RESEARCH ARTICLE
1875
Nuclear dispositions of subtelomeric and
pericentromeric chromosomal domains during
meiosis in asynaptic mutants of rye (Secale cereale L.)
Elena I. Mikhailova1, Svetlana P. Sosnikhina1, Galina A. Kirillova1, Oxana A. Tikholiz1, Victor G. Smirnov1,
R. Neil Jones2 and Glyn Jenkins2,*
1Dept of Genetics, Saint-Petersburg State University, Russia
2Institute of Biological Sciences, University of Wales Aberystwyth,
Penglais, Aberystwyth, Ceredigion, SY23 3DD, UK
*Author for correspondence (e-mail: [email protected])
Accepted 2 March 2001
Journal of Cell Science 114, 1875-1882 © The Company of Biologists Ltd
SUMMARY
The nuclear dispositions of subtelomeric and
pericentromeric domains in pollen mother cells (PMCs)
were tracked during meiosis in wildtype and two asynaptic
mutants of rye (Secale cereale L.) by means of fluorescence
in situ hybridization (FISH). Homozygotes for sy1 and sy9
non-allelic mutations form axial elements during leptotene
of male meiosis, but fail to form synaptonemal complexes.
Consequently, recombination is severely impaired, and
high univalency is observed at metaphase I. Simultaneous
FISH with pSc200 subtelomeric tandem repeat and CCS1
centromeric sequence revealed that at pre-meiotic
interphase the two domains are in a bipolar Rabl
orientation in both the PMCs and tapetal cells. At the onset
of meiotic prophase, the subtelomeric regions in PMCs
of wildtype and sy9 cluster into a typical bouquet
conformation. The timing of this event in rye is comparable
with that in wheat, and is earlier than that observed in
other organisms, such as maize, yeast and mammals. This
arrangement is retained until later in leptotene and
zygotene when the pericentromeric domains disperse and
the subtelomeric clusters fragment. The mutant phenotype
of sy9 manifests itself during leptotene to zygotene, when
the pericentromeric regions become distinctly more
distended than in wildtype, and largely fail to pair during
zygotene. This indicates that difference in the nature or
timing of chromosome condensation in this region is the
cause or consequence of asynapsis. By contrast, sy1 fails to
form comparable aggregates of subtelomeric regions at
leptotene in only half of the nuclei studied. Instead, two to
five aggregates are formed that fail to disperse at later
stages of meiotic prophase. In addition, the pericentromeric
regions disperse prematurely at leptotene and do not
associate in pairs at any subsequent stage. It is supposed
that the sy1 mutation could disrupt the nuclear disposition
of centromeres and telomeres at the end of pre-meiotic
interphase, which could cause, or contribute to, its
asynaptic phenotype.
INTRODUCTION
and facilitates regular segregation (Dernburg et al., 1996;
Karpen et al., 1996).
Because centromeres and telomeres are implicated in the
control of chromosome pairing, it is perhaps not surprising that
the nuclear dispositions of these domains have received
considerable attention, particularly at pre-meiotic interphase
and early prophase I when homologues may be making initial
contact. The ordered arrangement of these domains at premeiotic interphase into a typical bipolar Rabl orientation (Rabl,
1885) is apparent in some organisms, such as fission yeast
(Chikashige et al., 1997), budding yeast (Jin et al., 1998),
hexaploid bread wheat (Abranches et al., 1998) and other
members of the Triticeae (Martinez-Perez et al., 2000).
