Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710
Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro-activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions.
Meiosis is a specialized cell division that results in
the formation of haploid gametes instead of diploid daughter cells, as in mitosis. Special features of meiosis that differ from mitosis include pairing of
homologous chromosomes, recombination between homologues, and the occurrence of two successive divisions
that reduce the chromosomes to the haploid number. Oocytes of most organisms are arrested at specific stages of
meiosis that differ depending on the organism and can be
activated by ovulation or fertilization to re-enter the cell
cycle and complete the meiotic divisions.
Insights into the regulation of meiosis are anticipated
to come from an understanding of the assembly, arrest,
and reactivation of the meiotic spindle. Several structural
differences exist between meiotic spindles of some oocytes
and mitotic spindles of most animal cells. A striking difference is the absence of centrosomes at spindle poles of
oocytes of several organisms that have been examined,
including Drosophila (Sonnenblick, 1950 Thin-section electron microscopy has demonstrated that
meiotic spindles of mouse oocytes lack centrosomes and
centrioles, but electron-dense foci of microtubules are
present at the broad poles of the oocyte spindles (Szollosi
et al., 1972 The discovery that several of the recently identified kinesin microtubule motor proteins are spindle-associated has
led to the hypotheses that the kinesin motors function to generate the forces required for spindle assembly and maintenance of spindle bipolarity (for review see Walczak and
Mitchison, 1996 Assembly of the meiosis I spindle in wild-type oocytes
and oocytes of the cand null mutant has been examined by
injection of rhodamine-conjugated tubulin to visualize spindle microtubules (Matthies et al., 1996 Despite the emerging information regarding anastral
spindle assembly in oocytes of Drosophila, the events of
meiosis that occur after release from arrest and during
progression through the meiotic divisions have not been
adequately described. The classical account by Sonnenblick (1950) To provide further information about meiotic spindle
dynamics and Ncd function in the spindle, we fused the
Ncd motor to the green fluorescent protein (GFP) of the
jellyfish, Aequorea victoria (Prasher et al., 1992 Drosophila Stocks
Drosophila mutants and balancer chromosomes used in this study are described in Lindsley and Zimm (1992) Sequence Analysis of ncd 2
The ncd 2 mutant allele was sequenced using as template DNA fragments
amplified by PCR from genomic DNA of ncd 2 adult Drosophila, and
cloned into pBluescript KS(+) (Stratagene, La Jolla, CA). The overlapping DNA fragments spanned the length of the ncd 2 coding region, starting upstream of the first in-frame AUG and continuing past the UAA
stop codon (Endow et al., 1990 Construction of ncd-gfp Plasmids
An ncd-gfp gene fusion was constructed in the P element vector, pCaSpeR3
(Pirrotta, 1988 A plasmid encoding wild-type Ncd fused to the S65 A plasmid containing ncd with the G446 Germline Transformation and Genetic Tests for Null
Mutant Rescue
For P element-mediated germline transformation, the pCaSpeR/ncd-gfp,
pCaSpeR/ncd-gfp*, and pCaSpeR/ncd 2-gfp* plasmids were injected into
embryos of w1118 females crossed to y w; Tests of transgenes for rescue of cand and tests of ncd 2 mutant effects
on embryo viability and X chromosome segregation were carried out as
described previously (Komma et al., 1991 Localization of Transgene Insertions
The cytological sites of transgene insertion were determined by in situ hybridization using a biotin-11-dUTP-labeled ncd cDNA probe, as described (Yamamoto et al., 1989 Visualization of GFP in Live Oocytes
Nonactivated live mature stage 14 oocytes were dissected from ovaries of
ncd-gfp, ncd-gfp*, or ncd 2-gfp* females immersed in a pool of light halocarbon oil (halocarbon oil 27; Sigma Chemical Co., St. Louis, MO) and
transferred to a drop of light halocarbon oil on a slide (Theurkauf, 1994 For confocal microscopy, slides were prepared and oocytes were located under white light before laser scanning. Time lapse confocal images
were collected through the oocyte chorion using a laser scanning confocal
detector (MRC 600; BioRad, Richmond, CA) mounted on a microscope
(Axiophot; Zeiss, Inc., Thornwood, NY) and equipped with a krypton/argon laser. The BioRad BHS or GR2 filter set (used with the T3 trichroic
filter block; BioRad) or a custom confocal GFP filter set (Chroma Technology Corp., Brattleboro, VT; Endow and Komma, 1996 For activated live oocytes, mature stage 14 oocytes were dissected from
ovaries of ncd-gfp, ncd-gfp*, or ncd 2-gfp* females into Drosophila PBS
(Robb, 1969 Analysis of Time Lapse Images
Stack files of time lapse images were opened, converted to single images,
and saved as PICT or TIFF files using the public domain program, NIH
Image v 1.59. Image contrast and size was adjusted as necessary using
Adobe Photoshop v 3.0.4. Measurements of spindle length were carried
out with NIH Image calibrated with a 10 µm bar superimposed on the images using COMOS (BioRad). The montages shown in Figs. 6 and 7 were
made using the Crop and Make Montage stack macros of NIH Image. Image stacks were animated using NIH Image and recorded to videotape using NIH Image or Adobe Premiere v 4.0. Methods for making videotape
recordings and QuickTime movies from GFP time lapse images are described in detail elsewhere (Endow and Piston, 1997
Indirect Immunofluorescence Labeling of Fixed Oocytes
and Embryos
Nonactivated fixed oocytes were prepared by dissecting ovaries from
wild-type (Oregon R), ncd-gfp*, or ncd 2-gfp* females, partially submerged in modified Robb's medium (Theurkauf and Hawley, 1992 Normally activated fixed eggs at various stages of the meiotic divisions
were obtained by collecting embryos at 12-15-min intervals from wildtype females or ncd 2-gfp* mutant females, followed by dechorionation,
removal of the vitelline membrane, and fixation in MeOH/EGTA without
taxol, as described previously (Hatsumi and Endow, 1992b Antibody-stained oocytes and embryos were mounted in anti-fade solution consisting of 90% glycerol plus 0.1 vol of 10 mg/ml p-phenylenediamine in PBS, pH 9 (Stearns et al., 1991 Laser Scanning Confocal and Fluorescence Microscopy
Laser scanning confocal microscopy of fixed oocytes and embryos stained
with rhodamine-conjugated anti- Confocal or CCD camera images taken at several planes of focus were
merged into single files using Adobe Photoshop v 3.0.4 to display the entire meiotic spindle or set of meiotic chromosomes in a single image. The
CCD camera DAPI images were adjusted to the same size as the confocal
spindle images by comparing confocal and CCD camera spindle images to
one another. The DAPI images were then superimposed onto the confocal spindle images so that the relative positions of the chromosomes with
respect to the spindles could be determined. These composite confocal
plus CCD camera images were almost identical to merged CCD camera
spindle and chromosome images, except that the confocal spindle images showed less out-of-focus detail.
ncd-gfp Transgenes
A plasmid encoding an Ncd-GFP fusion protein was constructed by fusing a wild-type ncd cDNA in-frame to wildtype gfp. The ncd-gfp gene fusion is regulated by the native ncd promoter (Yamamoto et al., 1989 Transformants with single insertions of ncd-gfp were recovered, and the ncd-gfp transgenes were transferred into
Drosophila carrying cand. The two ncd-gfp transgene insertions reported here, F24M1 and F24M3, were mapped
by in situ hybridization to 44B,C and 2A1,2, respectively,
on chromosome 2R and the tip of the X chromosome. The
cytological map positions were confirmed by recombination analysis of so and blo with F24M1 and y and scute
with F24M3.
