In embryos, the timing and orientation of cell division are critical for both the spatial organization of cells and for the proper segregation of cell fate determinants. In C. elegans embryos, the 1-cell and cells of the P1 lineage undergo asymmetric divisions; prior to these divisions, nuclear rotation aligns the spindle with the axis of cellular polarity. In the early AB lineage, nuclei do not rotate and division is symmetric. We have used time-lapse video-microscopy to examine cleavage patterns in embryos produced by maternal effect lethal mutants, and we have identified a number of mutations that alter spindle orientations. Maternal effect lethal mutations in the
let-99 gene cause alterations in spindle orientation in many cells of the early embryo. In most 2-cell
let-99 embryos, there is a failure of rotation in the P1 cell. In about 40% of embryos this is coupled with nuclear rotation in the AB, leading to a complete reversal of spindle orientations. All other aspects of polarity that have been examined at the 1 and 2-cell stage appear normal. In addition to the orientation defects, nuclear-centrosome complexes appear unstable in position in the 1-cell and in both cells of the 2-cell embryo. Our current model for LET-99 function is that it plays a general role in spindle orientation that is needed for both rotation in P1 and inhibition of rotation in AB (Rose and Kemphues, 1998). The
let-99 gene encodes a predicted protein of 698 amino acids with a potential transmembrane domain, but no striking similarity to proteins of known function. A C. elegans gene, which we call
lrg-1 for
let-99 related gene, is 90% identical at the nucleotide level. However, the predicted LRG-1 protein is only 382 amino acids due to a frameshift relative to the
let-99 sequence that introduces a stop codon. Transcripts from both
let-99 and
lrg-1 are detectable in mRNA from gravid adults. The
let-99 transcript is SL1 spliced, while the
lrg-1 transcript is not. We have used affinity purified antibodies (raised against a peptide present in LET-99 but not LRG-1) to stain early embryos from wild-type and
let-99 mutant strains. Based on these studies, LET-99 appears to be present in the cytoplasm and at the cell periphery from the 1-cell to at least the 32-cell stage; the protein may be present at higher levels in regions of cell-cell contact. This pattern is absent in most embryos produced by several different
let-99 mutants. The presence of LET-99 at the cell periphery would be consistent with a role for LET-99 in influencing interactions between the cortex and the microtubule asters which mediate spindle orientation. In addition, during the completion of meiosis, LET-99 is localized asymmetrically in a peripheral patch next to the meiotic spindle. This localization brings up the intriguing possibility that LET-99 also plays a role in positioning the meiotic spindle. We have raised additional polyclonal antisera against a LRG-1 fusion protein which should cross react with LET-99. We will use antibodies affinity purified from this sera to try to confirm the LET-99 distribution pattern, and to determine if LRG-1 protein is present in embryos. We are also using reverse genetics to obtain deletions in
let-99 and
lrg-1 to determine the null phenotype of each gene. We will report on the results of these experiments, as well as our ongoing screen for other genes involved in the control of spindle orientation.