Furthermore, pre-meiotic association of homologues has been
observed in Saccharomyces cerevisiae (Weiner and Kleckner,
1994; Loidl et al., 1994) and wheat (Aragon-Alcaide et
al., 1997b; Schwarzacher, 1997; Mikhailova et al., 1998;
Martinez-Perez et al., 1999), which has been interpreted as an
important component of chromosome pairing. The dynamic
reorganization of telomeres into a tight cluster bound to the
Meiosis is a specialized cell division of sexually reproducing
eukaryotes, which halves the somatic chromosome number
during gametogenesis and thereby ensures that the
chromosome number of an organism does not double at
each generation. Integral parts of the process include the
recognition, pairing and synapsis of homologues, which in
most eukaryotes are pre-requisites for genetic recombination
and balanced segregation of half-bivalents at anaphase I. It is
clear from several studies that the integrity of these early
meiotic phenomena is largely invested in the behaviour and
interaction of two chromosomal domains, the centromeres
and telomeres, because perturbations of their nuclear
dispositions can dramatically impact upon the successful
completion of meiosis I (reviewed by Zickler and Kleckner,
1998; Walker and Hawley, 2000). The importance of
the centromeric domain is well illustrated in two previous
studies showing that, in achiasmate meiosis, centromeric
heterochromatin fulfils a role in the pairing of chromosomes
Key words: Meiosis, Rye, Asynapsis, FISH, Pericentromeric regions,
Subtelomeric regions.
1876
JOURNAL OF CELL SCIENCE 114 (10)
nuclear periphery has also received considerable attention
(reviewed by Zickler and Kleckner, 1998; Walker and Hawley,
2000). In maize (Bass et al., 1997), humans (Scherthan et al.,
1996), budding yeast (Trelles-Sticken et al., 1999) and other
species, the bouquet arrangement of telomeres occurs at the
transition between leptotene and zygotene and may be transient
or persist throughout zygotene (Scherthan et al., 1996). In
maize and humans, where there is no discernible pre-meiotic
association of homologues, the clustering of the telomeres of
chromosomes is considered to be the first step in the pairing of
homologues (Dernburg et al., 1995; Scherthan, 1997; Zickler
and Kleckner, 1998; Bass et al., 2000). However, it does not
necessarily guarantee subsequent regularity of meiosis,
because the bouquet forms in recombination-defective mutants
spo11 and Rad50s of yeast (Trelles-Sticken et al., 1999).
Hexaploid wheat also forms a bouquet, but this occurs earlier
at the onset of meiotic prophase and persists until the end of
zygotene (Martinez-Perez et al., 2000).
This investigation compares the nuclear disposition of
pericentromeric and subtelomeric domains at pre-meiotic
interphase and meiosis I in two asynaptic mutants of rye. It
constitutes part of an ongoing research programme to
characterize genetically and cytologically 21 existing,
spontaneous meiotic mutant stocks (Sosnikhina et al., 1994),
with the aims of understanding the genetic control of meiosis,
and of ultimately isolating genes responsible for specific
meiotic events in this organism. Microsporocytes of asynaptic
mutant sy1 form axial elements in the homozygote during
leptotene, but these do not assemble into synaptonemal
complexes (SCs), with the result that 99% of pollen mother
cells (PMCs) have only univalents at metaphase I (Sosnikhina
et al., 1992). Homozygotes for asynaptic mutation sy9 also
form axial cores but, in contrast to sy1, 33% of PMCs form
between 1 and 7 effective bivalents (Sosnikhina et al., 1998).
Mutations sy1 and sy9 are not allelic, and the latter is epistatic
(Sosnikhina et al., 1998). The more specific aim of this project
is to determine whether or not the differences in meiotic
phenotypes and epistatic relationship between the two mutants
could be interpreted in terms of differences in the disposition
and behaviour of pericentromeric and subtelomeric domains.
MATERIALS AND METHODS
Asynaptic mutant sy1 was originally isolated from an individual plant
of weedy rye (Sosnikhina et al., 1992) and is maintained in an
inbreeding population of diploid winter rye (Secale cereale L;
2n=2×=14). Asynaptic mutant sy9 was recovered from an individual
plant of the rye variety Vyatka (Sosnikhina et al., 1998) and is
propagated in the same way. Homozygotes for sy1 and sy9 were
isolated from segregating progenies of selfed heterozygotes and were
identified on the basis of their meiotic phenotypes. One plant
homozygous for sy1 and three for sy9 were used in this analysis. All
four plants had a characteristically high frequency of univalents at
metaphase I (Table 1), and other cytological abnormalities were
entirely consistent with published observations (Sosnikhina et al.,
1992; Sosnikhina et al., 1998). Three fertile plants from families
segregating for the mutants were used as wildtype (Sy1- or Sy9-)
controls. These had slightly elevated univalent frequencies compared
with those in rye taken from populations or varieties, and some minor
irregularities as a result of inbreeding (Table 1; Sosnikhina et al.,
1994).
