|
|
|
|
|
|
|
||
* European Molecular Biology Laboratory, D-69117 Heidelberg, Germany; Max-Planck-Institut für Biochemie, D-82152
Martinsried, Germany; and § Max-Planck-Institute for Cell Biology and Genetics, D-01307 Dresden, Germany
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.
Key words: genetics; video microscopy; mitosis; centrosomes; microtubules| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
PROPER cell division requires the faithful distribution of chromosomes and cytoplasmic material to daughter cells. In eukaryotes, this is achieved by a series of coordinated cytoskeletal processes which include spindle assembly, chromosome segregation, spindle positioning and cytokinesis. Although the mechanisms governing such processes have been investigated in a number of systems, they remain incompletely understood, and there persists a need for identifying novel participating molecules, especially in complex eukaryotes.
Mutational analysis has played an important role in
achieving our current understanding of cell division processes, owing in particular to extensive genetic screens
conducted in yeast and other simple eukaryotes (Hartwell
et al., 1974
; Morris, 1975; Nurse et al., 1976; Hoyt et al.,
1990). However, some aspects of cell division are not conserved between simple and complex eukaryotes. For instance, the nuclear envelope does not break down during
mitosis in yeast, in contrast to the situation in complex eukaryotes. Moreover, components required for specific cell
division processes in simple eukaryotes do not necessarily govern solely the analogous process in complex eukaryotes. For example, whereas cytoplasmic dynein appears to
be essential only for spindle positioning in Saccharomyces
cerevisiae (Eshel et al., 1993; Li et al., 1993), it is also required for other processes in mammalian cells (reviewed
in Hirokawa et al., 1998), including proper spindle assembly (Vaisberg et al., 1993; Heald et al., 1996, 1997; Gaglio
et al., 1997). Such differences underscore the importance
of performing a mutational analysis of cell division processes directly in a complex eukaryote.
Drosophila has proven especially valuable in this regard.
Indeed, genetic and cytological analysis of meiotic and mitotic mutants has led to the identification of many loci required for aspects of cell division (Baker and Carpenter,
1972; Gatti and Baker, 1989; Castrillon et al., 1993; reviewed in Gatti and Goldberg, 1991; Fuller, 1993; Hawley
et al., 1993). For instance, the minus-end-directed kinesin
ncd was originally discovered as a locus required for proper chromosome segregation during female meiosis
(Baker and Carpenter, 1972; Zhang et al., 1990). However,
these primary screens have not always allowed the identification of the exact cell division process affected in mutant animals, because they relied on the analysis of terminal phenotypes. In general, detailed observations in live
cells appear better suited for analyzing defects in cell division processes. Thus, in the syncytial Drosophila embryo, injection of fluorescent tracer molecules has greatly enhanced the resolution with which these processes can be
studied (reviewed in Sullivan and Theurkauf, 1995). For
instance, time-lapse observations of daughterless-abo-like
mutant embryos injected with labeled tubulin and histone
revealed specific defects in centrosome separation (Sullivan et al., 1990). However, although appropriate for analyzing select mutants, such techniques are probably too
cumbersome to become the basis of a large scale primary screen.
The one cell stage Caenorhabditis elegans embryo offers
an attractive alternative for undertaking a mutational
analysis of cell division processes in a complex eukaryote
for a number of reasons. First, division of the one cell
stage C. elegans embryo resembles that of most other complex eukaryotic cells. The cell cycle is mitotic, not meiotic,
and cytokinesis is conventional, unlike, for instance, that
seen in the syncytial Drosophila embryo. Thus, homologues of genes required for cell division processes in the
one cell stage C. elegans embryo will likely play similar roles in other metazoans. Second, cell division processes
can be observed in real time with great detail by differential interference contrast (DIC)1 microscopy in this 50-µm-long cell (Nigon et al., 1960; Sulston et al., 1983). Thus,
a simple visual screen can allow the identification of the
exact cell division process affected in a given mutant strain. Importantly, a large number of strains can be analyzed by such a straightforward primary screen. Third, the
early C. elegans embryo is amenable to experimental manipulations, including the use of cytoskeletal drugs and localized laser irradiations (Strome and Wood, 1983; Hyman
and White, 1987; Hyman, 1989). In combination with mutant analysis, this offers a unique potential for a thorough
analysis of cell division processes. Fourth, the recent completion of the genome sequence allied with the powerful genetics available in C. elegans will greatly facilitate the
molecular characterization of loci identified by mutational
analysis. This will be significantly aided by the advent of
RNA-mediated interference (RNAi), whereby expression
of a given gene can be silenced via microinjection of a corresponding fragment of double-stranded RNA (Fire et al.,
1998). Thus, candidate genes can be rapidly tested by
RNAi to determine whether they correspond to loci identified by mutational analysis.
