Fig. 5. MEX-3 immunolocalization in wild-type and mutant embryos. Genotypes and embryo stages are indicated (see Fig. 2C for quantitative summary of staining experiments). (A) In wild-type embryos, MEX-3 is present through the eight-cell stage and essentially disappears by the 14- to 18-cell stage, except for weak staining in the P₂ blastomere descendants. (B) In mex-6(RNAi) embryos, MEX-3 is undetectable by the eight-cell stage. (C,D) In spn-4(RNAi) embryos and mex-6(RNAi); spn-4(RNAi) embryos, MEX-3 persists through to the 14- to 18-cell stage. (E) In par-1 embryos, MEX-3 persists through the eight-cell stage but is undetectable by the 14- to 18-cell stage. (F,G) Defects in par-1 do not effect the MEX-3 phenotype of mex-6(RNAi) embryos or spn-4(RNAi) embryos. (H) In par-4 embryos, MEX-3 persists through the eight-cell stage but is undetectable by the 14- to 18-cell stage. (I) par-4 is required for the premature degradation of MEX-3 seen in mex-6(RNAi) embryos. (J) Defects in par-4 do not affect the MEX-3 phenotype of spn-4(RNAi) embryos. Slight variations in the intensity of MEX-3 signal in the micrographs are often the result of the embryo position in the focal plane, and may not reflect actual asymmetries in protein distribution. Only clear asymmetries that are visible in all focal planes, as in A2, were scored as patterned (see Fig. 2C). Representative embryos are shown. Owing to the high intensity of the signal, (C,D,G,J) spn-4(RNAi) two- and four-cell embryos are generally underexposed relative to other embryos. All eight-cell embryos are shown at the same exposure, with the exception of C3 and G3, which are underexposed relative to the other eight-cell embryos. All 14- to 18-cell embryos are shown at the same exposure with the exception of C4, G4 and J4, which are underexposed relative to the other 14- to 18-cell embryos. In two-cell (B1) mex-6(RNAi) and (C1) spn-4(RNAi) embryos, the anterior cell is typically larger than the posterior cell as in (A1) wild type, demonstrating that mex-6 and spn-4 RNAi embryos can maintain asymmetries in cell size. mex-6(RNAi) disrupts P-granule localization as monitored by MEX-3 localization to P-granules (Draper et al., 1996). Ten out of 10 mex-6(RNAi) embryos mis-segregate P-granules to all blastomeres of the four-cell embryo, while 10/10 of spn-4(RNAi) embryos segregate P-granules normally. (D) mex-6(RNAi); spn-4(RNAi) double mutant embryos show persistent MEX-3 expression characteristic of spn-4(RNAi) embryos, while 8/10 four-cell embryos mislocalize P-granules like mex-6(RNAi) embryos, indicating that both injected dsRNAs were effective in RNAi.