(A) Photomicrographs of live wild-type (mCherry::MEX-5 and GLH-1::eGFP) or fixed meg-4 zygotes (MEG::3 OLLAS and PGL-1) at three different stages: before polarization (pronuclear formation), during polarization (pronuclear migration) and after polarization (mitosis). meg-4(ax3052) zygotes were co-immunostained for MEG-3::OLLAS (anti-OLLAS, Novus Biological), and PGL-1 (K76, DSHB). meg-4 is required redundantly with meg-3 for P granule assembly, and each is sufficient to support localized granule assembly (Wang et al., 2014). In this and subsequent figures, dashed lines outline each embryo, embryos are oriented with anterior to the left and posterior to the right and are ~50 μM long. At least three embryos were examined per genotype shown. (B) MEG-3::meGFP levels in the anterior and posterior halves of the zygote during polarization. Values represent average fluorescence intensity over time (relative to initial levels) in the anterior (red) and posterior (blue). Averages come from values measured from three different embryos. Error bars represent standard deviation of the mean. (C) Photomicrographs of fixed zygotes after polarization immunostained for OLLAS, PGL-1, or FLAG. mex-5/6 zygotes were derived from wild-type hermaphrodites treated with mex-5 and mex-6 RNAi. meg-3/4 zygotes were derived from meg-3(ax3055); meg-4(ax3052) hermaphrodites. pgl-1/3 zygotes were derived from pgl-3(bn104) hermaphrodites treated with pgl-1 RNAi (see Figure 1—figure supplement 1 for additional examples of pgl-1(RNAi);pgl-3(bn104) zygotes also stained for PGL-1 to verify loss of PGL-1).