Because of these advantages, we have screened an extensive collection of C. elegans maternal-effect embryonic
lethal strains on chromosome III using time-lapse DIC
video microscopy. Maternal-effect strains were analyzed
since events in the one cell stage are thought to rely exclusively on the maternal genome (Edgar et al., 1994; Seydoux and Fire, 1994). We have identified and mapped 34 loci which are required for specific cell division processes
in the one cell stage embryo, including spindle assembly,
chromosome segregation, anaphase spindle positioning,
and cytokinesis. By carefully analyzing mutant phenotypes
in live and fixed specimens, we have gained novel mechanistic insights into several aspects of cell division in this
metazoan model system.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Culture Conditions and Strains
Basic methods of C. elegans culture and handling were as previously described (Brenner, 1974; Wood, 1988). Strains carrying the following
mutations were used: unc-32(e189), qC1 dpy-19(e1259ts) glp-1(q339),
him-3(e1147), zyg-8(b235) (Wood et al., 1980), zyg-11(b2) (Wood et al.,
1980), cyk-1 (or36) (Swan et al., 1998), mel-27(e2561) and mel-28(e2567)
(Ahringer, J., personal communication), let-733(s2621), let-748(s2578) and
let-771(s2442) (Stewart et al., 1998), emb-30(g53) (Cassada et al., 1981),
par-3(it62) (Kemphues et al., 1988b), and emb-8(hc69) (Schierenberg et
al., 1980). The following deficiencies were used: sDf121 and sDf125 (Stewart et al., 1998), sDf110 (from the Genetic Toolkit Project), nDf16, nDf20,
nDf40, tDf2, tDf5, tDf6, tDf7, and tDf9. Information on these strains and
deficiencies can be found in the C. elegans database Acedb which is available by anonymous FTP from , directory repository/ acedb or can be accessed online at the following Internet address: http://www.sanger.ac.uk/Projects/C_elegans/webace_front_end.shtml
Mutant Collection and Deficiency Mapping
Mutations were induced on an unc-32(e189) labeled third chromosome.
L4 hermaphrodites of genotype unc-32(e189)/qC1 III; him-3(e1147) IV
were mutagenized with 20 mM ethyl methane sulfonate according to standard procedures (Brenner, 1974). F1 progeny were placed on individual
plates and several F2 Unc L4 hermaphrodites were picked to a separate
plate and placed at 25°C. Strains giving rise to adult Unc hermaphrodites
which produced eggs that did not hatch were kept for further analysis. 254 strains carrying parental-effect embryonic lethal mutations were thus recovered from 15,600 mutagenized genomes (Schnabel, H., T. Kaletta, and R. Schnabel, manuscript in preparation). The mutations were outcrossed twice, and a male stock of each was established. This was facilitated by the
him-3 mutation which causes 3.5% male production (Hodgkin et al.,
1979).
The 254 strains were subdivided into sets by deficiency mapping (see Fig. 1). Complementation analysis was performed among mutations within each set, and with loci previously mapped to each of the corresponding regions. Six strains failed to complement more than one deficiency, or members of more than one complementation group, and are thus considered to carry two mutations, although no attempt has been made to separate them. In some cases, this behavior could be due to the presence of a small deficiency, rather than a double mutation. Thus, the collection comprised 260 mutations, which fell into 125 complementation groups, 81 of which had a single allele, and 44 of which had 2 or more alleles (see Tables II and III; H. Schnabel, T. Kaletta, and R. Schnabel, manuscript in preparation).
|
|
|
|
|
|
|
|
|
Strict and Paternal Tests
The "strict" test was conducted to determine whether there is a strict requirement for maternal contribution, or, alternatively, whether a wild-type paternal allele is sufficient to rescue lethality. Five homozygous mutant hermaphrodites (mut unc-32/mut unc-32) were crossed with plg-1
males at 25°C. After mating with plg-1 males, hermaphrodites have a visible plug in the vulva, indicating that mating took place (Barker, 1994). As
a control, five homozygous mutant hermaphrodites were allowed to self
fertilize without males. If only nonviable eggs were produced in the cross
and at least some animals showed a mating plug, the mutation was called
strict (see "s" in Tables II and III). In contrast, if live cross-progeny was
produced, the mutation was deemed nonstrict.
The "paternal" test was conducted on nonstrict strains to determine whether zygotic expression or, alternatively, sperm cytoplasm can rescue lethality. Five homozygous mutant hermaphrodites (mut unc-32/mut unc-32) were crossed with heterozygous animals from the same strain (mut unc-32/qC1). If only non-Unc progeny (mut unc-32/qC1) were produced, then homozygous mutant progeny (mut unc-32/mut unc-32) must have died, and zygotic expression is needed to rescue lethality. Such strains are marked "z" for zygotic rescue (see Tables II and III). In contrast, if both non-Unc progeny (mut unc-32/qC1) and Unc progeny were produced, then the rescue is independent of the genotype of the embryo, and sperm cytoplasm rescues lethality. Such strains are marked "p" for paternal rescue (see Tables II and III).
Number of Strains Analyzed
We attempted to analyze each single-allele locus of the collection, two independent alleles for loci represented by two or three alleles, and three independent alleles for loci represented by four or more alleles. However, 13 strains representing mutations in 10 loci could not be analyzed for the following reasons. First, in two strains, including a double-mutant chromosome, adult Unc hermaphrodites could no longer be found in the population at the time of analysis, even though they were present when the strains were initially derived. Second, in seven strains, including a double-mutant chromosome, homozygous mutant hermaphrodites gave rise to no or very little progeny, precluding analysis of events in one cell stage embryos. Third, for two double-mutant chromosomes, the phenotype was identical to that observed with single-mutant alleles at one of the two loci and may have precluded scoring the phenotype of the mutation at the other locus. Fourth, for one double-mutant chromosome, other single-mutant alleles at both loci could not be examined, making it impossible to ascribe the phenotype observed in the double-mutant combination to either locus with certainty. Fifth, we found during preparation of the manuscript that one strain carried two mutations; although this double-mutant chromosome had a pronuclear meeting phenotype (see Table III), it is not included in this study because no attempt has been made as of yet to determine which of the two mutations is responsible for the phenotype. In addition, 10 strains representing 10 loci were not included in the data set because they gave rise to unfertilized embryos (3 strains) or were extremely slow to progress through the one cell stage (7 strains).
All together the phenotypes in one cell stage embryos were analyzed for 160 mutations in 106 loci. 2 of the 160 mutations (t1463, t1539) were on double-mutant chromosomes (as indicated in Tables II and III).
