Worm Breeder's Gazette 15(1): 66 (October 1, 1997)

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.


Stephen E. Basham, Lesilee S. Rose

Section of Molecular and Cellular Biology, University of California at Davis, Davis CA 95616

Orientation of cell division plays a critical role in the development
of both plants and animals.  In early C. elegans embryos, two basic
patterns of division occur.  The AB lineage displays a typical
orthogonal pattern of cleavage.  In the P lineage, divisions repeatedly
occur on the same axis due to a 90 degree rotation of the
nuclear-centrosome complex.  In the P1 cell, rotation depends on an
interaction between astral microtubules and a site on the anterior
cortex.  We are studying maternal effect lethal mutations that alter
spindle orientation in early embryos.  In most embryos from animals
homozygous for mutations in ooc-5 (for abnormal oocyte formation), the
P1 nucleus migrates to the anterior cortex but fails to rotate.  This
results in P1 dividing transverse instead of along the
anterior-posterior axis.
        As an initial step in understanding the ooc-5 phenotype, we
have compared the distribution of cytoskeletal elements in wild-type
and ooc-5 mutant embryos. In the P1 cell of wild-type embryos, both
actin and CP accumulate in an anterior cortical dot which correlates
strongly with nuclear rotation (Waddle et al., 1994, Devel. 120,
2317-2328).  The percentage of ooc-5 embryos with a cortical
accumulation of CP in the P1 cell is comparable to what we observe in
wild-type embryos. These results suggest that the failure of rotation
is not due to a lack of CP accumulation.  In addition, the microtubule
cytoskeleton appears normal in ooc-5 embryos as observed using standard
immunofluorescence microscopy.  Specifically, astral microtubules are
seen projecting toward the cell periphery and appear long enough to
interact with the cortex, suggesting that failure of rotation is not
caused by microtubules being too short.
        Early C. elegans embryos are highly polarized.  To determine
whether ooc-5 mutations disrupt this polarity, we examined ooc-5
embryos for both P granule and PAR-3 localization.  In one and two-cell
embryos, P granules are localized to the posterior pole of  ooc-5
embryos, just as in wild type. In the majority of 4-cell ooc-5 embryos
examined however, P granules are seen in two of the four cells.  This
is presumably due to the failure of nuclear rotation resulting in P1
dividing transverse to the axis of P granule localization.  The
PAR-3protein is required for embryonic polarity and becomes localized
to the anterior periphery of wild-type one-cell embryos
(Etemad-Moghadam et al., 1995, Cell 83, 743-752).  Our preliminary
results show that PAR-3 protein is properly localized to the anterior
periphery of one-cell ooc-5 embryos.  Together, these observations
suggest that altered spindle orientation in ooc-5 embryos is not a
result of an overall polarity defect.
        Mutations in ooc-5 are pleiotropic however, resulting in a
germline defect in addition to disrupting spindle orientation in P1.
In ooc-5 mutant animals, the germline contains a double row of oocytes
that are smaller than wild-type oocytes.  Embryos produced by ooc-5
mutants are approximately half the size of a wild-type embryo while
their nuclei are approximately 80% wild-type size.  This observation
raises the possibility that failure of nuclear rotation in these
embryos is due to the steric effect of having a near normal size
nucleus or microtubule array in a small cell. We have examined other
strains that produce small embryos to see if reduced embryo size
generally correlates with a failure of nuclear rotation.  Homozygous
ooc-3  mutants also have a double row of oocytes; the embryos produced
are smaller than wild type and nuclear rotation fails to occur in the
P1 cell.  One interpretation of the ooc-5 and ooc-3 phenotypes is that
the reduced distance between centrosomes and the anterior cortex
prevents nuclear rotation,  for example because microtubules from both
asters make stabilizing connections with the anterior cortical site.
However, small embryos produced by ceh-18 hermaphrodites can undergo
nuclear rotation.  In two ceh-18 embryos examined, the distance from
the center of the nucleus to the anterior cortex was shorter than in
several ooc-5 and ooc-3 embryos.
In addition, we have observed several cases in wild-type embryos where
the P1 nucleus moves up against the anterior cortex before rotating.
These data suggest that lack of rotation in ooc-5 and ooc-3  embryos is
not a result of having a shorter distance between the centrosomes and
the anterior cortical site.  However, we can not rule out other steric
effects since ceh-18 and wild-type embryos are larger than ooc-5
embryos in other dimensions.
        We are beginning a molecular analysis of the ooc-5 gene to gain
more insight into its role in germ line formation and spindle