the
mutation in the SHO cell line or in the spontaneous
production of monocentric anaphases in the newt). In
theory, the complete lack of function of a gene essential
for either function (replication or segregation) should
result in the formation of a single giant cell with as
many chromosomes as the continuous cycle 'chromosome duplication-monopolar mitosis' could permit.
The difference between.this theoretical phenotype and
what is found in mutant individuals could be due, at
least in part, to the action of wild-type products left by
the maternal heterozygous genome in the oocyte:
mutant cells would divide normally until these maternal products are diluted out and/or degraded. If the
mutant mgr phenotype is only due to persisting wildtype products, all the cells found in mitosis should
show similar phenotypes, i.e. true polyploid metaphases and anaphases (CMFs). This is what happens
during meiosis in testes homo- or hemizygous for mgr,
where practically all the nuclei show the expected
phenotype. However, this is not the case in mutant
brains, where a variety of mitotic figures are found.
Although the phenotypes shown by larval brains
homo- and hemizygous for mgr deviate from the
phenotype expected for the complete lack of function of
the gene, the latter is closer to the amorphic phenotype.
The same happens with the homozygous brains grown
at low temperature. These observations clearly show
that the mutation is leaky (hypomorph), so that the
mutant allele is still capable of performing in part its
role during cell division. As a consequence, cells that in
a given cycle are unable to divide normally can behave
as either mutant or normal in subsequent cycles, and
vice versa. Owing to the combined action of persistence
of maternal products and hypomorphism, what we see
as the final mgr phenotype is only the addition of
defects progressively accumulating during brain development.
As discussed above, several phenotypes can be
explained by the complete absence of either centrosome
replication or separation: true polyploid cells, CMFs,
and number, size and chromosome complement of
spermatid nuclei. The abnormal shape of the mitochondrial derivatives in early spermatids (Fig. 3B) as
well as their lack of association with the adjacent nuclei
could be explained either as secondary effects of the
abnormal segregation of mitochondria during anaphase, or as due to failure in the development of the
axoneme, a derivative of the centriole. Other mutant
phenotypes can be explained as due to a delay in the
formation of a functional bipolar spindle, a phenomenon spontaneously occurring with relatively high
frequency in primary cultures of newt cells (Bajer,
1982). This late establishment of bipolarity could be
due to delayed replication, segregation or acquisition of
functionality of centrosomes, and it can explain the
existence of both asymmetric anaphases and aneuploid
cells. Starting from a typical CMF, the subsequent
appearance of bipolarity would result in the attachment
of a set of chromatids to the new functional pole. The
original pole would frequently keep its circular configuration, while the new pole would in most instances
form a normal anaphasic plate. Failure of some chromatids to attach to the second pole would explain
the presence of aneuploid cells. Finally, cells with
over-condensed chromosomes, typical of arrest at
metaphase, could result from absence of functional
centrosomes. A possible general explanation for this
abnormal centrosome behaviour could consist of a
diminution of its microtubule-nucleating ability, which
would affect both its segregation and the structure of
Monopolar spindles in Drosophila
45
the resulting spindle (Brinkley, 1985), as well as the
elongation of the axoneme (Tokuyasu, 1974).
Centrosomes have until now been resistant to
biochemical and genetic analyses, mgr provides a
promising starting point for the genetic analysis of
centrosomes in an organism where the power of genetics is known to everybody: the mutational analysis of
the locus will yield both new mutant alleles and, it may
be hoped, mutations in other loci related to the
centrosome that will interact in trans with mgr, similar
to the second site non-complementing mutations found
for B2t (Fuller, 1986).
We thank T. Fitzgerald and M. Suffness for their generous
gifts of MTC and taxol, respectively, and D. Mathog for
comments. We also thank C. Sunkel and D. Glover for their
interest and for sharing their unpublished results. Many of
the mutant strains used for this work were kindly provided by
the Drosophila Stock Centers in Oak Ridge and Bowling
Green. The work was supported by grants from Comision
Asesora para la Investigacion Cientifica y Tecnica, and an
institutional grant from Fondo de Investigaciones Sanitarias.
J.C. was supported by a fellowship from Plan de Formacion
del Personal Investigador.
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{Received 13 August 1987 - Accepted 8 September 1987)
Monopolar spindles in Drosophila
47