Sample Preparation and Recordings
Worms were grown at 16°C, picked as L4s, and shifted to 25°C for a minimum of 12 h. Gravid hermaphrodites were then dissected in M9 medium
(Brenner, 1974). Their embryos were collected with a mouth pipette and
mounted on a 2% agarose pad. A coverslip was placed onto the pad and
sealed with melted Vaseline (Sulston et al., 1983).
Embryos were analyzed at 25°C ± 1°C with a 100× (1.25 NA) Achrostigmat objective lens and standard DIC optics on a Zeiss Axioskop microscope. Under these conditions, pronuclei, asters, spindles, and nuclei can be observed in living C. elegans embryos. Early embryonic events were recorded with time-lapse video microscopy (one image every 5 s), typically from the time of pronuclear formation to the four cell stage, using a monochrome CCD camera (Hamamatsu C5405 or Sony CCD-IRIS).
In Utero Observations
In a few strains, the embryos lost structural integrity upon dissection or
mounting on the agarose pad. These strains were dubbed egg-osmotic sensitive (eos). For eos strains, gravid hermaphrodites were anaesthetized for
10 min in M9 medium containing 0.1% tricaine and 0.01% tetramisole,
and mounted intact on an agarose pad to observe embryogenesis in utero
(Kirby et al., 1990). Although early events could usually be scored with confidence in this manner, the resolution achieved by viewing through the body
wall does not equal that achieved by observing dissected embryos. Therefore, subtle phenotypic manifestations may have been missed in eos strains.
Phenotypic Classes
One cell stage embryos were scored for the following: number and position of female and male pronuclei, migration of pronuclei, position of pronuclear meeting, centration and rotation of centrosomes and associated pronuclei, spindle assembly, anaphase spindle positioning, and cytokinesis. The number and position of daughter nuclei in AB and P1 were also determined. Moreover, the size of the embryo, the appearance of its cytoplasm, and the general timing of events were noted.
Five or more embryos were analyzed for each strain, and a given phenotype was ascribed to the allele if it was observed in at least three out of the first five embryos analyzed. The observed phenotypes fell into different classes; mutations that affect one of the six cell division processes which are the focus of this report are found in Table II, mutations that affect other processes are in Table III.
The vast majority of strains (157/160) fell into only one phenotypic class. However, four strains (t1586, t1465, t1517, and t1714) displayed characteristics of two phenotypic classes. These strains have been classified according to their phenotype in one of the six cell division processes (see Table II), with a mention of the other phenotypic characteristic in the comments column.
Loci were named according to phenotypic classes (see abbreviations in Tables II and III). Multiple allele loci which displayed allele-specific phenotypes were not given name designations. Instead, they are referred to by gene numbers used during generation of the mutant collection. In addition, strains that fell in the "progression through the one cell stage" or "unique phenotypes" classes were not given name designations, as further analysis is required to identify the primary defect in most of these cases.
Other Deviations from Wild-Type
Some embryos were significantly smaller than wild-type. Being small, like being eos, was not considered as a phenotypic class per se; both traits are nevertheless indicated for the relevant strains in Tables II and III.
Aspects that were sometimes variable in wild-type and often variable in many mutant strains were not included in the phenotypic classification. These aspects include the position and the strength of the pseudocleavage furrow, the completeness of the rotation of the centrosomes and associated pronuclei, the extent of rocking of the posterior spindle pole during anaphase displacement, or the extent of cortical contractions in the AB blastomere after cytokinesis.
Other apparent phenotypic deviations from wild-type seemed too subtle to justify a placement in a phenotypic class at this stage of the analysis. These included a minor lack of yolk granules, a slightly smaller size of pronuclei, a hesitant rotation of the centrosomes and associated pronuclei, a delay for the AB or P1 nucleus to reach their appropriate positions, or an unstable AB nucleus.
Indirect Immunofluorescence
Indirect immunofluorescence was carried out with slight modifications of
standard procedures (Strome and Wood, 1983; Albertson, 1984). Slides
were coated with 1% poly-L-lysine (Sigma, P 1524) in PBS. 2 µl H2O was
pipetted onto the slide, and five gravid hermaphrodites were placed into
the drop and cut open with a scalpel to release embryos. An 18-mm2 coverslip was placed onto the drop and excess fluid was wicked away with
3MM Whatman paper. The slide was frozen on a block of metal precooled
on packed dry ice. After a few minutes, the coverslip was flicked off with a
razor blade, and the slide was plunged into 
20°C methanol for 15 min or
more. Slides were rehydrated in PBS for 5 min and incubated with 30 µl of
primary antibody in PBS for 45 min at room temperature in a wet chamber. The following primary antibodies were used: mouse anti-tubulin
(clone DM1A, used at 1:400; Sigma) and rabbit anti-ZYG-9 (used at
1:100; Matthews et al., 1998). Slides were washed for 5 min in PBT (PBS-0.05% Tween 20), 5 min in PBS, and incubated as described above for 45 min with the following secondary antibodies: 1:800 goat anti-mouse AlexaTM 488 (Molecular Probes) and 1:1,000 donkey anti-rabbit Cy3 (Dianova). Slides were placed for 5 min in PBT containing 1 µg/ml Hoechst
33258 (Sigma), washed 5 min in PBT, 5 min in PBS, and mounted in 6 µl
of 90% glycerol containing 4% n-propyl-gallate in PBS. An 18-mm2 coverslip was placed onto the sample and sealed with nail polish. Immunofluorescence microscopy was performed with a Zeiss Confocal Microscope
(LSM 510), and images were processed with Adobe Photoshop 4.0.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Screening of an Extensive Mutant Collection Leads to the Identification of 34 Loci Required for Cell Division Processes
We set out to identify novel components required for cell division processes in a complex eukaryote by mutational analysis, using the one cell stage C. elegans embryo as a model system. To this end, we screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy (see Materials and Methods). 160 mutations in 106 loci were analyzed; an overview of the screen is given in Table I.
