Worm Breeder's Gazette 12(5): 32 (February 1, 1993)

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.

Searching for Mutations that Alter the Cleavage Pattern

Lesilee Simpson Rose[1], Ken Kemphues[2]

Figure 1

The initial devclopment of the C. elegans embryo is characterized by

a stereotypical cleavage pattern: The first cleavage is unequal and

oriented on the anterior-posterior axis. During second cleavage, the

larger anterior cell, AB, divides equally and transversly to the

anterior-posterior axis. The Pl cell divides after AB and thc Pl

cleavage is unequal and oriented on the anterior-posterior axis (l;

Flg. lA). The orientation of the Pl division results from a rotation of

the centrosomes and nucleus that aligns them on the the same axis

as the previous division (2). We are interested in understanding the

mechanisms which specify this pattern. The previously identifiedpar

genes are required for the cytoplasmic localization of factors, some

of which act to control the cleavage pattern (3). We are ca~Tying out

a screen to identify new maternal effect lethal mutations that

disrupt the normal cleavage pattern.

L4 woqms were mutatgenized with EMS and their Fl progeny picked

singly to plates. Of 3073 Fl worms screened to date, 442 segregated

F2 maternal effect lethals. In 198 maternal effect lines, mutant

hermaphrodites p~oduced embryos that arrested with large numbers

of cclls, but little or no morphogenesis. Embryos from those lines

were filmed using time-lapse video microscopy to identify

mutations affecting the cleavage pattern. Three mutadons with

phenotypes resembling those of par mutations were recovered, as

wcll as dght mutations with novd phenotypes. Based on mapping and

complcmentation data, these dght mutations represent fivc or more

genes. At least three of the mutations appear to specifically affect

cleavage onentations.

Hermaphrodites homozygous for itl41 (LGIV, between unc-22 and

dpy4 )produce embryos in which the earliest visible defect is the

orientation of second cleavage spindles. In all embryos, the Pl

division is transverse to the antenor-posterior axis. In about half

the em~ryos, the AB division is also incorrectly oriented, on the

anterior-posterior axis (Fig. lB). Ihe relative timing of ~e AB and Pl

cleavages is nomlal, however. In addition, P granules are localized

normally prior to the first and second cleavages. P granules are

prescnt in two cells at the four cell stage, presumably due to the

incorrect orientation of the Pl division. Third cleavage oricntations

are also abnormal.

The itl43 mutation causes a similar but more variable phenotype:

Embryos from itl43 homozygous mutant mothers appear normal up to

the second cleavage, at which time both the Pl and AB orientations

can be incoTrect ~Fig lC). Some embryos look normal at the second

cleavage, but then show altered orientations at third cleavage. This

allele complements itl41 and maps to LGI between dpy-S and unc-13 .

Hermaph~dites homozygous for mutation 173 produce many embryos

in which both AB and Pl divide on the anterior-posterior axis, but

with the normal timing. The remaining embryos show either the

reversal phentoype described above (AB dividing on the anterior-

posterior axis, Pl dividing transversly), or they look normal at

second cleavage but have third cleavage abnormalities (Fig. lD).

Onc complementation group consists of two alleles which were

identified because the Pl cleavage is transverse to the anterior-

posterioT axis. This phenotype is probably an indirect effect of

oogenesis defects, however. In mutant hesmaph~odites, oocytes are

produced from both sides of the proximal gonad (instead of from only

the outer side). This results in a double r~w of oocytes which are

about half the size of wildtype. After fer~lization, multiple female

pronucld are often visible and ccll cycles are slower.

We are furdur charactenzing the mutations that appear to

specifically affect cleavage crientation. We will determine if the

altered cleavage onentalions are due to failures of rotation of the

nucleus and centrosomes in Pl, accompanied by rotation in the AB.

Also of interest is the bchavior and fate of the daughtÇ cells derived

from an incorrecdy oriented Pl division. Fmer mapping of the

mutations, and analysis of the phenotypes of mutations in ~ans to

dcficiencies is in progress.

Figure 1. Schematic representations of wildtype and mutant

cleavage patterns. Centrosomes, microtubules, and DNA are indicated

by circles, lines, and shading. Cells that show incorrect cleavage

orientations are stippled.

Literature Cited:

2. Hyman and White, 1987. JCB 105:2123-2135.

3. Kemphues et al., 1988. Cell 52:311-320.

Figure 1