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.
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.