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 daught 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.