To understand the mechanisms governing the cell division processes of the early embryo, we have conducted genetic screens to identify temperature sensitive, maternal effect, embryonic lethal mutations. From these screens we have identified a class of mutants with spindle orientation defects at the one-cell stage P0 blastomere (see posters by Jen Phillips, Greg Ellis and Sandra Encalada). One of these mutations,
or404ts , was positioned by meiotic mapping near the
dnc-1 locus.
dnc-1 , the orthologue of the vertebrate gene
p150Glued, is a component of the multi-subunit dynactin complex, which is required for all known functions of the microtubule-associated dynein motor protein. Sequence analysis of
dnc-1 from
or404ts animals identified a missense mutation in the C terminus of the protein. To our knowledge,
or404ts is the only known mutant allele of
dnc-1 in C.elegans. Roles for dynein and dynactin in the early embryo were revealed by studies in which known components, including
dnc-1 , were inhibited by RNAi (1,2). These studies show that dynein and dynactin are required for multiple microtubule dependent events in the early embryo; these include pronuclear migration, centrosome migration, and rotation of the centrosomal/nuclear complex. In wild type embryos, the sperm contributed centrosome duplicate and migrate to opposite poles of the male pronucleus, transverse to the anterior-posterior (a-p) axis of the embryo. Meanwhile, the female pronucleus migrates to meet the male pronucleus at the posterior of the embryo. This centrosomal/nuclear complex then rotates 900 to lie along the polarized a-p axis of the embryo before the completion of the cell cycle. In
dnc-1 (
or404ts ) mutant embryos pronuclear migration and centrosome migration occur as in wild-type, but centrosomal/nuclear complex rotation does not occur, resulting in a mitotic spindle which aligns transverse to the a-p axis. Since dynein and dynactin RNAi mutant embryos exhibit early defects in embryogenesis, a role for dynein and dynactin in spindle orientation past the one cell stage has not been addressed. In both the P1 blastomere and the EMS blastomere at the 2 and 4 cell stage, respectively, the mitotic spindle rotates to align along the a-p axis. It is not known if dynein or dynactin are required for these rotations. To address this we are performing temperature up-shift experiments with
dnc-1 (
or404ts ) to determine its role in these rotations. Preliminary experiments show that P1 spindle orientation is aberrant when
dnc-1 (
or404ts ) embryos are shifted to the restrictive temperature after a wild-type first division (1/3 embryos). This suggests that dynactin and dynein may function in P1 spindle orientation. Further characterization of this function and a possible requirement for
dnc-1 (
or404ts ) in EMS spindle orientation will be covered in this poster. 1. Skop A. and White J. (1998) Current Biology 8: 1110-1116. 2. Gonczy et al., (1999) JCB,14(1) : 135-150.