Figure 3.

Knockdown of Piwi in the germline causes PHeMAAH defects, which can be rescued by wild-type, but not mutant, Piwi expression. (A) Western blot comparing Piwi levels in knockdown (KD) and control (C) ovary extracts from flies harboring the Piwi V20-1 or the V20-2 hairpin. For each lane, the Piwi signal intensity was normalized to the total protein in the corresponding lane of the blot, and the relative levels of Piwi protein in knockdown and control extracts are shown below the blots. (B) Matα-driven expression of two hairpins targeting different regions of the Piwi mRNA both result in PHeMAAH defects for FM7a/X chromosomes. Sixty ovarioles per genotype were scored for the V20-1 hairpin, and 30 ovarioles were scored for each V20-2 genotype. (C) FM7a/X PHeMAAH defects caused by Piwi knockdown can be rescued by expression of a wild-type (WT) Piwi transgene but not a transgene encoding the V30A Piwi mutant protein. “Control” oocytes carry the Piwi RNAi^V20-1 hairpin transgene but lack the Gal4 driver, so the hairpin is not expressed. “KD” oocytes express the RNAi^V20-1 hairpin under control of the matα driver. “KD + WT” oocytes express the Piwi UAS-RNAi^V20-1 hairpin and also contain a wild-type Piwi transgene in which Piwi expression is controlled by native Piwi regulatory sequences. “KD + V30A” oocytes express the RNAi^V20-1 Piwi hairpin and also harbor a transgene in which native Piwi regulatory sequences control expression of Piwi protein containing a V30A mutation. Ninety ovarioles of each genotype were scored. For B and C, the number of individual oocytes scored for each genotype is indicated in white, and P-values are shown above the bars. Error bars represent 95% confidence intervals. Complete data for FISH experiments are provided in Table S3.