Spikelets from meiotic inflorescences were numbered from the base
of the spike, fixed individually in a fresh 3:1 (v/v) mixture of ethanol
and acetic acid, and stored at −20°C.
Meiotic stage determination and delineation
The accurate determination of meiotic stage is crucial to the
comparisons made between the three phenotypes in this study.
Traditional staging methods based upon progress of synapsis are not
applicable in this case, because the pairing of chromosomes in
asynaptic mutants is compromised. Similarly, stage determination at
early meiosis based upon the relative clustering of centromeres and
telomeres is inappropriate, because the behaviour and nuclear
disposition of these domains are also affected in the mutants. Staging
of meiocytes was therefore accomplished by adhering to the following
criteria applied equally and without bias to each phenotype:
(1) Immature inflorescences (spikes) of rye have a developmental
gradient along their lengths, with younger spikelets towards the base
and more advanced stages in the middle. The intervals between
adjacent spikelets along the length of the spike are predictable, which
has particular use in selecting stages before leptotene. (2) Each
spikelet contains three anthers, which are approximately synchronous
in development. One of these was staged by standard squashing in 1%
aceto-carmine, and recorded for future reference by phase-contrast
microscopy under a Zeiss Axioplan microscope equipped with an
MC100 camera and black-and-white film. Pre-meiotic interphase is
characterized by two or more nucleoli centrally located in the nucleus
(Bennett et al., 1973), and the absence of DAPI-positive threads in
FISH preparations. The onset of meiotic prophase is typically
heralded by the aggregation of nucleoli into one large nucleolus,
which adopts a peripheral location in the nucleus. The nuclei contain
distinct threads, reflecting the progressive condensation of the
leptotene chromosomes. (3) Mononucleate tapetal cells stained with
aceto-carmine or DAPI indicate that pollen mother cells are at stages
before leptotene. Synchronous division of tapetal cells coincides with
transition between leptotene and zygotene, and binucleate tapetal cells
correspond with zygotene (Bennett et al., 1973; Bennett et al., 1979;
Martinez-Perez et al., 1999).
All of the anthers used in this study have been staged by applying
the above criteria, and only individual anthers were prepared for
FISH. However, because there is some variation in stage even within
the same anther, we have not rigidly assigned a stage to each cell
analyzed. Rather, we have defined meiotic stage intervals, which have
been applied to the three phenotypes.
Preparation of anther cells
Fixed anthers were prepared for FISH following procedures
Table 1. The mean frequencies and frequency distributions of univalents at metaphase I in PMCs of wildtype and the two
asynaptic mutants
Genotype
Sy1- or Sy9- (wildtype)
sy1 sy1
sy9 sy9
Number
of plants
Number
of cells
Mean univalent
frequency per cell
(x±mx)
0
2
4
6
8
10
12
14
3
1
3
200
100
400
0.8±0.09
14.0±0.03
10.3±0.14
69.0
−
−
24.5
−
1.3
6.5
−
3.5
−
−
7.2
−
−
20.0
−
−
26.7
−
2.0
17.8
−
98.0
23.5
Frequency (%) of cells with univalents
Tracking meiosis in asynaptic rye
1877
essentially as previously described (Schwarzacher and HeslopHarrison, 2000; Zhong et al., 1996), with some modifications (Jenkins
et al., 2000). Because the method was optimized to preserve
chromatin and to increase the resolution of FISH at early stages of
prophase I, the salient features of the method are recorded below.
Individual anthers were washed for 20 minutes in 10 mM citrate buffer
(pH 4.5) before digestion for 2 hours and 15 minutes in an enzyme
mixture comprising 0.5% (w/v) cellulase (Calbiochem), 0.65% (w/v)
cellulase (Onozuka RS), 10% (v/v) pectinase (Sigma), 0.15%
pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd) and 0.15%
cytohelicase (Sigma) in 20 mM citrate buffer (pH 4.5). After
digestion, the anthers were washed for 15 minutes in 10 mM citrate
buffer, followed by sterile distilled water (SDW), before maceration
with a fine needle and passage through a pipette tip in 20 µl of SDW.