|
We identified 48 mutations in 34 loci affecting one of six major processes which normally contribute to proper distribution of chromosomes and cytoplasmic material at cell division. These 48 strains are listed by phenotypic class in Table II. Each of the six phenotypic classes is described in the main text and is illustrated by a figure (see Figs. 4-9), with the top three panels (A-C) representing the relevant wild-type sequence, and the bottom three panels (D-F) the corresponding mutant sequence. In addition, Quicktime movies corresponding to these figures can be viewed on the Hyman lab web site (http://embl-heidelberg.de/ ExternalInfo/). We also identified 47 mutations in 34 loci which had other phenotypic manifestations detectable by DIC microscopy in the one cell stage embryo. These 47 strains are listed by phenotypic class in Table III. Finally, 65 mutations in 51 loci had either no phenotype or no penetrant phenotype in the one cell stage embryo (data not shown).
All 106 loci analyzed were mapped by deficiencies to distinct regions of chromosome III (see Materials and Methods). The location of the loci reported in Tables II and III is given in Fig. 1.
Time-Lapse DIC Video Microscopy Reveals Six Major Classes of Mutants in Cell Division Processes
Analysis of one cell stage embryos typically started ~20
min after fertilization. In wild-type at this stage, the male
and female pronuclei are clearly visible by DIC microscopy at opposite poles of the embryo (Fig. 2 A, arrows).
The male pronucleus is tightly apposed to the cortex (Fig.
2 A, right arrow), while the female pronucleus lies slightly
off the cortex (Fig. 2 A, left arrow). Since the position of
the male pronucleus defines the future posterior of the
embryo (Goldstein and Hird, 1996), the location of the
male and female pronuclei serve to orient the embryo,
which is shown with anterior to the left and posterior to
the right in all figures.
|
The sperm contributes the single centrosome of the one
cell stage embryo (Albertson, 1984). This centrosome duplicates and the two resulting centrosomes then separate,
while remaining associated with the male pronucleus (Albertson, 1984; Hyman and White, 1987). The cell division
that follows entails a sequence of six major processes
which can be monitored by DIC microscopy: pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, anaphase spindle positioning, chromosome segregation, and cytokinesis (Figs. 2 and 3; Nigon
et al., 1960; Sulston et al., 1983; Albertson, 1984). Screening of the mutant collection with time-lapse DIC video microscopy led to the identification of the first point in time
when a given mutant deviates from the wild-type sequence. Mutant strains affecting cell division processes were thus classified into six phenotypic classes, corresponding to defects in one of the six major processes which
take place in wild-type. For each of these processes, we
first describe the events in wild-type and then those in the
corresponding mutant strains.
|
Pronuclear Migration Mutants
The fiirst process to be considered is pronuclear migration. In wild-type, the female pronucleus migrates away from the anterior region, slowly at first and faster thereafter; meanwhile, the male pronucleus migrates slightly away from the posterior cortex (Figs. 3 A and 4, A and B). As a result of these migrations, the female and male pronuclei meet at 70% egg length (0%: anterior-most; 100%: posterior-most).
Pronuclear migration was defective in seven mutants representing six loci in the collection. We define as pronuclear migration mutants those strains in which the female and male pronuclei are initially positioned similarly to wild-type (Fig. 4 D, arrows), but fail to migrate towards each other (Fig. 4 E). In two pronuclear migration mutants (see Table II), a spindle which appears smaller than wild-type assembles around the male pronucleus at the very posterior of the embryo and perpendicular to the longitudinal axis (Fig. 4 F, arrowheads). This spindle moves off the posterior cortex by the end of mitosis, and is bisected by a cleavage furrow ingressing from the posterior and along the longitudinal axis. In the remaining five pronuclear migration mutants (see Table II), the spindle is usually barely detectable or not visible at all after breakdown of the male pronucleus. Consistent with the absence of a functional spindle, there is usually no cleavage furrow ingression from the posterior of the embryo. In occasional pronuclear migration mutant embryos from either set, the female pronucleus undergoes part of its migration towards the posterior. In others, there is more than one female pronucleus, indicating that the female meiotic divisions can be affected as well.
Interestingly, in all pronuclear migration mutants, the nuclear envelope of the male pronucleus breaks down ~1 min before that of the female pronucleus (see Fig. 4 F). This asynchrony in pronuclear envelope breakdown may reflect a wave in cell cycle progression along the longitudinal axis of the embryo (see Discussion).
Rotation Mutants
After pronuclear migration in wild-type, the centrosome pair and associated pronuclei move to the embryo center, while undergoing a 90° rotation that aligns the centrosomes along the longitudinal axis (Figs. 3 B and 5, A and B).
Rotation was defective in three mutants representing three loci in the collection. We define as rotation mutants those strains in which the pronuclei migrate as in wild-type (Fig. 5 D), but in which the centrosome pair and associated pronuclei then fail to rotate. In two rotation mutants (see Table II), the centrosome pair and associated pronuclei also fail to center, and the spindle thus assembles at 70% egg length, perpendicular to the longitudinal axis (Fig. 5 E). In the third rotation mutant (see Table II), the centrosome pair and associated pronuclei do center, and in fact move towards the anterior more so than in wild-type; as a result, the spindle assembles at 30-40% egg length, perpendicular to the longitudinal axis. In all three rotation mutants, the spindle is reoriented along the longitudinal axis by the end of anaphase (Fig. 5 F), perhaps because of the physical constraints of the eggshell. As a consequence of this reorientation, roughly normal-sized daughter blastomeres are generated. However, these blastomeres often have more than one nucleus, indicative of problems in chromosome segregation.