Tests of function showed almost complete rescue of cand
for meiotic and mitotic chromosome segregation by two
copies of the ncd-gfp F24M1 transgene, resulting in a frequency of 0.004 X chromosome missegregation compared
to 0.294 for cand and <0.001 for wild type (Table I). Embryo viability (0.617) was partially rescued compared to
0.113 for cand and 0.944 for wild type. Two copies of the
F24M3 transgene partially rescued cand both for chromosome segregation and embryo viability (Table I). The incomplete rescue of cand by F24M1 and F24M3 indicates a
position effect on ncd-gfp expression or interference by
GFP with Ncd function. Drosophila with four copies of the
ncd-gfp transgene (two copies each of F24M1 and F24M3)
were also tested for rescue of cand. These females showed
no significant differences from wild type with respect to
chromosome segregation (0.002 missegregation compared with <0.001 for wild type; P = 0.06) or embryo viability
(0.933 compared with 0.944 for wild type; Table I.
Rescue of the cand Null Mutant by the ncd-gfp Transgene
), Xenopus (Gerhart, 1980
), and the mouse (Szollosi et al., 1972
). The absence of centrosomes raises the question of how microtubules are nucleated and organized for assembly of these
spindles.
). A centrosomal protein, pericentrin, has been
localized to multiple foci at the poles of the mouse oocyte
spindle (Doxsey et al., 1994
), consistent with the interpretation that the foci constitute dispersed microtubule organizing centers. Assembly of the meiotic spindle could occur from these dispersed centers in a manner similar to
that of a single center. In contrast, known centrosomal
components have not been identified in the Drosophila
meiosis I spindle and are thought to be absent. The centrosomal protein, DMAP190 (CP190), was not found at
the poles of the meiosis I spindle (Theurkauf and Hawley
1992
), but it has recently been reported to localize to a
ring- or disk-shaped structure between the two tandem
meiosis II spindles of Drosophila oocytes (Riparbelli and
Callaini, 1996
). The ring-shaped structure in the central
pole region of the Drosophila meiosis II spindles was observed previously (Puro, 1991
) and was suggested to function in pole organization for the second meiotic division.
Nucleation and organization of microtubules for assembly of the Drosophila meiosis I and II spindles could therefore
differ from one another and from anastral spindles of
other organisms.
). Drosophila Nonclaret disjunctional (Ncd;1
Yamamoto et al., 1989
; Endow et al., 1990
; McDonald and
Goldstein, 1990
) is a minus-end directed kinesin motor
protein (McDonald et al., 1990
; Walker et al., 1990
) that,
when mutant, causes the formation of abnormal meiotic
spindles in oocytes (Wald, 1936
; Kimble and Church, 1983
;
Hatsumi and Endow, 1992b
; Matthies et al., 1996
). The
wild-type Ncd motor protein has been proposed to function in establishing poles for assembly of bipolar meiosis I
spindles (Kimble and Church, 1983
; Hatsumi and Endow,
1992a
,b; Matthies et al., 1996
), replacing centrosomes in
the spindle. A role in establishing bipolarity during assembly of the meiosis I spindle is consistent with the effects of
null mutants on chromosome segregation (Sturtevant,
1929
; Lewis and Gencarella, 1952
), the abnormal spindles
that have been observed in oocytes of the claret nondisjunctional (cand) null mutant, and the association of the
Ncd motor with meiotic spindle fibers (Hatsumi and Endow, 1992a
; Matthies et al., 1996
).
). The results indicate that Ncd is required for normal kinetics of spindle assembly, as well as stabilization of the meiosis I spindle
after assembly. Spindles were followed in this study from nuclear envelope breakdown through meiosis I spindle assembly and arrest of the meiosis I spindle.
begins well into oocyte activation, with the
spindle perpendicular to the oocyte surface. Spindles in
mature arrested oocytes, however, are positioned parallel to the cortex (Theurkauf and Hawley, 1992
; White-Cooper et al., 1993
; Matthies et al., 1996
). This raises the questions of how the spindle becomes oriented vertical to the
cortex and when this occurs. There also exists a gap in our
knowledge of spindle dynamics during re-entry into the
meiotic cell cycle and completion of the meiotic divisions,
which have not been directly visualized because of their
rapid occurrence. The role of the Ncd motor during the
meiotic divisions is also not known, although both the meiosis I and II spindles have been reported to be abnormal in
ncd mutant oocytes (Wald, 1936
; Hatsumi and Endow,
1992b
).
; Chalfie et
al., 1994
). An initial report of Ncd-GFP in mitosis in early
embryos, including the rescue of an ncd null mutant by
one of the first ncd-gfp gene fusions, has been published
elsewhere (Endow and Komma, 1996
). Here we show that
Ncd-GFP is spindle-associated during meiosis in oocytes, like wild-type Ncd. The binding of Ncd-GFP to spindle
microtubules provides a highly effective method for visualizing the meiotic spindle in live oocytes, resulting in new
information regarding spindle dynamics during re-entry
into meiosis and completion of the meiotic divisions. A
mutant form of Ncd fused to GFP shows loss of function
but binds to oocyte meiotic spindles, permitting spindle
dynamics and the genesis of abnormal spindles in the presence of a loss-of-function Ncd motor to be observed. The
results provide evidence that the Ncd microtubule motor
is required for spindle elongation and, unexpectedly, stability of spindle fibers during the meiotic divisions. The
Ncd motor probably also functions in polar body formation after the meiotic divisions, which are needed to prevent continued spindle-associated divisions of the maternal chromosomes after the initial two divisions.
Materials and Methods
. The classical ncd mutant allele
(O'Tousa and Szauter, 1980
) is designated ncd 2 by Lindsley and Zimm
(1992)
. ncd 2 was obtained from J. Kennison (National Institutes of
Health, Bethesda, MD) in 1985 and has been maintained in our stock collection. For the present studies, chromosome 3 proximal to ebony (e,
3-70.7) in the ncd 2 stock was replaced by recombination, and the X chromosome and chromosome 2 were replaced using balancer chromosomes
to remove any modifiers that may have accumulated in the stock. The recloned e ncd 2 chromosome was used as a control in genetic tests of the
ncd 2-gfp* transgene.
) and included the two introns present in
the ncd coding region. Changes in the DNA sequence that resulted in
amino acid changes relative to wild-type Ncd were confirmed by sequence
analysis of both DNA strands of two independent PCR clones.
; Thummel et al., 1988
), by insertion into BglII plus EcoRIdigested plasmid of a 3.1-kb BamHI-EcoRI fragment containing the promoter and 5
region of ncd (Yamamoto et al., 1989
), and a 2.1-kb EcoRI
cDNA fragment encoding the remainder of Ncd (Endow et al., 1990
). The
XbaI site in the plasmid at the 5
end of the insertion was removed by digestion with NotI and StuI, followed by repair and religation. The plasmid
was then digested with BamHI and XbaI, and the 3
end of the ncd gene in the plasmid was replaced with a BamHI-AflII ncd fragment and an AflII-
XbaI gfp fragment. The ncd fragment was synthesized by PCR using an
ncd cDNA plasmid as template and the primers, 5
ACA ATG GAC
GGA GTG 3
and 5
TCA TCT TAA GGA AAT TGC CG 3
, followed by digestion with BamHI and AflII, and gel purification. The gfp fragment
was synthesized by PCR using a gfp-exu plasmid (Wang and Hazelrigg,
1994
) as template and the primers, 5
GTG CTT AAG ATG AGT AAA
GGA GAA 3
and 5
C CTT CTA GAA TTC TTT GTA TAG TTC 3
,
followed by digestion with AflII and XbaI, and gel purification. The ncd
PCR insertion in the recombinant plasmid and all but the last 50 bp of
the gfp insertion were sequenced to confirm the absence of PCR synthesis
errors. The plasmid, pCaSpeR/ncd-gfp, consists of a wild-type ncd cDNA
fused in-frame to wild-type gfp and encodes residues 1-700 of Ncd (Endow et al., 1990
) followed by residues 1-238 of GFP (Prasher et al., 1992
),
with a change of D699
L in Ncd. Transcription of ncd-gfp is under the
regulation of the ncd promoter.
