The orientation of the mitotic spindle is thought to be a crucial during development for proper segregation of cytoplasmic determinants upon asymmetric cell division. Nonetheless, the mechanisms by which cells control the orientation of the spindle remain to be understood. One example of asymmetric cell division with an oriented mitotic spindle occurs in P 1 , the posterior blastomere of the 2-cell stage C. elegans embryo. In P 1 the centrosomes, after duplication and migration, are positioned transversely to anterior-posterior (a-p) axis of the embryo, subsequently the centrosome-nucleus complex rotates roughly 90 degrees, orienting the P 1 mitotic spindle along the a-p axis of the embryo. It has been suggested that this rotation involves interactions between astral microtubules and a remnant site from Cytokinesis, however the underlying mechanism remains unknown. We have identified maternal effect embryonic lethal mutations in a gene, we call
spn-4 , required for orientation of mitotic spindle in the P 1 blastomere, in these mutants the P 1 spindle stays transverse to the a-p axis. Moreover the ectopic longitudinal orientation of the spindles observed in
par-3 mutants at the 2-cell stage requires the function of the
spn-4 gene, and the distribution of PAR-2 protein is normal in
spn-4 mutant embryos. Thus
spn-4 probably acts downstream of, or in parallel with, the par genes to positively regulate rotation of the spindle in P 1 . Although spindle orientation is abnormal in P 1 , this cell division remains remarkably asymmetric in
spn-4 embryos: the daughter blastomeres are unequal in size with the bigger daughter dividing before the smaller one, and the P granules and PIE-1 protein are still segregated to the smaller daughter cell, like in wild type. In addition, and apparently independently of the spindle orientation phenotype,
spn-4 mutant embryos also exhibit defects in cell fate patterning.
spn-4 embryos fail to produce endoderm and pharyngeal mesoderm, like
skn-1 mutants, and also produce ectopic anterior body wall muscle, as in
mex-3 mutants. Futhermore in
spn-4 embryos the germline-specific P granules are still segregated to a single P 4 -like blastomere, but its daughters proliferate abnormally during embryogenesis, instead of staying quiescent like in wild type. Interestingly PIE-1 protein levels decline prematurely in
spn-4 , as it is not detectable in most of these P 4 -like blastomeres or in its daughters, while in wild type it is present in P 4 and transiently in its daughters Z 2 and Z 3 . This premature loss of PIE-1 might explain loss of germ cell quiescence in
spn-4 mutants. We determined the molecular identity of
spn-4 , and it encodes a putative RNA binding protein containing a single RNA Recognition Motif (RRM). SPN-4 is closely related to another C. elegans RRM protein, FOX-1, a component of the dosage compensation and sex determination machinery and it's known to act post-transcriptionally. We therefore suspect that SPN-4 also acts post-transcriptionally, by regulating the stability or translation of mRNAs encoding components more specifically implicated in the multiple developmental pathways that are defective in
spn-4 mutants. Thus SPN-4 seems to be mediating a subset of asymmetries downstream of the polarity initiated by the PAR proteins.