The suspension of anther cells was spun briefly at 13,000 rpm, and
the pellet was washed in 10 µl of 45% acetic acid, spun down again
and resuspended in 12 µl of fresh 45% acetic acid. 4 µl aliquots of
the suspension were spotted onto clean slides and, for squash
preparations, a 20×20 mm coverslip lightly applied and excess fixative
removed. Following removal of the coverslips by freezing at −80°C,
the preparations were post-fixed in a few drops of fresh 3:1 methanolacetic acid. For spread preparations, the droplets of suspension were
surrounded by fresh, ice-cold 3:1 ethanol-acetic acid and the cells
allowed to precipitate onto the glass. The spreads were then washed
in fresh fixative. Both squash and spread preparations were prepared,
because the preservation of 3D organization was considered to be
better in the latter. However, it was found subsequently by optical
sectioning that nuclei prepared in either way had approximately the
same finite depths.
All preparations were dehydrated in 100% ethanol, air-dried and
stored at 4°C (short-term) or −20°C (long-term). Before FISH, slides
were pre-treated with RNAse A (10 µg/ml) in 2× SSC for 1 hour at
37°C, followed by washing in 2× SSC, refixing for 10 minutes at room
temperature in 1% formaldehyde in PBS (pH 7.0), further washing in
2× SSC, dehydration in an ethanol dilution series and air-drying.
DNA. The mixture was denatured at 70°C for 10 minutes, snapcooled on ice, pipetted onto the preparations of anther cells and
overlaid with plastic coverslips. The slides were sealed into the
humid chamber of an Omnislide thermal cycler (Hybaid), denatured
at 70°C for 5 minutes and incubated overnight at 37°C. They were
then washed stringently in 0.1× SSC at 50°C for 30 minutes and 2×
SSC for 15 minutes at room temperature. The preparations were
blocked with 5% (w/v) nonfat dry milk/4× SSC solution for 30
minutes at room temperature in the dark, before detection of the
digoxygenated pericentromeric sequence by incubation for 1-2
hours at 37°C with sheep anti-digoxygenin antibodies conjugated
to fluorescein. After three washes in 0.05% Tween-20 in 4× SSC,
the slides were dehydrated in an ethanol dilution series, air-dried
and mounted in Vectashield (Vector Laboratories) containing DAPI
(20 µg/ml) and propidium iodide (0.4 µg/ml). Fluorescent images
were visualized, processed and stored using either a Zeiss Axioplan
microscope and MC100 camera, or a Zeiss Axiovert microscope
coupled to a Bio-Rad MRC-1024 MPR confocal laser scanning
microscope. To be sure of visualizing all FISH signals, the confocal
micrographs shown are composites of optical section series through
individual nuclei.
DNA probes
pSc200 is a 521 bp insert in pUC18 comprising a 380 bp tandem
repeat unit of subtelomeric DNA from rye (Vershinin et al., 1995).
The sequence localizes to 18 major subtelomeric sites and four minor
sites in the chromosome complement of wild-type rye and the
asynaptic mutants. The insert was amplified and labelled by PCR
using M13 forward and reverse primers and FluoroRed (rhodamine4-dUTP) or FluoroLink Cy3-dCTP (Amersham Pharmacia). First, the
insert was amplified using the following conditions: 1 cycle of 93°C
for 5 minutes, 35 cycles of 94°C for 30 seconds, 55°C for 40 seconds,
72°C for 1 minute 30 seconds, and 1 cycle of 72°C for 5 minutes.
Then the insert itself was labelled as follows: 18 cycles of 94°C for
30 seconds, 55°C for 30 seconds, 72°C for 1 minute 30 seconds, and
1 cycle of 72°C for 5 minutes.
CCS1 is a 260 bp region (Aragon-Alcaide et al., 1996) of a
centromere-specific clone (Hi-10) originally isolated from
Brachypodium sylvaticum (Abbo et al., 1995). It is localized
exclusively to the pericentromeric regions of all rye chromosomes.