T mutant GFP
(Heim et al., 1995
), denoted GFP*, was constructed by replacing the
AflII-XbaI gfp fragment in pCaSpeR/ncd-gfp with an AflII-XbaI PCR
fragment encoding the mutant gfp. The S65
T mutation in the pCaSpeR/
ncd-gfp* plasmid was confirmed by DNA sequence analysis.
R mutation of the classical
ncd 2 mutant allele fused to the S65
T mutant gfp, ncd 2-gfp*, was constructed by replacing the SphI-BamHI fragment of pCaSpeR/ncd-gfp*
with an SphI-BamHI fragment encoding the G446
R mutation. The
SphI-BamHI fragment of the new plasmid, pCaSpeR/ncd 2-gfp*, was sequenced to confirm the presence of the G446
R mutation and the absence of PCR synthesis mutations.
2-3 Sb/TM6 Ubx males. Transformant lines were established and made homozygous in cand null mutant
females for cytological analysis of live or fixed oocytes and embryos. Females from the lines were tested genetically for rescue of cand.
; Endow et al., 1994
). Parental
females were mated to tester +/BSYy+ or y2 wbf/BSY males. Regular offspring of these matings are + females and BS males. Meiotic nondisjunction of the X chromosome in oocytes results in BS (X/X/Y) females and X/0
males; meiotic loss of the X results in X/0 males. Mitotic loss of the X in
early embryos gives rise to gynandromorphs (X/X:X/0 mosaics). Minute
offspring, haploid for chromosome 4, were scored but are excluded from
calculations of total chromosome missegregation because of their highly
variable recovery (Lindsley and Zimm, 1992
). Calculations of meiotic
nondisjunction and chromosome loss were performed as described
(Komma et al., 1991
; Endow et al., 1994
). Statistical tests of significance
were carried out using standard
2 and Poisson distribution tests, assuming the null hypothesis that the offspring being compared were from the
same population (Komma et al., 1991
).
). The F24M1 and F24M3 ncd-gfp insertions were localized to 44B,C on 2R and 2A1,2 on the X chromosome,
respectively. Recombination analysis of sine oculis (so, 2-57.1, 43B1-2)
and bloated (blo, 2-58.5, 44F1-2 to 45E1-2) with F24M1, and yellow (y, 1-0.0, 1B1) and scute (sc, 1-0.0, 1B3) with F24M3 was carried out to confirm the
cytological map positions. The ncd-gfp* transgene insertions, M3M1 and
M9F1, were localized by in situ hybridization to 42A,B at the base of 2R
and 75C,D on 3L, respectively. The map positions were confirmed by recombination analysis of so and blo with M3M1 and blistery (by, 3-48.7, 85D11-E3) with M9M1.
).
Oocytes were positioned dorsal side up, and a coverslip fragment was
mounted over the oocytes onto two layers of double-stick tape placed on either side to form a channel. Initial observations were carried out using a
fluorescence microscope (Leitz Dialux 22; Leica, Inc., Rockleigh, NJ)
equipped with a standard FITC or custom GFP (HQ470/40 excitation,
Q495LP dichroic, HQ525/50BP emission; Chroma Technology Corp.) filter set.
) was used to
image GFP. The custom GFP filter set increased the GFP signal over that
collected with the BHS or GR2 sets by ~1.3 and ~1.8 times, respectively. The bandpass emission filter in the custom GFP and GR2 sets suppresses autofluorescence of the chorion and vitelline membrane when imaging GFP (Endow and Piston, 1997
), improving image quality. A 10% neutral density filter was used for imaging Ncd-GFP and a 1% filter for Ncd-
GFP*. The 488-nm line of the laser and an objective (63×/1.4 NA Planapochromat; Zeiss, Inc.) were used for image collection. Images were
collected into stacks of 60 at 16.5 s intervals using the time lapse feature of
COMOS software (BioRad) and 3 Kalman-averaged slow scans per image.
) and transferred to a drop of light halocarbon oil on a glass
slide. The chorion was partially removed under oil by pulling on the dorsal
appendages with fine-tipped watchmaker's forceps, and a coverslip fragment was mounted over the oocytes, as described above for nonactivated oocytes. In some cases the halocarbon oil was bubbled briefly with O2
before use. The meiotic spindles of activated live oocytes could be effectively visualized without removal of the chorion, although sharper images
were usually obtained by removing the chorion over the spindle. Laser
scanning confocal microscopy was carried out using the 488-nm line of the
laser, the GR2 or custom GFP filter block, and a neutral density filter that
transmitted 1 or 3% of the laser light. Images were collected into stacks at 16.5 or 23 s intervals with 3 or 5 Kalman-averaged slow scans for each
image.
). Video sequences of
the figures can be viewed at http://abacus.mc.duke.edu
Fig. 6.
Meiotic divisions in an activated live ncd 2-gfp* mutant oocyte. Images from a time lapse sequence of an activated live ncd 2-
gfp* mutant oocyte show, from left to right and top to bottom, the spindle consisting of closely apposed separate spindles (A and B), separation of the spindle poles (C, arrows), and the formation of a small focus of Ncd2-GFP* (C, arrowhead) that marks the position of the
central spindle poles. Movement of the new spindle poles away from the rest of the spindle (D-H) caused a short multipolar spindle to
form (H) due to the attachment of the microtubules to the original poles. Time in minutes and seconds is indicated on each frame. Bar, 10 µm.
[View Larger Version of this Image (77K GIF file)]
Fig. 7.
Release of spindle poles in an activated live ncd 2-gfp* mutant oocyte. Time lapse images of an activated live ncd 2-gfp* mutant oocyte show, from left to right and top to bottom, three spindles that were initially separated from one another moving in different
directions to become somewhat more widely separated (A-C), fragmentation of spindle fibers in the centers of the spindles (D-H), and
release of the spindle poles (G and H). Fluorescent particles are present in the cytoplasm and form a hazy network in the center of the
spindles (E-H). These are probably microtubule fragments bound to Ncd2-GFP*. Time in minutes and seconds is indicated on each
frame. Bar, 10 µm.
[View Larger Version of this Image (80K GIF file)]
) containing 4.7% (vol/vol) formaldehyde. Mature oocytes were teased from
the ovaries, fixed for a total of 1.5 min at room temperature in the formaldehyde/saline solution, and then transferred into PBS. The chorion and vitelline membranes were removed from the fixed oocytes manually using
fine-tipped watchmaker's forceps. Fixed oocytes were then incubated
overnight at room temperature in TBST (TBS plus 0.3% Triton X-100;
Williams et al., 1992
) before blocking in TBST plus 10% fetal calf serum for 1-2 h. Oocytes were incubated in rhodamine-conjugated anti-
-tubulin monoclonal antibody (a gift of W. Sullivan; University of California, Santa Cruz, CA), as described (Endow et al., 1994
). 5 µg/ml of DAPI
(Boehringer Mannheim Corp., Indianapolis, IN) was added to one of the
washes after antibody incubation to stain the chromosomes.
). Some collections of wild-type or ncd 2-gfp* eggs were dechorionated manually before
removal of the vitelline membrane rather than by treatment with Clorox,
to save time. Rehydration of embryos, antibody staining, and subsequent
washes were performed as previously described (Hatsumi and Endow,
1992b
), except that TBST (TBS plus 0.1% Triton X-100) was used
throughout instead of PBST (PBS plus 0.05% Triton X-100).
) for visualization of fluorescence.