Spindle Assembly Mutants
After rotation in wild-type, the nuclear envelopes of the apposed pronuclei break down and a bipolar spindle is assembled along the longitudinal axis, in the center of the embryo (Figs. 2 D and 3 C).
Spindle assembly was defective in five mutants representing three loci in the collection. We define as spindle assembly mutants those strains in which the pronuclear envelopes break down similarly to wild-type, but in which a bipolar spindle is not apparent thereafter (Fig. 6 E). Although there is an area in the center that appears clear by DIC microscopy, this area lacks bipolarity. Consistent with the absence of a functional spindle, the cleavage furrow does not form and many small nuclei, presumably containing nonsegregated chromosomes, form as the cell returns to interphase (Fig. 6 F, arrows). In a minority of spindle assembly mutant embryos, prior rotation of the centrosome pair and associated pronuclei is also defective.
Anaphase Spindle Positioning Mutants
After spindle assembly in wild-type, the posterior spindle
pole is displaced towards the posterior during anaphase,
resulting in an asymmetric spindle position along the longitudinal axis (Figs. 3 D and 7, A and B). Since the cleavage furrow in animal cells is specified by the end of
anaphase to form at equal distance from the spindle poles
(reviewed in Rappaport, 1971; Glotzer, 1997; Oegema and
Mitchison, 1997), this posterior displacement in the one
cell stage C. elegans embryo results in an asymmetric placement of the cleavage furrow (Fig. 7, B and C).
As expected from previous work (Kemphues et al.,
1988b), we found that mutations at the par-3 locus abolish
posterior anaphase displacement, resulting in a symmetric
first cell division (Table III). However, this lack of posterior displacement is probably a secondary consequence of
the known requirement for par-3 in setting up antero-posterior embryonic polarity (Kemphues et al., 1988b; reviewed in Kemphues and Strome, 1997). To indicate this fact, par-3 is not included among the loci strictly required
for cell division processes which are reported in Table II;
instead, par-3 is mentioned in Table III.
We found that anaphase spindle positioning was defective in a novel manner in nine mutants representing six loci in the collection. In these anaphase spindle positioning mutants, the spindle is usually assembled in the cell center similarly to wild-type (Fig. 7 D), but is displaced to aberrant locations on the cortex during anaphase. In five anaphase spindle positioning mutants (see Table II), the spindle exhibits an exaggerated and abrupt movement towards the posterior during anaphase (Fig. 7 E). The spindle thus comes in contact with the posterior cortex and is reoriented perpendicular to the longitudinal axis by the end of anaphase, resulting in an aberrant first cleavage (Fig. 7 F). In four other anaphase spindle positioning mutants (see Table II), the spindle drifts slowly during anaphase, and ends up being positioned towards the posterior or lateral cortices. In a minority of anaphase spindle positioning mutant embryos from either set, prior rotation of the centrosome pair and associated pronuclei is defective, in which case the spindle sets up perpendicular to the longitudinal axis and tends to drift towards the anterior of the embryo.
Chromosome Segregation Mutants
In wild-type, the spindle segregates one chromosome complement to each daughter of the first cell division (Fig. 3, D and E). In contrast to the other processes described here, chromosome segregation cannot be directly observed by DIC microscopy in C. elegans embryos because individual chromosomes cannot be seen. However, proper chromosome segregation can be inferred from the presence of two equally sized daughter nuclei, one in each daughter blastomere (Fig. 8 C, arrows).
According to this criterion, chromosome segregation was defective in 18 mutants representing 12 loci, comprising the largest phenotypic class in the collection. We define as chromosome segregation mutants those strains in which a bipolar spindle is assembled similarly to wild-type (Fig. 8 D), but generates more than one nucleus in daughter blastomeres (Fig. 8 F, arrows). This indicates that the spindle is unable to properly segregate the entire complement of chromosomes to daughter cells. Perhaps some chromosomes are lagging during anaphase, and are thus not incorporated in the larger nucleus that reforms after telophase. In 15 chromosome segregation mutant strains, the first division spindle looks indistinguishable from wild-type by DIC microscopy, while it has a somewhat unusual appearance in the other three (see Table II; compare Fig. 8, B and E). In seven chromosome segregation mutant strains (see Table II), some embryos have more than one female pronucleus, indicative of defects during the female meiotic divisions.
Cytokinesis Mutants
Cytokinesis is the last cell division process to be observed in the one cell stage embryo. In wild-type (Fig. 3 E), the cleavage furrow is first specified at equal distance from the spindle poles (Fig. 9 A, small arrowheads), then ingresses (Fig. 9 B, small arrowheads) and finally cleaves the cell into two unequally sized blastomeres (Fig. 9 C).
Cytokinesis was defective in six mutants representing four loci in the collection. We define as cytokinesis mutants those strains in which the spindle is positioned by the end of anaphase similarly to wild-type (Fig. 9 D), but in which the cell fails to cleave in two. In two cytokinesis mutants (see Table II), the cleavage furrow is not visible by DIC optics and may thus not be properly specified. In the remaining four cytokinesis mutants (see Table II), the cleavage furrow is specified in the correct location (Fig. 9 D, small arrowheads), starts to ingress (Fig. 9 E, small arrowheads), but then regresses before going to completion (Fig. 9 F). In both sets of cytokinesis mutant embryos, daughter nuclei then appose in the undivided cell (see Fig. 9 F, two right-most arrows). Cytokinesis during the female meiotic divisions seems also affected: additional nuclei, most likely corresponding to nonextruded polar body material, join the daughter nuclei from the anterior of the embryo (Fig. 9, E and F, left-most arrow).