The sequence was amplified by PCR in the presence of digoxygenin11-dUTP (Roche) as described previously (Aragon-Alcaide et al.,
1996). Both probes were purified by precipitation under ethanol.
Patterns of aggregation
To compare more objectively the nuclear dispositions of
pericentromeric and subtelomeric domains in the three
phenotypes throughout meiotic prophase, the following
categories of aggregation were adopted:
(1) The telomeric/centromeric domains are considered
‘clustered’ if they comprise a single, amorphous and indivisible
mass closely adpressed to the nuclear periphery at one of the
poles of the nucleus. (2) ‘Multiple clusters’ describes 2-5
distinct aggregates in the same region of the nucleus. (3)
‘Grouped’ refers to largely or wholly separate telomeric/
centromeric domains that occupy a distinctly polarized region
of the nucleus but mainly unattached to the nuclear periphery.
(4) ‘Dispersed’ indicates that largely or wholly separate
telomeric/centromeric domains are distributed without obvious
pattern throughout the nucleus.
The relative frequencies of the four classes of subtelomeric
and two classes of pericentromeric distribution throughout
meiotic prophase of the three phenotypes are presented in Fig.
2. It is not considered likely that the patterns of distribution of
these domains are disturbed to any great extent by the
preparatory techniques, because it has already been shown that
clustering is essentially the same in spread cells compared with
those in which the 3D order has been preserved (TrellesSticken et al., 1999). Furthermore, in the unlikely event that
disturbances occur, it is probable that the three phenotypes
would be affected in the same way and comparisons would still
be valid.
Fluorescence in situ hybridization
FISH was performed according to an amalgam of procedures
(Leitch et al., 1994; Aragon-Alcaide et al., 1996; Fransz et al.,
1996; Zhong et al., 1996; Schwarzacher and Heslop-Harrison,
2000). For clarity, the salient features of the method adopted are
given below. The two probes were mixed to a final concentration
of 1-2 ng/µl in a hybridization solution containing 50% (v/v)
formamide, 2× SSC, 10% (w/v) sodium dextran sulphate, 50 mM
phosphate buffer (pH 7.0) and 125 ng/µl sonicated salmon sperm
Cell identification at the pre-meiotic interphase-leptotene
interval
Discrimination between meiocytes and tapetal cells in
heterogeneous cell populations at this interval was effected as
follows. Meiocytes from sister anthers have a characteristic
triangular form and granular chromatin when stained with acetocarmine (Fig. 1A). In FISH preparations stained with DAPI,
meiocytes have egg-shaped nuclei with evenly distributed
chromatin, whereas tapetal cells have more spherical nuclei with
polarized chromatin (Fig. 1B).
RESULTS
1878
JOURNAL OF CELL SCIENCE 114 (10)
Fig. 1. (A) Light micrograph of PMC nuclei of wildtype at the onset
of meiotic prophase, and stained with aceto-carmine. Note the
triangular shape of the cells. (B) DAPI-stained PMC nucleus (large
arrow) and tapetal cell nuclei (small arrows) at pre-meiotic
interphase of wildtype. Note that the two types of nuclei can be
easily distinguished on the basis of shape at this stage. The figure
corresponds to that in Fig. 3A. Bars, 5 µm.