-tubulin antibody was carried out using
a scanning confocal detector (MRC 600; BioRad) equipped with a krypton/argon laser and attached to a microscope (Axiophot; Zeiss, Inc.). A
63X/1.4 NA Planapochromat objective and COMOS software (BioRad) were
used to collect images of rhodamine-labeled spindles. Images of DAPIstained chromosomes were collected using a cooled CCD camera (PentaMax-1317-K; Princeton Instruments, Trenton, NJ) interfaced to a Power
Macintosh 8100/100 PC and mounted on a fluorescence microscope (Dialux 22; Leica Inc., Deerfield, IL) with an Hg light source. A 63X/1.4 NA
Planapochromat objective and IPLab Spectrum software (Signal Analytics Corp., Vienna, VA) were used for image collection.
Results
) and encodes
full-length Ncd (amino acids 1-700) followed by full-length
GFP (residues 701-938). Amino acid 699 of Ncd was changed
from aspartic acid to leucine during construction of the
gene fusion. The GFP is at the COOH terminus of Ncd,
adjacent to the motor domain, which contains ATP- and
microtubule-binding sites that are required for motor
function. Two introns that are present in the coding region
of wild-type ncd were deleted in the ncd-gfp construct. An
ncd cDNA transgene without the two introns was tested
previously and shown to fully rescue the chromosome missegregation of the ncd null mutant, cand, when present in
one copy (Komma, D.J., and S.A. Endow, unpublished observation) or two copies (Endow et al., 1994
). cand, originally isolated after X irradiation (Lewis and Gencarella, 1952
), contains a deletion of the ncd promoter and 5
end
of the ncd mRNA coding region and does not produce detectable ncd RNA transcripts (Yamamoto et al., 1989
).
Rescue of cand by the ncd cDNA transgene indicates that
the two introns present in the ncd gene are not required
for normal expression of ncd.
2 = 1.79, 1 degree of freedom, 0.5 > P > 0.1; Table I). The results demonstrate that the ncd-gfp F24M1 and F24M3 insertions together can rescue cand and replace the function of the wildtype Ncd microtubule motor protein.
Drosophila carrying the ncd-gfp* transgene, with wildtype gfp replaced by the S65 T mutant gfp (Heim et al.,
1995
), were also recovered. The ncd-gfp* transgenes,
M3M1 and M9F1, were localized by in situ hybridization
to 42A,B at the base of 2R and 75C,D on 3L, respectively.
The cytological map positions were confirmed by recombination analysis of so and blo with M3M1 and blistery with
M9F1. Tests of homozygous M3M1 or M9F1 showed partial rescue of cand, while four copies of ncd-gfp*, two each
of M3M1 and M9F1, showed essentially complete rescue
of cand both for chromosome segregation and embryo viability (Endow and Komma, 1996
).
Association of Ncd-GFP with Oocyte Meiotic Spindles
Initial observations using a cooled CCD camera or laser
scanning confocal microscopy to visualize GFP fluorescence in live ncd-gfp oocytes revealed green fluorescent
spindles, demonstrating that the ncd-gfp transgene is expressed and can be detected in live oocytes. The spindle
fluorescence indicated that the Ncd-GFP fusion protein is
associated with the oocyte meiotic spindle, as observed
previously for wild-type Ncd using antibody staining (Hatsumi and Endow, 1992a; Matthies et al., 1996
). Association of Ncd-GFP with meiotic spindles permits analysis of meiotic spindle dynamics in live oocytes of Drosophila.
Live ncd-gfp oocytes were prepared for observation by
dissection from ovaries either under halocarbon oil or into
Drosophila PBS (Robb, 1969), followed by mounting under oil. Oocytes dissected under oil were interpreted to be
nonactivated, by comparison with nonactivated fixed oocytes prepared by dissecting ovaries directly into fixative.
Oocytes dissected into Drosophila PBS showed dramatic changes in the spindle, including meiosis I spindle elongation and assembly of tandem meiosis II spindles. These oocytes are interpreted to be activated since the cytological
changes in the spindle are consistent with oocyte activation, as evidenced by analysis of normally activated fixed
eggs. These results are described below.
Meiotic Spindle Dynamics in Nonactivated Live Oocytes
Spindles of nonactivated live ncd-gfp oocytes, prepared by
dissection from ovaries of females under halocarbon oil,
could be observed through the chorion by laser scanning confocal microscopy. The tapered bipolar spindles, located
near the base of the dorsal appendages, were brightly fluorescent with Ncd-GFP (Fig. 1). The dark region in the
center of the fluorescent spindles was presumed to correspond to the chromosomes, which exclude Ncd-GFP. This was confirmed by antibody staining of nonactivated fixed
oocytes. Spindles of nonactivated live oocytes imaged
without removal of the chorion were relatively stable, remaining in the same position throughout the 15-30 min
periods of image collection with little or no net change in
length or orientation. The spindle shown in Fig. 1 was
from a cand oocyte with four copies of the ncd-gfp* transgene. Stable metaphase I meiotic spindles were also observed in nonactivated live oocytes with one copy of the
ncd-gfp* transgene or four copies of the ncd-gfp transgene.
Despite their relatively stable positions, dynamic changes in the spindles of nonactivated live oocytes were occurring that could be detected over time. These changes comprised small lengthwise extensions and contractions of the spindles and alternating clockwise and counter clockwise rotational movements around their long axis, as diagrammed in Table II. These slight movements of the spindle were observed in all of the nonactivated live oocytes that were imaged (n = 13) and were best analyzed by animating the time lapse images and determining differences in orientation and position of the spindles over time. The spindles of most nonactivated live oocytes showed both extensions/contractions and rotational movements, but no net change in length or position over time (Table II). Spindles of several oocytes showed more extensive movements: one spindle rotated two to three revolutions around its long axis during the first 4 min of image collection before assuming a more stable position, and one spindle rotated approximately a quarter turn over the 16.5-min observation time. Another spindle moved lengthwise a quarter of its length and then returned to its original position, and one spindle elongated ~75% of its length. The oocyte containing this last spindle may have been unintentionally activated by the continuous laser irradiation. With the exception of this oocyte, none of the nonactivated live oocytes with intact chorions showed changes in the spindle that resembled completion of meiosis I and progression into meiosis II. These oocytes were therefore interpreted as arrested in meiosis I.
Table II. Spindle Movement in Nonactivated Live Oocytes |
The spindles of nonactivated live oocytes were typically observed through the chorion. Removal of the chorion from the live oocytes resulted in changes that included elongation of the spindle followed by the formation of two tandem meiosis II spindles. These changes are consistent with oocyte activation and occurred even though the oocytes were dissected under oil and the chorion was removed under oil. These changes were observed in 3/3 oocytes that were prepared in this manner and observed. Perturbation of nonhydrated Drosophila oocytes can therefore cause oocyte activation, resulting in re-entry into meiosis and completion of the meiotic divisions.
Chromosomes associated with the meiotic spindles
could not be visualized in nonactivated live ncd-gfp oocytes in the absence of further perturbating treatments,
such as injection of fluorescent DNA dyes, that could cause
oocyte activation. Nonactivated fixed ncd-gfp and wildtype oocytes were therefore prepared and stained with
anti--tubulin antibody and DAPI for comparison with
spindle images from nonactivated live ncd-gfp oocytes and
determination of the chromosome configurations. Metaphase I-arrested spindles in nonactivated fixed wild-type
oocytes, merged with DAPI-stained chromosomes, are
shown in Fig. 2. The fluorescent spindles visualized by
tubulin antibody staining are similar in appearance to the Ncd-GFP fluorescent spindles. The highly condensed metaphase I chromosomes are present in the center of the spindle, in a position corresponding to the dark regions in the
spindles visualized with Ncd-GFP. The spindles stained
with anti-tubulin antibody also showed a dark region in
the center corresponding to the chromosomes. The small
dotlike chromosome 4 could be observed either associated with the remainder of the chromosomes and identified as a
small protrusion at the ends of the chromosome mass (Fig.
