Figure 5. Transient JNK activation in the germarium boosts the IIS-Myc signaling.

(A) Representative germaria with bsk¹ FRT clones showing EdU incorporation (green) to visualize mtDNA replication. bsk¹/+ cells are positive for mRFP (red), while bsk¹ mutant cells are negative for mRFP. Arrows indicate EdU incorporated into mtDNA, arrowheads point out EdU incorporated into the nuclear genome. Scale bars, 5 μm. (B) Percentage areas of EdU incorporated into mtDNA relative to total areas of the germline cysts at indicated germarium stages from bsk¹/+ and bsk¹ clones. n = 11, 14, 15, and 10 cysts each column (left to right). Error bars represent SEM. *p<0.005. (C) Germarium from ovaries expressing LacZ driven by the puc promoter stained with anti-β-galactosidase (green) and anti-Vasa (red). Germarium is outlined with dotted lines. Scale bar, 10 μm. (D) Germaria from ovaries of ctrl and bsk^RNAi driven by nos-Gal4 stained with anti-p-AKT (green) and anti-1B1 (red). Note that IIS activity is markedly reduced when JNK signaling is decreased by bsk RNAi. Scale bars, 10 μm. (E) Quantification of p-AKT intensity from cysts in germarium region 2A and 2B of ovaries with indicated genotypes. p-AKT intensity is normalized to that of region 2B cysts from the ctrl line. n = 11 germaria for each genotype. Error bars represent SEM. *p<0.005. (F) Visualization of InR-EGFP in germaria from ovaries of ctrl and bsk^RNAi driven by nos-Gal4. Note that InR-EGFP level on the region 2B cyst membrane is decreased by bsk RNAi. Germaria are outlined with dotted lines. Scale bars, 10 μm. (G) Quantification of background subtracted InR-EGFP intensity on cell membrane of the region 2B cyst. n = 7 germaria for each genotype. Error bars represent SEM. *p<0.005. (H) Germaria from ovaries of bsk^RNAi; bsk^RNAi, InR^OE; jra^RNAi; jra^RNAi, InR^OE driven by nos-Gal4 endogenously expressing Myc-GFP co-stained with anti-GFP and anti-1B1. Note that Myc-GFP level in germ cells is markedly lower than that in follicle cells pointed out by arrows. Scale bars, 10 μm. (I) COX activity in region 2B cysts from ovaries with indicated genotypes. The activities are normalized to that of region 2B cysts from the ctrl line. n = 10 germaria for each genotype. Error bars represent SEM. *p<0.005.

Figure 5—figure supplement 1. Other IIS components are not altered by bsk RNAi.

(A) Confocal images for germaria expressing Chico-GFP, myc-Dp110 or PDK1-GFP in the background of ctrl or bsk^RNAi from ovaries stained with anti-GFP or anti-myc. Scale bars, 10 μm. (B) Ovaries expressing LacZ driven by the puc promoter stained with anti-β-galactosidase, anti-1B1, and DAPI. Note that JNK activity is moderately induced in late germarium and decreased in the following stages, while JNK activity is much stronger in follicle cells of a maturing egg. Scale bars, 20 μm. (C) Germaria from ovaries of ctrl and bsk^RNAi driven by nos-Gal4 stained with anti-AKT and anti-1B1. Scale bars, 10 μm. (D) Quantification of AKT intensity from cysts in germarium region 2A and 2B of ovaries with ctrl and bsk^RNAi driven by nos-Gal4. Intensities are normalized to the value of ctrl at region 2B. n = 7 and 8 germaria for ctrl and bsk^RNAi, respectively. Error bars represent SEM. *p<0.005. (E) Visualization of the InR mRNA by FISH in germaria from ovaries of ctrl and bsk^RNAi driven by nos-Gal4. Note that InR mRNA level is decreased by bsk RNAi. Germaria are outlined with dotted lines. Scale bars, 10 μm. (F) Quantification of InR mRNA density in region 2A and 2B germ cells from ovaries with indicated genotypes. n = 9 germaria for each genotype. Error bars represent SEM. *p<0.005. (G) Quantification of relative Myc-GFP intensity in region 2B cysts of ovaries illustrated in Figure 5H. n = 10 germaria for each genotype. Error bars represent SEM. *p<0.005.