contain dispersed sites (Fig. 3G). Pericentromeric clusters
become more relaxed and the pericentromeric regions
themselves become distended (Fig. 3G). A minority of these
regions are clearly associated in parallel pairs in some nuclei,
which is possibly indicative of presynaptic alignment (not
shown). The phenotype of sy9 at this stage bears some similarity
to in that the highest proportion of nuclei has multiple clusters
of subtelomeric regions. In sy1 the majority of nuclei have
multiple clusters of subtelomeric regions. The pericentromeric
sites are loosely grouped at the opposite pole, and comprise
pairs of fuzzy dots or short distended regions (Fig. 3I). There
is no obvious alignment of pairs of pericentromeric regions as
Pre-meiotic interphase-leptotene
At pre-meiotic interphase in wildtype and both asynaptic
mutants, the pericentromeric and subtelomeric domains adopt a
typical Rabl orientation, which is consistent with similar
observations of other species of the
Triticeae (Abranches et al., 1998;
Aragon-Alcaide et al., 1997a; A
Martinez-Perez et al., 1999; Martinez%
Perez et al., 2000). In all three
phenotypes
the
pericentromeric
regions are clustered at one pole of the
Subtelomeric
100
nucleus and the subtelomeric regions
Patterns of
grouped at the opposite pole (Fig. 3AAggregation
C). This distinctive pattern of
Cluster
distribution of these domains is clearly
50
visible in tapetal nuclei also (Fig. 3AMultiple clusters
C). At the onset of meiotic prophase,
Grouped
marking the transition between preDispersed
meiotic interphase and leptotene, the
0
subtelomeric but not pericentromeric
Phenotype/
WT sy9 sy1
WT sy9 sy1
WT sy9 sy1
WT sy9 sy1
5
15
12
14
10
28
22
41
16
15
16
7
regions
undergo
considerable
No. of nuclei
reorganization in wildtype and sy9 to
PMI-L
L-Zg
Zg-Pa
Di
Stage
form a tight, polar cluster in all or
most nuclei (Fig. 3D,E). By contrast,
sy1 fails to form a bouquet in half of B
its nuclei. Instead, the subtelomeric
%
DNA forms multiple clusters (Fig.
3F), and in 71.4% of the nuclei studied
the pericentromeric regions are
dispersed or interspersed between the
100
subtelomeric aggregates (Fig. 2).
Pericentromeric
Thus, the sy1 mutation exhibits
Patterns of
incomplete
penetrance
in
Aggregation
50
homozygous condition, since four out
of 14 PMC nuclei have aggregations
Clusters
of pericentromeric/subtelomeric DNA
Dispersed/distended
comparable with wildtype (Fig. 3F).
0
Phenotype/
In all three phenotypes tapetal nuclei
WT sy9 sy1
WT
sy9 sy1
WT sy9 sy1
15
12
14
10
28
22
41
16
15
No. of nuclei
retain the Rabl orientation of these
domains.
PMI-L
L-Zg
Zg-Pa
Stage
Leptotene-zygotene
Fig. 2. The percentages of PMC nuclei of the three phenotypes with (A) the four patterns of
As leptotene progresses, subtelomeric subtelomeric aggregation and (B) two patterns of pericentromeric aggregation during the intervals
clusters dissipate in to a point at pre-meiotic interphase-leptotene (PMI-L), leptotene-zygotene (L-Zg), zygotene-pachytene (Zgwhich a large proportion of nuclei Pa), and diplotene (Di).
Tracking meiosis in asynaptic rye
in wildtype, although this may be attributable to the small
sample size of nuclei taken. In some cells, 14 discrete signals
reflect the asynaptic condition of this mutant.
Zygotene-pachytene
During zygotene the tight aggregation of subtelomeric DNA
lapses in wildtype (Fig. 4A) so that, by pachytene, broken
aggregates are dispersed seemingly randomly throughout the
nuclei (Fig. 4D). At the latter stage, the centromere regions
have a distinctive tripartite structure comprising two condensed
pericentromeric subdomains flanking the centromere proper
(Fig. 4D). Mutant sy9 has 50% of nuclei with dispersed
subtelomeric regions (Fig. 2), as in (41.5%), but retains a
sizeable proportion of subtelomeric regions (31.2%), which
form multiple clusters at zygotene (Fig. 4B). Fourteen
pericentromeric regions can now be easily counted and reveal
the same tripartite structure as wildtype (Fig. 4B). However, at
a later stage they appear more distended and less discrete (Fig.
4E) than wildtype (Fig. 4D). A large majority
(73.3%) of nuclei of sy1 still have multiple
clusters of subtelomeric regions at this interval
(Fig. 4C,F) with no nuclei showing dispersal of
these domains as in wildtype and sy9 (Fig.
4D,E). It appears that the resolution of the
bouquet is hampered, with the possible
consequence that multiple clusters of
subtelomeric regions persist throughout this
period. Pericentromeric regions are fuzzy and
differ in the degree of distension within the
complement (Fig. 4F).