2 A, arrows), or separated from the rest of the chromosomes and positioned closer to the poles (Fig. 2 B, arrows).
The spindle and chromosome configurations of nonactivated fixed ncd-gfp oocytes, prepared using the same methods as nonactivated fixed wild-type oocytes and stained
with anti-tubulin antibody and DAPI, were similar in appearance to the spindles and chromosomes shown in Fig. 2.
Rotation of the Meiotic Spindle in Activated Live Oocytes
Oocytes were also prepared by dissection from ovaries of
ncd-gfp or ncd-gfp* females into Drosophila PBS (Robb,
1969). Mature oocytes, visibly swollen by the brief (<3
min) immersion in the undiluted saline solution, were
transferred to a drop of halocarbon oil on a slide, the
chorion was removed from the anterior of the oocyte, and
a coverslip was mounted over the oocytes. Time lapse images collected for periods of 15-50 min by laser scanning
confocal microscopy showed that the meiotic spindles of
live oocytes prepared by immersion in Drosophila PBS exist in a highly dynamic state. As an example, the spindle
shown in Fig. 3 was first observed parallel to the oocyte
surface (Fig. 3 A). The spindle elongated (Fig. 3 B), contracted, and then underwent an acute pivoting movement (Fig. 3 C) that reoriented the spindle from its initial position parallel to the oocyte surface into a position perpendicular to the oocyte cortex (Fig. 3 D). Fig. 3 D shows an
image looking down the long axis of the spindle formed by
the spindle poles, with the dark "holes" corresponding to
the chromosomes, based on fixed oocytes and embryos
stained for tubulin and DNA. The spindle remained in this
position for at least 9.2 min, rotating around its long axis.
Rotations of the spindle around its long axis, after pivoting vertical to the cortex, were either clockwise or counter clockwise, or rapidly alternating clockwise and counter clockwise rotations. Spindles of 11 oocytes were analyzed after pivoting. Six spindles showed only rapidly alternating rotations, three rotated clockwise accompanied by rapidly alternating rotations, one only rotated clockwise, and two rotated counter clockwise and also showed rapidly alternating rotations.
Spindles of oocytes prepared by immersion in Drosophila PBS could rotate around their long axis before or after pivoting vertical to the oocyte surface, and several spindles elongated or contracted, as shown in Fig. 3. Microtubules, either single fibers or bundles, could be seen in images in which the spindles were cross-sectioned, projecting out from the spindles. Comparison of successive images in the time lapse sequences gave the impression that rapid shortening and elongation of the spindle microtubules was occurring and that the spindle was in a highly dynamic state.
Meiosis I to II Progression in Activated Live Oocytes
Spindles of oocytes that were prepared by immersion in
Drosophila PBS could spontaneously complete the meiosis I and II divisions, permitting, for the first time, visualization of spindle dynamics during the transition from meiosis I to II. The meiosis I spindle shown in Fig. 4 A was
positioned obliquely to the oocyte surface and was viewed
from an angle that allowed imaging of the entire spindle.
The spindle extended into an elongated, tapered meiosis I
spindle (Fig. 4 B), measuring ~30 µm from pole to pole,
and then rapidly reassembled into two tandemly arrayed
meiosis II spindles (Fig. 4, C and D). Assembly of the tandem meiosis II spindles occurred by the formation of a
bright focus of Ncd-GFP in the center of the elongated
meiosis I spindle (Fig. 4 B, arrow), followed by the establishment of two central spindle poles. The tandem meiosis
II spindles were interpreted to assemble by rapid sliding of
the spindle microtubules against one another, based on the
movements of the spindle observed in the time lapse sequence. The transition into meiosis II occurred without
disassembly of the meiosis I spindle and involved reorganization of the spindle fibers into the two tandem meiosis
II spindles.
The time required to complete the meiotic divisions was
determined by analysis of oocytes for which a complete sequence of meiosis I and II had been recorded. Meiosis I
and II in time lapse sequences of three oocytes with four
copies of the ncd-gfp transgene each lasted ~2.5-5 min,
giving an estimate of ~5-10 min for completion of both divisions. In a previous study by other workers, eggs were
collected for 5 min and fixed after an additional 5 min for a
total development time of 5-10 min, and then stained and
analyzed (Riparbelli and Callaini, 1996). 37% of the eggs
were in meiosis I, 51% were in meiosis II, and 12% were in
mitosis. This distribution is consistent with our estimate of
~5-10 min for completion of both meiotic divisions.
To determine if the spindles observed in activated live
ncd-gfp oocytes resembled those of normally activated
eggs, wild-type embryos collected at 0 to 12-15-min intervals were fixed and stained with anti-tubulin antibody and
DAPI. The meiosis II spindles of normally activated fixed
eggs were typically perpendicular to the cortex and aligned
in tandem (Fig. 5). A faint ring-like central spindle pole
body consisting of foci of tubulin with radiating microtubules could be observed between the two meiosis II spindles (Fig. 5 A, arrow). Other spindles showed only diffuse
tubulin staining in the region between the two meiosis II
spindles (Fig. 5 B). The central pole body of meiosis II
spindles of normally activated fixed eggs was also stained
by anti-Ncd antibody (not shown).
A Loss-of-function ncd Mutant and ncd-gfp Transgene
ncd2 is a classical mutant of ncd that was originally isolated
after EMS mutagenesis. The mutant was shown to fail to
complement cand for chromosome missegregation (O'Tousa
and Szauter, 1980), but a large-scale genetic analysis of the
effects of ncd2 has not been reported previously. Tests of
ncd2 mutant females showed markedly reduced embryo viability and elevated chromosome nondisjunction and loss
compared to wild type (Table III). ncd 2 shows a slightly
more severe effect on embryo viability (0.098) than cand
(0.113), but there is no significant difference in total X
chromosome missegregation between ncd 2 and cand (
2 = 0.371, 1 degree of freedom, 0.5 < P < 0.9). Chromosome
missegregation is fully rescued in heterozygous ncd 2/+ females, but embryo viability (0.810 compared to 0.944 for wild type) is only partially rescued (Table III). ncd 2 therefore behaves like a loss-of-function mutant of ncd with respect to chromosome segregation and shows a small semidominant effect on embryo viability. The mutant ncd 2
gene is transcribed (Yamamoto et al., 1989
), and the mutant protein is spindle-associated (Hatsumi and Endow,
1992a
). The cytological effects of ncd 2 on meiotic spindles
were found previously to be the same as those of cand
(Hatsumi and Endow, 1992b
).
Table III. The ncd 2-gfp* Transgene Fails to Rescue the cand Null Mutant |
Sequence analysis of the coding region of ncd 2 revealed
four amino acid changes, G446 R, A566
S, G696
A,
and N697
S, compared to wild-type Ncd. The G446
R
missense mutation affects a glycine residue in the ATPbinding motif of Ncd, IFAYGQTGSGKTYTMDG, that is
highly conserved among the kinesin proteins. This amino acid change is the likely cause of the loss of function of
ncd 2. Of the other changes in ncd 2, A566
S is a conservative amino acid change, G696
A lies outide the motor domain, and N697
S is a polymorphism found in some presumed wild-type strains. No other nucleotide substitutions were found in ncd 2 that result in missense mutations, or
changes in the introns or intron/exon boundaries compared to ncd+.
The G446 R missense mutation, but not the other 3 amino acid changes in ncd 2, was introduced into an ncd-
gfp* plasmid containing the S65
T mutant gfp, and the
plasmid was injected into embryos for germline transformation. Transformants carrying the ncd 2-gfp* transgene
were recovered, and the transgenes were made homozygous in cand females. The ncd 2-gfp* transgene that was analyzed in this study is designated M4F1. The ncd 2-gfp*
cand females showed poor fertility, high egg inviability, and
elevated chromosome missegregation that paralleled the
effects of ncd 2 (Table III). The effects of the M4F1 ncd 2-
gfp* transgene on chromosome segregation were not significantly different from those of ncd 2 (
2 = 0.944, 1 d.f.,
0.5 < P < 0.9), although M4F1 showed a slightly more severe effect on embryo viability (0.085) than ncd 2 (0.098).