Immunofluorescence Microscopy Reveals that Two Pronuclear Migration Loci Are Required for Generating Normal Microtubule Arrays and Four for Centrosome Separation
Secondary screens customized for each phenotypic class will enhance understanding of the underlying cellular defect. While it is beyond the scope of this study to perform secondary screens on every phenotypic class, we wanted to examine pronuclear migration mutants further to elucidate the basis of a subdivision that was apparent from our observations with time-lapse DIC video microscopy. We had noted that a small but detectable spindle assembled around the male pronucleus and moved off the posterior cortex during mitosis in two pronuclear migration mutants. In contrast, the spindle was barely detectable or seemed altogether absent in five mutants of this class. To better understand the basis for this subdivision, we analyzed fixed wild-type and pronuclear migration mutant embryos with anti-tubulin and anti-ZYG-9 antibodies, as well as with the DNA dye Hoechst 33258.
We examined the distribution of microtubules because
migration of both male and female pronuclei is abolished
in the presence of the microtubule-depolymerizing agent
nocodazole (Strome and Wood, 1983), as well as in zyg-9
mutant embryos, which exhibit short astral microtubules
(Albertson, 1984). Therefore, short or otherwise abnormal
microtubules may be causing the phenotype in some pronuclear migration mutants identified here. In addition, we
simultaneously examined the distribution of ZYG-9 protein, a convenient centrosomal marker which maintains
this localization even after nocodazole treatment (Matthews et al., 1998).
We examined embryos both during prophase, when pronuclear migration normally takes place, and during mitosis, when a potential difference in spindle morphology may
be apparent. In wild-type, the single centrosome lies
initially between the male pronucleus and the posterior
cortex (Albertson, 1984; Hyman and White, 1987). After
centrosome duplication, the two resulting centrosomes
separate and move towards the anterior, seemingly along
the surface of the male pronucleus. As a result, both centrosomes lie on the anterior of the male pronucleus, facing
towards the female pronucleus (Fig. 10 C, arrows). The
cell is in prophase at this stage, as can be seen from condensing DNA in both male (Fig. 10 A, arrow) and female
(Fig. 10 A, arrowhead) pronuclei. During prophase, astral microtubules emanating from the centrosomes become
numerous and some of them are fairly long (Fig. 10 B, arrows). The dense mesh of cortical microtubules which is
predominant in earlier stages (Albertson, 1984) is still
present during prophase, but begins to disappear (Fig. 10
B). During anaphase (Fig. 10 D), an extensive array of astral microtubules extends towards the anterior and posterior cortices (Fig. 10 E, arrows), while spindle microtubules extend centrally towards the chromosomes (Fig. 10
E, arrowhead).
|
We found that astral microtubules were short in the first set of two pronuclear migration mutants, representing two loci (see Table II). During prophase, astral microtubules emanating from the centrosomes were consistently shorter than in wild-type (Fig. 10 H, arrow, compare with 10 B, arrows). This was still the case during anaphase (Fig. 10 K, arrows, compare with 10 E, arrows). At this stage, a spindle which contained less microtubules than in wild-type was apparent (Fig. 10 K, arrowhead, compare with 10 E, arrowhead). This attenuated spindle was perpendicular to the longitudinal axis and located slightly off the posterior cortex, as had been suggested by the DIC observations. Chromosomes coming from the male pronucleus were found to lie on this attenuated spindle in 19 out of 20 embryos examined for the two mutants combined (Fig. 10 J, arrows). In contrast, chromosomes coming from the female pronucleus were left towards the anterior of the embryo (Fig. 10 J, arrowhead).
Interestingly, ZYG-9 protein distribution was affected in these two mutants, in a similar manner. First, ZYG-9 was localized to the anterior cortex (Fig. 10, I and L, arrowheads), where it is not detected in wild-type (see Fig. 10, C and F). In some embryos, ZYG-9 was present at the anterior cortex in patches (see Fig. 10 I, arrowheads). Second, compared with wild-type, ZYG-9 signal appeared stronger at centrosomes and spindle poles (for example, compare Fig. 10 L, arrows, and 10 F, arrows). Taken together, these observations suggest that these two pronuclear migration loci are required for generating normal microtubule arrays and for proper subcellular localization of ZYG-9 protein.
Somewhat unexpectedly, we found that centrosomes failed to separate in the second set of five pronuclear migration mutants, representing four loci (see Table II). In prophase, the centrosome had duplicated, but the resulting daughter centrosomes failed to separate and remained positioned posterior of the male pronucleus (Fig. 10 O, arrows). During mitosis (Fig. 10 P), the two centrosomes were still in close proximity to one another and located at the very posterior of the embryo (Fig. 10 R, arrows). Probably as a consequence of this failure in centrosome separation, no bipolar spindle was assembled, as had been suggested by the DIC observations. Chromosomes were not located between the spindle poles in 42 out of 43 embryos examined during mitosis for the five mutants combined (see for example Fig. 10 P, arrowhead, and 10 R, arrows). Microtubules seemed to grow preferentially or to be stabilized in the vicinity of chromosomes (Fig. 10 P, arrowhead), and were occasionally fairly long (Fig. 10 Q, arrow). In some mutant embryos, centrosomes were separated to a greater extent towards the end of mitosis (data not shown). Taken together, these observations suggest that these four pronuclear migration loci are required for centrosome separation.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Large Scale Mutational Analysis Reveals Spectrum of Phenotypic Classes Affecting Cell Division Processes in the One Cell Stage Embryo
Mutational analysis has been successfully used to dissect a
number of biological processes. Often, success has relied
on having a mutant collection sufficiently extensive to
recognize the full spectrum of phenotypic classes. Thus,
analysis of a large number of cell division cycle mutants
in S. cerevisiae was instrumental for unraveling the dependence between successive events in the cell cycle (Hartwell et al., 1974). In Drosophila, a large scale screen revealed that a limited number of gene classes pattern the
early embryo (Nüsslein-Volhard and Wieschaus, 1980).