1879
has enabled the tracking before and during meiosis of two
important domains implicated in the control of chromosome
pairing, synapsis and recombination. The results presented
above are summarized in Fig. 6, which compares the essential
behaviour of the two domains in the three phenotypes. Fig. 6
embodies a large number of observations and represents by
necessity the average picture of nuclear organization at the
various sub-stages. At pre-meiotic interphase the two domains
adopt a typical Rabl configuration, in which the bipolar
distribution of centromeres and telomeres reflect the
orientation of chromosomes in the previous mitotic anaphase.
The beginning of meiosis, however, heralds the tight clustering
of telomeres, which become adpressed to the nuclear periphery.
The formation of a characteristic bouquet is specific to pollen
mother cells and occurs earlier in rye than in other organisms
(Zickler and Kleckner, 1998). Indeed, in a diversity of
organisms such as maize (Bass et al., 1997; Bass et al., 2000),
yeast (Trelles-Sticken et al., 1999) and mammals (Scherthan et
Diplotene-diakinesis
During this period of meiosis, most of the
subtelomeric regions of wild-type chromosomes
are dispersed throughout the nucleus.
Progressive contraction of the seven bivalents
fuses the three pericentromeric subdomains into
one by the end of diakinesis. All of the nuclei
of sy9 have dispersed subtelomeric regions at
this stage (Fig. 4G). Mutant sy1 still has
multiple clusters of subtelomeric regions, but
some are evidently dispersing by this stage.
Fourteen pericentromeric regions of sy9 and sy1
can now be easily counted, representing the
characteristic number of univalents at this stage
of meiosis. The morphology and contraction
status of the pericentromeric DNA of the 14
univalents are indistinguishable from wildtype
at this stage (Fig. 4H,I).
Metaphase I
As expected, wildtype regularly forms seven
bivalents at metaphase I, each of which
displays
discrete
pericentromeric
and
subtelomeric signals (Fig. 5).
DISCUSSION
The fluorescent labelling of pericentromeric
and subtelomeric regions of rye chromosomes
Fig. 3. Confocal micrographs of anther nuclei in wildtype (A,D,G), sy9 (B,E,H) and sy1
(C,F,I). The PMCs (large arrows) are at pre-meiotic interphase (A,B,C), the transition
between pre-meiotic interphase and leptotene (D,E,F) and at leptotene (G,H,I). Tapetal
nuclei are indicated by small arrows. Pericentromeric sequences are green and
subtelomeric repeats are red. Note the same Rabl orientation of domains at pre-meiotic
interphase in both meiocytes and tapetal cells of all three phenotypes. A full bouquet
fails to form at the onset of meiotic prophase in most PMCs of sy1 (F). Bars, 10 µm.
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JOURNAL OF CELL SCIENCE 114 (10)
Fig. 4. Confocal micrographs of PMC nuclei in
(A,D,G), sy9 (B,E,H) and sy1 (C,F,I). The PMCs are
at zygotene (A,B,C), pachytene (D,E,F) and at
diakinesis (G,H,I). Pericentromeric sequences are
green and subtelomeric repeats are red. Note the
tripartite structure (arrowheads) of the seven paired
pericentromeric regions in wildtype at pachytene,
contrasting with the aberrations of the two mutants.
The arrow in (I) depicts a tapetal cell nucleus of sy1
in which the Rabl orientation of the two domains is
still apparent and unaffected by the asynapsis of the
chromosomes of the PMCs. Bars, 10 µm.
al., 1996) this major reorganization of
telomeres occurs at the transition between
leptotene and zygotene and is coincident with
the onset of synapsis. The timing of bouquet
formation is similar to allohexaploid bread
wheat (Moore, 1998), with the one notable
difference that centromeres are not associated
in pairs at this stage in rye (Aragon-Alcaide,
1997b; Martinez-Perez et al., 1999; MartinezPerez et al., 2000). Clearly, the formation of the
bouquet at the transition between premeiotic
interphase and leptotene is not dependent upon
prior association of homologous chromosomal
domains. The association of pairs of
pericentromeric regions in this material
becomes evident later as these domains prealign or undergo synapsis.