Females carrying one copy of M4F1 together with one ncd+ allele (M4F1; cand/+ females) showed complete rescue for chromosome segregation, but embryo viability
(0.886 compared with 0.944 for wild type) was only partially rescued (Table III). The M4F1 ncd 2-gfp* transgene
therefore closely parallels the ncd 2 mutant allele in its effects on chromosome segregation and embryo viability.
Meiotic Divisions in Loss-of-Function Mutant Oocytes
Green fluorescent spindles were not detected in nonactivated live ncd 2-gfp* oocytes prepared by dissection under
halocarbon oil and observed by laser scanning confocal
microscopy. Nonactivated fixed oocytes stained with -tubulin antibody and DAPI to visualize spindles and chromosomes contained unstained or faintly stained spindles, or
partially formed abnormal spindles (not shown). Fully formed meiosis I spindles were not observed in the nonactivated fixed mutant oocytes (n = 104), even though mature oocytes were selected for the cytological analysis. The
condensed meiotic chromosomes associated with the spindles also appeared to be at an earlier stage than chromosomes in nonactivated fixed wild-type mature oocytes. It is
therefore probable that spindle assembly and maturation of the ncd 2-gfp* mutant oocytes is delayed relative to wild
type.
Live ncd 2-gfp* oocytes, prepared by dissection into Drosophila PBS, contained green fluorescent spindles, providing evidence that the ncd 2-gfp* transgene is expressed and the Ncd 2-GFP* protein is associated with meiotic spindles. The basis of the ability to visualize spindles in activated live oocytes but not nonactivated live oocytes is not certain, but is likely to be due to changes in the oocyte that occur upon activation. The spindles in the live mutant oocytes were activated to complete the meiotic divisions, but the divisions were highly abnormal. In the time lapse series shown in Fig. 6, a spindle was initially observed that appeared to be formed of closely apposed spindles (A and B). The spindle was interpreted to be in meiosis I, based on the subsequent events in the time lapse sequence. Multiple poles arose by separation of the spindles at the poles (Fig. 6 C, arrows). The spindles failed to extend into the highly elongated meiosis I spindles typical of wild-type oocytes. A small focus of Ncd2-GFP* formed in part of the spindle (Fig. 6 C, arrowhead), marking the position of the meiosis II-like central spindle poles, but normal meiosis II spindles failed to form. Instead, a short multipolar spindle arose by movement of the newly formed central spindle poles away from the rest of the spindle, pulling on microtubules attached to the original poles (Fig. 6, D-H).
Although spindles in most of the activated live mutant oocytes failed to elongate into typical meiosis I spindles, spindles in 2 of the 23 mutant oocytes that were recorded did show elongation typical of wild-type meiosis I spindles. Elongation of spindles in these two oocytes was followed by the formation of two independent pairs of central spindle poles in the closely apposed spindles that comprised the spindle; movement of the new poles in opposite directions resulted in the formation of cruciform spindles.
Multiple spindles were present in some activated live mutant oocytes that arose by separation of meiotic spindles into several spindle-like components. As an example, the time lapse series in Fig. 7 shows three spindles that were separated from one another when initially observed. Movement of the spindles in different directions caused them to become somewhat more widely separated (Fig. 7, A-C). The spindle fibers in the centers of the spindles then began fragmenting (Fig. 7, D-H), releasing the poles from the spindles (G and H). Fluorescent particles, presumed to be microtubule fragments bound to Ncd2-GFP*, were observed in the cytoplasm and formed a hazy network in the center of the spindles (Fig. 7, E-H). The released poles of the spindles moved far from the original site of the spindles and could be observed in the oocyte after the time lapse imaging, separated from one another by as much as 75-80 µm.
Progression of the meiotic divisions was slow in the activated live mutant oocytes compared with wild-type oocytes, requiring 30 min or longer for completion of two divisions, and spindles in some mutant oocytes continued dividing after the initial two divisions without undergoing disassembly.
Spindles in normally activated fixed mutant eggs were
examined for comparison with spindles in activated live
mutant oocytes. Mutant eggs in various stages of the meiotic divisions were collected at 0-15 min intervals, fixed,
and stained with -tubulin antibody and DAPI to visualize
the meiotic spindles and chromosomes. Anaphase I and
metaphase II spindles in normally activated fixed ncd 2-
gfp* mutant eggs are shown in Fig. 8. Several of the normally activated fixed mutant eggs exhibited a haze of microtubules associated with the meiotic spindles (Fig. 8 A),
as observed in activated live mutant oocytes. Multiple
spindles were present, both in meiosis I and II, with each
of the separated chromosomes, including the small chromosome 4 (Fig. 8 A, arrows), associated with a spindle spur or spindle. The spindles were oriented obliquely or
vertically with respect to the embryo cortex. The arrow in
Fig. 8 B indicates a cross-sectioned spindle that is vertical,
associated with a spindle spur, indicating that the mutant
Ncd-GFP does not prevent reorientation of the spindles
perpendicular to the cortex. One or more of the spindleassociated meiotic chromosomes in some eggs was widely
separated from the others. For example, one of the 8 halfbivalent chromosomes was missing from the cluster of
spindle-associated metaphase II chromosomes in one egg.
The missing chromosome was found 85-90 µm from the
nearest chromosome in the cluster, associated with a small
spindle remnant.
Normally activated fixed early eggs of ncd 2-gfp* mutant females frequently contained multiple spindle-associated chromosomes that exceeded the 4N = 16 number expected upon completion of the two meiotic divisions. The spindles lacked centrosomes, like the oocyte meiotic spindles, and, in many eggs, were abnormal (either multipolar, branched, or spurred). The fixed mutant egg shown in Fig. 8 C contained 18 chromosomes or pairs of nondisjoined sister chromatids. Many of the chromosomes were separated from one another and associated with separate spindles, and some of the chromosomes showed anaphase configurations, as if they were in the process of dividing. One of these is indicated by an arrow. The spindles were located near the cortex of the egg, where the polar bodies are found in wild-type embryos. The egg was unfertilized, and no mitotic spindles were observed. The >4N chromosomes in this and other eggs probably arose by continued meiotic divisions of the maternal chromosomes. This interpretation is consistent with the more than two meiotic divisions that were observed in activated live mutant oocytes.
GFP as a Fluorescent Tag to Monitor Meiotic Spindle Dynamics
The meiotic divisions in oocytes of most organisms, including Drosophila, have not been directly visualized previously. Several steps after oocyte activation occur very rapidly and have not been observed even in rapid collections of eggs analyzed by fixation and antibody staining. These steps include the expansion/contraction of the spindle, spindle rotations, and the acute pivoting movement that reorients the spindle into a position perpendicular to the cortex before completion of the meiotic divisions.
Fusion of ncd to gfp and expression of the gene fusion in Drosophila under the control of the wild-type ncd promoter targets the fluorescent motor-GFP fusion protein to the meiotic spindle, permitting visualization of meiotic spindle dynamics in live oocytes. The Ncd-GFP fusion protein can replace the function of the wild-type Ncd motor protein, rescuing the cand null mutant both for chromosome segregation and embryo viability. Visualization of meiotic spindles using a microtubule-associated motor protein fused to GFP is a highly effective method for monitoring changes that occur in the oocyte spindle. The meiotic spindles can be visualized through the chorion of nonactivated or activated live oocytes, minimally perturbing the oocyte.