The availability of an extensive mutant collection was
also of utmost importance in this work. This allowed us to
recognize that mutations affecting cell division processes
in the one cell stage C. elegans embryo fall into a limited
number of characteristic phenotypic classes when analyzed by DIC microscopy. The spectrum of phenotypic
classes was not apparent from earlier studies relying on
smaller mutant collections. Maternal-effect embryonic lethal mutations affecting aspects of C. elegans development
have been identified previously, notably among three sets
of temperature-sensitive mutant collections (Schierenberg
et al., 1980; Wood et al., 1980; Isnenghi et al., 1983), and
one set of nonconditional mutants on chromosome II
(Kemphues et al., 1988a). However, these studies did not
focus solely on the one cell stage embryo nor on cell division processes, in contrast to what was done here. More
recently, a screen specifically designed to identify mutants
in cell division processes has generated 19 temperature-sensitive mutants, 10 of which display defects in early embryos (O'Connell et al., 1998). However, it is clear that this
screen was far from saturation, as all 19 mutations were
single alleles of different loci. In contrast, 39 loci of the
chromosome III collection analyzed here are represented
by multiple alleles, arguing that this collection is significantly closer to saturation. Thus, analyzing the most extensive mutant collection to date has allowed us to build on earlier studies and recognize a well-defined spectrum of
phenotypic classes affecting cell division processes in the
one cell stage embryo.
100-400 True Maternal-Effect Genes May Be Required Genome-wide for Cell Division Processes in the One Cell Stage Embryo
Screening an extensive mutant collection has also allowed
us to estimate the number of true maternal-effect genes
that are required genome-wide for cell division processes
in the one cell stage embryo. We found that mutations in
34 loci on chromosome III affect one of these processes.
These mutations probably alter two different sets of genes;
the first of these corresponds to true maternal-effect genes,
which encode components required solely in the early embryo. Such genes should mutate to a maternal-effect lethal
phenotype with reasonable frequency. It follows that many of the 39 multiple-allele loci analyzed in the chromosome
III collection may correspond to true maternal-effect
genes. Mutations in 15 (38%) of these multiple-allele loci
affect a cell division process in the one cell stage embryo.
The number of genes in C. elegans which mutate with reasonable frequency to maternal-effect embryonic lethality
is postulated to be 300-1,000 (Feichtinger, 1995). Assuming an even distribution of true maternal-effect genes in the
genome, one can extrapolate that some 100-400 of them
may be required for cell division processes genome-wide.
The second set comprises genes which encode components required not only in the early embryo but also later
during development. The null phenotype in such genes is
usually zygotic lethal. However, rare mutations in these
genes may still be isolated as maternal-effect alleles if, for
instance, they generate sufficient gene product to sustain a
given process in the small larval and adult cells, but not
in the larger embryonic blastomeres. Thus, several single- allele loci in maternal-effect mutant collections turned out
to be hypomorphic alleles of essential genes (Perrimon et al.,
1986; Kemphues et al., 1988a; Schüpbach and Wieschaus,
1989). Such rare mutations are drawn from a very large pool
of genes, whose size cannot be estimated with accuracy.
Time-Lapse DIC Video Microscopy Permits Identification of Mutations Affecting Specific Cell Division Processes
Detailed time-lapse analysis was crucial during our screen for determining the first deviation from the wild-type sequence of events, thus identifying the earliest cell division process for which a given component may be required. It is worth noting that an early phenotypic manifestation may mask a potential requirement in a later process. For example, if a mutation results in a failure in pronuclear migration, a requirement for the corresponding locus in subsequent rotation may not be assessed. Importantly, how early the first deviation from wild-type occurs can yield clues about the type of component encoded by the corresponding locus. Indeed, one might expect that mutations affecting fundamental aspects of cytoskeletal function, such as microtubule integrity, would interfere with many cell division processes, and, thus, exhibit a very early defect. Conversely, mutations affecting components more specifically required for a later cell division process should not exhibit a very early defect.
In fact, our analysis demonstrates that the vast majority of mutations do not exhibit a very early defect, and may thus identify loci required for specific cell division processes. Importantly, we note that for 29 of 39 (74%) multiple allele loci, all alleles examined fall in the same phenotypic class, indicating that the encoded genes may indeed be primarily required for the corresponding cell division process. However, whether this will truly be the case remains to be determined for each locus. Indeed, some of the mutations are likely not null, and additional alleles may reveal that some loci are required for other processes as well. Moreover, in some cases, the function of a molecule in a given process may be difficult to unravel by mutational analysis. For instance, a small protein domain required for a given process may be rarely hit by mutagen.
Even in the event that a mutation is not null, the availability of mutations that specifically disrupt a given process
can provide important insights. For example, in Drosophila,
although the null phenotype of mutations in the actin binding protein profilin is embryonic lethality, female-sterile alleles at this locus revealed the role of the actin cytoskeleton
in regulating transport of cytoplasmic material during oogenesis (Cooley et al., 1992; Verheyen and Cooley, 1994).
Pronuclear Migration: An Early Process Which Relies on Fundamental Aspects of Cytoskeletal Function
The earliest defect in a cell division process that we uncovered is a failure in both male and female pronuclear migration. Analysis of this phenotypic class demonstrates that the corresponding genes are required, as expected, for fundamental aspects of cytoskeletal function. In addition, analysis of the timing of nuclear envelope breakdown in this mutant class reveals the existence of a wave in cell cycle progression in the one cell stage embryo.
