Salsa is required for dorsal-anterior expression of Gurken. (A,B) Salsa is required for oocyte dorsal-anterior localization of gurken mRNA. (A) Germline-specific depletion of Salsa impaired dorsal-anterior localization of gurken mRNA during mid-oogenesis. Fragmented digoxigenin-labeled antisense RNA probes were used to detect gurken mRNA in situ. Visualization of probes was done using an anti-Digoxigenin Cy3 secondary antibody. DNA (blue) and gurken mRNA (red). Scale bar, 10 µm. (B) Quantification of anterior dorsal localization defects of gurken mRNA in stage 8/9 egg chambers using three phenotypic classes: “normal localization,” “partial localization,” and “no localization.” Additional examples of scored phenotypes are shown in Supplemental Figure S11. Negative control (mCherry RNAi): 100% “normal DV localization” (n = 19); Salsa depletion (salsa RNAi-1): 16% “normal DV localization,” 56% “partial DV localization,” and 28% “no DV localization” (n = 18); Salsa depletion (salsa RNAi-2): 0% “normal DV localization,” 28% “partial DV localization,” and 72% shows “no DV localization” (n = 25). (C) Salsa is not required for stability of gurken mRNA. Real-time qPCR analysis detected no significant reduction of total levels gurken mRNA after depletion of Salsa. Relative normalized expression corresponds to values normalized with two distinct reference genes (β-actin and GAPDH) and relative to negative control (mCherry RNAi). At least three biological replicates were used for all data sets. Error bars indicate standard deviation. (D,E) Salsa is required for oocyte dorsal-anterior localization of Gurken. (D) Immunostaining of Gurken during mid-oogenesis (stage 8/9 egg chambers). Gurken was detected with an anti-Gurken monoclonal antibody and DNA was visualized with DAPI staining. DNA (Blue) and Gurken (green). Scale bar 10 µm. (E) Relative oocyte Gurken dorsal anterior signal (arbitrary units [a.u.]) corresponds to Gurken signal pixel intensity (average of three different measurements taken from the oocyte dorsal-anterior region with the highest perinuclear signal) divided by the respective background signal (average of three different measurements from the respective nurse cells cytoplasm). To minimize sample variation, all measurements were obtained from maximum intensity projections obtained from confocal Z stacks of stage 8/9 egg chambers. Each dot represents an individual stage 8/9-egg chamber. Horizontal lines specify mean values and asterisks indicate significant difference (two-tailed unpaired t-test; P < 0.001).