Meiosis I and II Spindle Dynamics in Wild-Type Oocytes
Spindles of nonactivated live oocytes were observed after
dissection under oil, conditions that prevent oocyte hydration and consequent activation (Theurkauf, 1994). Most of
the spindles were stable, showing only slight movements
or changes in position over periods of 15-30 min observation. The spindles appeared to be undergoing dynamic
changes, as evidenced by the detection of slight extensions/contractions in length and alternating clockwise and
counter clockwise rotational movements. The spindles of
nonactivated live oocytes were positioned parallel to the
oocyte cortex and could be observed as early as stage 13 (King et al., 1956
; Spradling, 1993
) in oocytes that were
still associated with degenerating nurse cells.
In contrast to the nonactivated live oocytes, live oocytes
that were observed after brief immersion in Drosophila
PBS contained spindles that were highly dynamic. The
spindles extended and contracted, rotated around their
long axis, and pivoted from their initial position parallel to
the oocyte cortex into a vertical position, where they continued to rotate. We interpret the highly dynamic state of
the meiotic spindle that we observe upon hydration of oocytes as representing the state of the spindle immediately
after activation. The dynamic changes in the meiotic spindle after oocyte activation are shown schematically in the
model in Fig. 9 and can be summarized as follows: (a) rotation of the spindle, (b) pivoting of the spindle vertical to
the cortex, followed by rotations, (c) completion of meiosis I, and (d) assembly of meiosis II spindles and completion of meiosis II. Several of these changes can also be detected or observed in normally activated fixed eggs.
Rotation and pivoting of the Drosophila meiotic spindle
have not been observed previously, although reorientation
of the meiosis I spindle has been inferred to occur based
on positional differences in spindles in metaphase-arrested
and activated fixed oocytes (Riparbelli and Callaini, 1996).
The rotations and acute pivoting movement of the spindle
represent two of the initial changes that occur in the spindle after oocyte activation. The ability of the spindle to rotate and reorient before completing the meiotic divisions explains why the spindle in nonactivated live (Fig. 1) and
fixed oocytes (Fig. 2; Theurkauf and Hawley, 1992
; WhiteCooper et al., 1993) can be observed parallel to the cortex,
while the meiotic divisions occur with the spindle perpendicular to the cortex (Sonnenblick, 1950
; Figs. 4 and 5).
Our observations indicate that the meiotic spindle reorients soon after oocyte activation by an acute pivoting movement, accompanied by spindle rotations.
Upon reorientation perpendicular to the oocyte cortex,
the spindles of in vitro-activated Drosophila oocytes frequently elongated and completed the meiosis I and then
the meiosis II division. The transition into meiosis II after
the completion of meiosis I, including assembly of the tandem meiosis II spindles, has not been observed previously,
although the second meiotic division is known to occur
with the two spindles tandemly aligned (Sonnenblick, 1950).
The observations reported here indicate that assembly of the meiosis II spindles occurs by the formation of new
spindle poles in the center of the anaphase I spindle, as anticipated by the work of others (Puro, 1991
; Riparbelli and
Callaini, 1996
). The newly formed spindle poles serve as
the central poles for the two meiosis II spindles. Assembly
of the tandem meiosis II spindles appears to involve reorganization of the meiosis I spindle fibers, possibly by rapid
sliding of the spindle microtubules, without disassembly of
the meiosis I spindle.
The mechanism by which the central meiosis II spindle
poles form is not known, although the ring- or disk-shaped
central spindle pole body that lies between the two meiosis
II spindles has been suggested to organize the poles (Puro,
1991). The recent observation that a centrosomal protein,
CP190 (DMAP190), localizes to the central spindle pole
body (Riparbelli and Callaini, 1996
) suggests that centrosomal proteins are involved. In contrast, centrosomal proteins such as
-tubulin, DMAP60, and DMAP190 have not
been found associated with meiosis I spindles of Drosophila oocytes (Theurkauf and Hawley, 1992
; Matthies et
al., 1996
). These observations raise the possibility that the
mechanism by which spindle poles are formed for meiosis
I and II spindle assembly in Drosophila oocytes differs,
and that assembly of meiosis II spindles requires centrosomal proteins that are found in mitotically dividing cells.
Rotation of Meiotic Spindles in Xenopus Oocytes
In addition to the present work with Drosophila, meiotic
spindle dynamics have been followed in activated live oocytes of Xenopus after injection of FITC-conjugated tubulin (Gard, 1992). The Xenopus meiosis I and II spindles assembled with the long axis parallel to the oocyte surface
and then rotated into a vertical position before undergoing
division. Reorientation of the meiotic spindle in Xenopus
oocytes was inhibited by treatment of oocytes with cytochalasin B during maturation (Gard et al., 1995
), indicating the requirement for an intact actin cytoskeleton. Based
on our present observations, meiosis I spindles of Drosophila and Xenopus oocytes undergo an analogous acute
pivoting rotation before division. Division with the spindle
perpendicular to the cortex positions one of the daughter
nuclei more internally and the other closer to the surface.
After completion of the second meiotic division, the innermost nucleus in Drosophila or the more internal nucleus in
Xenopus becomes the female pronucleus, and the nuclei
or nucleus near the cortex undergo polar body formation.
The basis for the initial assembly of spindles parallel to the
cortex is not certain, but may have to do with the anchoring
or attachment of the spindle to the cortical actin cytoskeleton.
Despite their overall similarity, some aspects of spindle dynamics in Xenopus and Drosophila oocytes differ. For example, Xenopus meiotic spindles were not reported to rotate around their long axis before the acute pivoting rotation that reorients the spindle perpendicular to the cortex. The rotations of the Drosophila oocyte spindles may cause the pivoting and, after reorientation, lead to spindle elongation and completion of the meiotic divisions. The spindle pivotings in Xenopus and Drosophila could therefore be similar in effect, but differ in basis.
Oocyte Activation in Drosophila
The initial report of in vitro activation of Drosophila oocytes showed that hydration in hypotonic (diluted) Robb's
medium (Robb, 1969) induces physiological changes in oocytes that are consistent with normal activation by ovulation (Mahowald et al., 1983
). Tests of undiluted Robb's
medium did not result in the ultrastructural changes typical of normally ovulated eggs that were also observed in
oocytes treated with hypotonic medium. These results
have led to the belief that hypotonic solution is required to
activate oocytes. Activation by the criterion of completion of the meiotic divisions was apparently not tested in oocytes treated with undiluted medium. The Robb's medium
used in this previous report was probably the chemically
defined medium (Robb, 1969
), which is similar in osmolarity and ionic composition to the Drosophila PBS developed at the same time (Robb, 1969
), which was used in the
present study.
Our observation that oocytes immersed briefly in undiluted Drosophila PBS undergo changes in the spindle that are consistent with activation, was therefore unexpected. The oocytes become visibly swollen upon immersion in the solution, resembling normally ovulated eggs, in contrast to nonactivated oocytes which appear wrinkled and shrunken. The oocytes treated with undiluted Drosophila PBS are activated by the criteria of re-entry into the meiotic cell cycle and completion of the meiotic divisions. Not all of the oocytes completed the meiotic divisions, however. Spindles in some oocytes reoriented perpendicular to the oocyte cortex and remained in this position for periods of 15-40-min observation, rotating around their long axis.
Perturbation of nonhydrated Drosophila oocytes can
also cause activation, since 3/3 oocytes that were dissected
under oil completed the meiotic divisions after their chorions were partially removed. Hydration of oocytes is therefore not required for activation. Activation of ncd-gfp oocytes using methods recently developed for efficient mass
activation of Drosophila oocytes (Page and Orr-Weaver,
1997) may prove of value in future studies.