When newly fertilized embryos are treated with nocodazole, migration of both male and female pronuclei is abolished (Strome and Wood, 1983). Therefore, this phenotype is the first deviation from wild-type expected for
mutations in loci generally required for microtubule structure or function. Accordingly, our immunofluorescence data show that astral microtubules are shorter than wild-type in mutations at two of the pronuclear migration loci
identified here. Short astral microtubules likely prevent
migration of the male and female pronuclei in different
ways, as distinct mechanisms are thought to normally govern these processes. In wild-type, migration of the male
pronucleus away from the posterior cortex may result
from the movement of the associated centrosomes (Albertson, 1984). Polymerization forces from astral microtubules would act against the posterior cortex to push centrosomes and associated male pronucleus away from it. In
contrast, migration of the female pronucleus is postulated
to result from its interaction with astral microtubules emanating from the centrosomes. Minus-end-directed motors
anchored on the female pronuclear membrane would
power movement along microtubules towards the centrosomes, and, thus, towards the male pronucleus (reviewed in Reinsch and Gönczy, 1998). Based on these
models, the failure in male pronuclear migration in the
two mutants with short astral microtubules likely results from insufficient polymerization forces acting against the
cortex to push centrosomes and associated male pronucleus away from it. Meanwhile, the lack of female pronuclear migration may be due to astral microtubules not
reaching all the way to the female pronuclear membrane.
Intriguingly, our immunofluorescence data raise the
possibility that ZYG-9 protein mislocalization is involved
in generating the short astral microtubule phenotype in
these two pronuclear migration mutants. Short astral microtubules are also observed in zyg-9 mutant embryos, and
ZYG-9 is a member of an evolutionarily conserved family
of microtubule-binding proteins which promote microtubule growth in vitro (Albertson, 1984; Gard and Kirschner, 1987; Matthews et al., 1998). Here, we found that
ZYG-9 protein localization is abnormal in these two mutant strains. Most notably, ZYG-9 protein is present at the
anterior cortex, where it is not detected in wild-type. Possibly ZYG-9 protein trapped at this location promotes abnormal microtubule growth. Consistent with this view,
cortical microtubules appear denser than in wild-type at
the anterior cortex in these mutant embryos (see Fig. 10
K). This may in turn result in less efficient microtubule
growth in the cytoplasm, perhaps because a rate-limiting
component is trapped at the anterior cortex along with
ZYG-9. Further analysis is needed to determine whether
ZYG-9 mislocalization indeed causes the short astral
microtubule phenotype. In the meantime, however, the
present observations demonstrate that these two pronuclear migration loci are required for proper ZYG-9 subcellular localization.
Our immunofluorescence analysis also led to the unexpected discovery that centrosome separation is a prerequisite for the migration of both male and female pronuclei, as separation is defective in mutations at the remaining four pronuclear migration loci. The unseparated centrosomes do not leave the vicinity of the posterior cortex, despite fairly long astral microtubules. Perhaps the overlap of astral microtubules emanating from two centrosomes in close proximity to one another causes steric hindrance which prevents polymerization forces from efficiently acting against the posterior cortex. Alternatively, the lack of centrosome separation may signal to prevent the subsequent migration of centrosomes away from the posterior cortex.
Asynchronous Breakdown of Pronuclei in Pronuclear Migration Mutant Embryos Reveals Possible Regional Differences in Cell Cycle Progression
Time-lapse observations revealed a remarkable property
of pronuclear migration mutants. In all mutant embryos of
this class, the nuclear envelope of the male pronucleus
breaks down ~1 min before that of the female pronucleus.
A similar asynchrony had been noted in zyg-9 mutant
embryos (Albertson, 1984), and is observed in embryos
treated with nocodazole (Gönczy, P., unpublished observations). The generality of this phenomenon suggests that
it reflects a fundamental feature of the one cell stage C. elegans embryo, rather than a peculiar behavior of particular mutant strains.
In sea urchin zygotes in which pronuclear fusion has
been prevented, the female pronucleus always breaks
down earlier than the male pronucleus (Sluder and Rieder, 1985; Sluder et al., 1994). In this case, nuclear envelope breakdown is controlled at the level of each individual pronucleus, rather than by regional differences of cell
cycle progression, as the female pronucleus breaks down earlier even if it is found by chance in the immediate vicinity of the male pronucleus (Sluder et al., 1995). In contrast,
in C. elegans, both pronuclei appear to undergo nuclear
envelope breakdown synchronously when they happen to
be juxtaposed in rare pronuclear migration mutant embryos (Gönczy, P., unpublished observations). Therefore,
the asynchrony observed under conditions where the two
pronuclei are distant from one another most likely results
from regional differences in cell cycle progression.
One possibility to explain such regional differences is
that par genes, which set up polarity along the antero-posterior axis shortly after fertilization (reviewed in Kemphues and Strome, 1997), simultaneously set up a wave of
cell cycle progression emanating from the posterior pole.
Another possibility is that centrosomes, which remain at
the posterior of pronuclear migration mutant embryos, are
preferred sites of cdc2 activation. In this context, it might
be interesting to note that cyclin B1, a component of cdc2
kinase, is associated with centrosomes in HeLa cells
(Bailly et al., 1992). Perhaps a component or an activator
of cdc2 kinase similarly localizes to centrosomes in the one
cell stage C. elegans embryo to start a wave of cdc2 kinase activity.
Centration and Rotation of Centrosomes Can Be Separated by Mutational Analysis
It has been proposed that centration and rotation of centrosomes result from capture and pulling of astral microtubules by the anterior cortex (Hyman and White, 1987; Hyman, 1989). One centrosome would be pulled preferentially,
thus generating a torque which induces rotation of the entire centrosome-pronuclear complex. However, to what
extent centration and rotation are separable processes was
previously unknown.
During this screen, we have identified two kinds of rotation mutants. In the first kind, represented by two mutant strains, both centration and rotation are defective. Possibly, these mutations correspond to weak alleles of loci more generally required for microtubule structure and function, since astral microtubules are required for centration and rotation (Hyman and White, 1987
