Using RNA interference (RNAi) to phenotypically screen randomly selected cDNA inserts from an early embryo library, we have identified a number of genes that are required for early embryonic cleavages and patterning in C. elegans embryos. We have focused on one gene,
mlc-4, that is required for cytokinesis and anterior-posterior patterning.
mlc-4 encodes the regulatory light chain of nonmuscle myosin. Blocking activity of
mlc-4 using RNAi does not affect the ability of myosin to organize, but does appear to block the force-generating activity of nonmuscle myosin, as assayed by staining for nonmuscle myosin (NMY-2). As a consequence, formation of cleavage furrows are blocked. In addition, early markers of anterior-posterior polarity are disrupted in
mlc-4(RNAi) embryos, including the distribution of PAR-2 and PAR-3. This polarity loss is correlated with a loss in cytoplasmic flux at the pronuclear stage in
mlc-4(RNAi) embryos. We also found that
nmy-2(RNAi) embryos, previously shown to have polarity defects (1), have defects in cytoplasmic flow. These results strengthen the idea that cytoplasmic flow at the pronuclear stage localizes factors that specify the future posterior of the embryo (2) and indicate that this flow is dependant on myosin motor activity. One exception to this model is the localization of P-granules; we found that P-granule localization to the posterior at the first cleavage can occur in the absence of cytoplasmic flows. In addition, we found that
par-1 activity, which is required to localize P-granules (3), has no effect on cytoplasmic flows. In contrast, we found that
par-2 activity, which is not required for posterior localization of P-granules at the first cleavage (3), is necessary for normal cytoplasmic flow. These results suggest that two independant pathways exist to localize factors along the anterior-posterior axis in C. elegans embryos. 1. Guo, S, Kemphues, K.J. Nature 382, 455-458 (1996). 2. Goldstein, B., Hird, S. N. Development 122, 1467-1474 (1996). 3. Kemphues, K. J. et al. Cell 52, 311-320 (1988). We thank Ken Kemphues for supplying NMY-2, PAR-2, and PAR-3 antibodies and Susan Strome for antibodies recognizing P-granules.