Worm Breeder's Gazette 10(1): 100

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

mei-1(b284): A Mutation that Specifically Affects Meiotic Spindles

S.A. Sprunger and K. Kemphues

Figure 1

Many maternal-effect lethal mutations have been shown to cause 
interesting alterations in early development.  For the recessive 
maternal-effect lethal allele mei-1 (b284) I, meiosis-defective, (
mapping between unc-1 3 and fer-1), the primary developmental defect 
is the specific disruption of the egg meiotic spindle.  This 
conclusion is based on the study of mutant embryos using both indirect 
immunofluorescence staining and time-lapse videotape recording.  
Hermaphrodites homozygous for mei-1(b284) and gravid with mutant 
embryos were squashed on slides to release the embryos, fixed, treated 
with DAPI to visualize chromatin, and stained first with monoclonal 
mouse anti-alpha-tubulin antibodies and then with fluorescein-
conjugated anti-mouse antibodies to show microtubules.  Slides of N2 
embryos were also made for comparison.  Based on the DAPI and anti-
tubulin staining, all the one- to three-cell embryos on each slide 
were scored for developmental stage, meiotic spindle structure, and 
the presence of polar bodies.  From this data, the sequence of early 
developmental events was reconstructed for both the wild type and 
mutant embryos.  In the N2 embryos, normal development was observed: 
formation of a barrel-shaped meiotic spindle without asters at both 
meiosis I (MI) and meiosis II (MII), production of two polar bodies, 
migration of the maternal and paternal pronuclei toward the center of 
the embryo, pronuclear conjunction, and the first mitotic division.  
In contrast to wild type, embryos from mei-1 (b284) hermaphrodites 
never formed a normal meiotic spindle.  Instead, the chromosomes 
appeared to be embedded in a localized cloud of diffuse anti-tubulin 
staining near the anterior end of the embryo, suggesting that tubulin 
subunits or short microtubules are recruited to the correct site in 
this mutant but not assembled into a spindle.  None of the mutant 
embryos had normal polar bodies: 60% of them made no polar body at all,
but 40% had formed one abnormally large polar body, indicating that 
the mutation does not block meiotic cytokinesis.  The one-cell embryos 
without polar bodies retained the entire 4n complement of maternal 
chromosomes, while those that made an abnormal polar body segregated a 
variable amount of chromatin into it.  The retained maternal 
chromosomes typically formed several pronuclei interspersed with 
smaller, condensed bits of chromatin.  At the pronuclear migration 
stage, the multiple maternal pronuclei and chromatin bodies were 
distributed between the anterior end and the site of pronuclear 
conjunction in a manner that suggested a processional mechanism for 
migration, capable of moving more than one pronucleus and of 
initiating movement over a period of time.  A processional pronuclear 
migration would result in some groups of chromatin reaching the 
conjunction site before others, and this interpretation is supported 
by the mitotic configuration at the first cell division.  The mutant 
embryos always formed a normal mitotic spindle and underwent wild type 
patterns of cell division, but varied in the inclusion of maternal 
chromosomes on the metaphase plate: generally, some of this chromatin 
was included, some was associated with the astral microtubules, and 
some was excluded entirely.  The death of mutant embryos is likely the 
result of the varying retention of maternal chromosomes in the zygote 
and abnormal segregation of those chromosomes at the first division.  
If the initiation of later developmental events depends on the 
successful completion of earlier events, one would expect a delay in 
the onset of pronuclear migration and the first mitotic division in 
mutant embryos from mei-1 (b284) hermaphrodites.  Comparison of the 
proportions of mutant embryos at each developmental stage with the 
corresponding data for N2 embryos, however, shows that there is no 
significant difference between the time required to complete each 
phase in mutant and in wild type embryos, suggesting that starting 
these events does not require the normal completion of the preceding 
event. (See figure below.  Since there is no consistent polar body 
formation in the mutants, it is difficult to distinguish MI from MII 
embryos, and these are grouped together as 'meiotic' embryos.)
[See Figure 1]
Observation of live mutant embryos with time-lapse videotape 
recording confirmed the immunofluorescence staining results.  Due to 
the difficulty of mounting fertilized embryos isolated from 
hermaphrodites in time to record meiosis, whole b284 homozygous 
hermaphrodites were mounted and their mutant embryos observed in utero.
To keep the worms still during microscopy, they were paralyzed with 
a solution of 0.1% tricane and 0.01% tetramisole in M9 salts, a 
treatment that was shown not to affect the viability of the paralyzed 
animal's embryos.  In a representitive mutant embryo we observed 
ruffling of the cell membrane at the anterior end, followed by the 
formation of an abnormally large polar body at a time consistent with 
meiosis I.  It is not yet known whether all the abnormal polar bodies 
in the mutant embryos appear at the time at which MI would normally 
occur, or whether some are formed at MII or at intermediate times.  
Multiple maternal pronuclei, processional migration of these pronuclei,
and normal mitosis, save for the varying inclusion of maternal 
chromatin as described above, were also observed in live mutant 
embryos.  In conclusion, the recessive allele mei-1 (b284) disrupts 
the formation of the meiotic spindle in the embryos of homozygous 
hermaphrodites, but does not affect the mitotic spindle.  Furthermore, 
since we have not seen obvious evidence of aneuploid sperm nuclei in 
mei-1 (b284) homozygotes, the effect of this mutation may be 
restricted to the meiotic apparatus of the egg.

Figure 1