Effects of Loss of Ncd Function on Oocyte Meiotic Divisions
The classical mutant allele, ncd 2, shows loss of function
based on its failure to rescue the cand null mutant. The basis of the loss of function of ncd 2 is a missense mutation in
a residue that forms part of the ATP-binding motif of Ncd:
an ncd 2-gfp* transgene containing the missense mutation
but not the other three amino acid changes of ncd 2 causes
mutant effects that parallel those of ncd 2. The G446 R
missense mutation of ncd 2 affects a glycine residue that is
highly conserved among the kinesin proteins and located
at the base of a loop, L5, on the surface of the Ncd motor
domain in the crystal structure (Sablin et al., 1996
). Together with two other surface loops, L5 forms the entry to
the nucleotide-binding pocket of Ncd. Replacement of
G446 with a positively charged arginine is likely to affect nucleotide binding or release from the motor, and alter the
ability of the motor to bind to or dissociate from microtubules. This is expected to impair the ability of the motor to
function in the spindle by causing defective movement on
microtubules and/or aberrant crosslinking activity.
The Ncd 2-GFP* fusion protein is associated with meiotic spindle fibers in ncd 2-gfp* mutant oocytes, as reported previously for Ncd2 (Hatsumi and Endow, 1992a),
but the meiotic spindles are abnormal. Previous reports
(Hatsumi and Endow, 1992b
; Matthies et al., 1996
) have focused on Ncd and the assembly and stability of the meiosis I spindle in Drosophila oocytes. Based on the observation
of multiple or multipolar spindles in mutant oocytes, the
Ncd motor has been proposed to crosslink and move on
the microtubules associated with the meiosis I chromosomes, forming a single bipolar spindle (Hatsumi and Endow, 1992a
,b; Matthies et al., 1996
). Analysis of live oocytes
injected with rhodamine-tubulin to visualize meiotic spindles indicates that the Ncd motor is required for maintenance, as well as assembly, of bipolar meiosis I spindles
(Matthies et al., 1996
).
The present study focuses on spindle dynamics after oocyte activation. Our observations demonstrate that multipolar spindles are formed by separation of spindle poles upon oocyte activation in the presence of a loss-of-function Ncd motor. Wild-type Ncd is therefore required to maintain spindle pole integrity during the meiotic divisions, probably by crosslinking and moving on microtubules, preventing sliding forces from disrupting the spindle and separating the microtubule-associated chromosomes from one another. Normal elongation of the meiosis I and II spindles was infrequently observed in ncd mutant oocytes, implying that the Ncd motor facilitates the microtubule sliding that is probably needed for spindle elongation and the rapid assembly of the meiosis II spindles in wild-type oocytes. Loss of Ncd function does not eliminate the ability of the central meiosis II spindle poles to form, but it is probably the impaired ability of the spindle fibers to slide against one another that causes failure of the meiosis II spindles to assemble, resulting instead in the formation of short tripolar or multipolar spindles.
Sliding of spindle microtubules against one another has
been proposed previously to contribute to poleward translocation, or flux, of microtubules in mitosis that may underlie poleward movement of chromosomes (Mitchison,
1989). Microtubule polymerization/depolymerization and
microtubule motors are both thought to produce forces
that result in poleward microtubule flux (Mitchison and
Salmon, 1992
). The observations reported here indicate
that microtubule sliding is an important aspect of meiotic
spindle dynamics as well as the dynamics of mitotic spindles,
and is probably facilitated by the Ncd microtubule motor.
In addition to defective spindle elongation and meiosis
II spindle assembly, the spindle microtubules appear to be
destabilized in ncd 2-gfp* mutant oocytes. Fragmentation
of spindle microtubules was observed in activated live
ncd 2-gfp* mutant oocytes, but not in activated live ncd-
gfp or ncd-gfp* oocytes. Fluorescent particles, probably
microtubule fragments bound to Ncd 2-GFP*, were associated with depolymerizing spindle fibers of ncd 2-gfp*,
forming a hazy network in the center of the meiotic spindles, where the more unstable microtubule plus ends are
expected to lie. Normally activated fixed ncd 2-gfp* mutant
eggs, stained with -tubulin antibody to visualize microtubules, also showed a hazy mass of tubulin-positive fragments in the central regions of the meiosis I or II spindles,
which was not observed in normally activated wild-type
eggs fixed and stained with
-tubulin antibody. These observations indicate that the mutant Ncd2-GFP* motor
causes destabilization of spindle fibers and imply that wildtype Ncd functions to stabilize spindle fibers during completion of the meiotic divisions. Destabilization of microtubules by Ncd2-GFP* could be due to altered ability of
the mutant motor to bind to and move on microtubules, or
to crosslink or bundle spindle fibers, as a consequence of
the mutational change in its nucleotide binding site. It is
also possible that Ncd2-GFP* exhibits gain-of-function effects, based on its small semidominant effect on embryo
viability. The implied role of Ncd in stabilizing microtubules therefore warrants substantiation by further evidence.
Dynamics of Spindle Pole Formation and the Ncd Microtubule Motor
A recent model for microtubule motor protein function in
spindle pole formation invokes the ability of the motor
both to crosslink microtubules and move along the crosslinked microtubules (Vernos and Karsenti, 1995). The proposed requirement for movement of the motor on the
crosslinked microtubules is consistent with the cytological
effects of the mutant Ncd2-GFP* motor. Ncd2 can bind to
spindle microtubules and could also bundle them by interactions with its highly charged tail region (Chandra et al., 1993
), but movement of the mutant motor on microtubules is probably impaired, as evidenced by its apparent
inability to mediate microtubule sliding needed for spindle
elongation. The proposed impaired ability of Ncd2-GFP*
to move on microtubules is correlated with the inability of
the motor to maintain spindle poles, thus movement of the
Ncd crosslinking activity along spindle microtubules is
likely to be required to maintain spindle bipolarity.
Loss of Ncd function in ncd 2-gfp* mutant oocytes
causes misregulation of the meiotic divisions, resulting in
continued divisions after the initial two divisions in in
vitro-activated live mutant oocytes. Maternal chromosomes are also associated with spindles instead of the normal polar bodies in normally activated fixed mutant eggs.
In wild-type embryos, the formation of polar bodies could
sequester the maternal chromosomes from cytoplasmic
factors, preventing further spindle-associated divisions.
Loss of regulation over the number of meiotic divisions
could be due to the inability of the mutant Ncd2 motor to
focus the meiotic chromosomes into polar bodies, which consist of chromosomes with centromeres oriented inward, surrounded by an array of short microtubules (Hatsumi and Endow, 1992b; Komma and Endow, 1997
; Page
and Orr-Weaver, 1997
). The polar bodies of Drosophila
resemble monopolar spindles and may undergo assembly in a manner similar to that of meiotic spindle poles. The
apparent requirement for Ncd to focus the microtubuleassociated chromosomes into polar bodies reinforces the
idea that both crosslinking activity and movement of the
crosslinking activity along microtubules are required to
maintain focused arrays of microtubules such as spindle poles and polar bodies. Spindle-associated maternal chromosomes are also observed in embryos of the cand null mutant (Hatsumi and Endow, 1992b
), evidence that the absence of Ncd, as well as the loss of Ncd function, results in
continued divisions of the oocyte chromosomes.
Several effects of the loss of Ncd function that are reported here were not detected in previous studies of antibody-stained fixed oocytes and embryos. These include failure of meiosis I spindle elongation, failure of meiosis II spindle assembly, and destabilization of spindle fibers. The ability to monitor meiotic spindle dynamics in live oocytes provides an important means of determining the effect of mutants on spindle assembly and dynamics. Further studies should lead to a complete picture of the role of microtubule motors like Ncd, and microtubule dynamics, in meiotic spindle assembly and function.
Received for publication 18 February 1997 and in revised form 8 April 1997.
1. Abbreviations used in this paper: blo, bloated; cand, claret nondisjunctional; GFP, green fluorescent protein; Ncd, Nonclaret disjunctional; so, sine oculis; y, yellow.This work is supported by grants from the National Institutes of Health and American Cancer Society to S.A. Endow.
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