Asynaptic mutant sy1 differs significantly
from wildtype in that, at the start of meiotic
prophase, subtelomeric DNA fails to fully
cluster and the pericentromeric regions disperse prematurely.
If, as has been suggested (Zickler and Kleckner, 1998), the
clustering of the telomeres and the centromeres is an integral
component of homologue recognition, alignment and
recombination, their aberrant behaviour at this stage could be
directly responsible for the synaptic phenotype of this mutant.
However, it has been shown that homologues in yeast will
synapse despite the failure of bouquet formation, indicating
that the bouquet is not absolutely necessary for meiotic
progression in this organism (Trelles-Sticken et al., 2000). It
could, of course, be possible that the failure to form a bouquet
is a consequence of a lesion in a different meiosis-specific
process. For example, the tardy resolution of subtelomeric
clusters in sy1 could be symptomatic of the paucity of
recombination intermediates, analogous to spo11 (Cao et al.,
1990) and Rad50s (Weiner and Kleckner, 1994) of budding
yeast. Differences between the nuclear disposition of these
domains in wildtype and sy1 could reflect differences in the
Wildtype
sy9
sy1
Fig. 5. Confocal micrograph of
metaphase I of wildtype
indicating the predictable
regularity of formation of
bivalents and chiasmata. Bar,
10 µm.
Premeiotic
Interphase
Leptotene
Zygotene
Pachytene
Diplotene
Fig. 6. The trends in behaviour of the subtelomeric regions (grey
circles) and pericentromeric regions (black circles) during premeiotic
interphase and prophase I of wildtype and the two mutants. For
clarity, only six regions of each are shown, and the change in the
morphology of pericentromeric regions is not illustrated.
Tracking meiosis in asynaptic rye
status of meiotic chromatin organization, although the
differences are manifested earlier compared with the chromatin
restructuring observed at the leptotene/zygotene transition in
maize (Dawe et al., 1994).
Asynaptic mutant sy9 bears closer similarity to wildtype in
terms of its timing of formation and resolution of the bouquet.
The presence of subtelomeric clusters during early prophase I
is inconsistent with previous observations under the electron
microscope of axial elements of surface-spread meiocytes, in
which no clusters of telomeres were detected (Sosnikhina et
al., 1998). A possible explanation for this discrepancy is that
telomeres are notoriously difficult to discern at leptotene
in these preparations and may have been overlooked.
Alternatively, the bouquet may have been at an advanced stage
of resolution at the meiotic prophase stages observed. Indeed,
in the one microsporocyte nucleus illustrated (Sosnikhina et al.,
1998) the telomeres form two distinct aggregates, which would
be interpreted as a multiple cluster in the present work. The
nuclear disposition of pericentromeric regions of sy9 at premeiotic interphase is similar to wildtype. However, at the onset
of meiotic prophase the pericentromeric regions become
distended and decondensed. It is not known whether this
change in morphology compromises the integrity of meiosis
from this point onwards, or whether it is a manifestation of
failure of another process. It has been speculated that the
abnormally diffuse centromeres in the ph1 mutant of wheat
could be responsible for altering the way in which homologues
interact during meiotic prophase (Aragon-Alcaide et al.,
1997b). Subsequently, pairs of centromeres seldom associate,
no synaptonemal complexes are formed and the majority of
homologues fail to form chiasmata.
We thank Trude Schwarzacher and Graham Moore (John Innes
Centre, Norwich) for supplying the subtelomeric and pericentromeric
clones, and Steve Taylor for his expert technical assistance with the
confocal microscopy. E. I. M. would like to thank J. Hans de Jong and
J. Wennekes van Eden for extensive training in FISH during
collaboration on an INTAS project in Wageningen. We acknowledge
with gratitude the receipt of a Royal Society ex-Agreement award for
E. I. M. to visit Aberystwyth, and support from the Russian
Foundation for Basic Research (grants 00-04-48522 and 99-0448182). The initial contact between the two laboratories was
sponsored by the Federation of European Genetical Societies.
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