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. 2019 Oct 15;8:e49309. doi: 10.7554/eLife.49309

Figure 2. A candidate RNAi screen reveals Myc as an essential regulator of mitochondrial biogenesis.

(A) Left panel: germarium of a fly endogenously expressing Myc-GFP stained with anti-GFP (Green) and anti-1B1 (Red). Right panel: germarium of a fly expressing LacZ driven by the myc endogenous promoter stained with anti-β-galactosidase (Green) and anti-1B1 (Red). Myc protein is expressed at low level in GSCs and dividing cysts, but markedly induced from region 2B germ cells. In contrast, myc promoter activity is uniform in the germarium. Scale bars, 10 μm. (B) COX activity in germaria from wt, mycP0, bam >ctrl, and bam >mycOE ovaries visualized by COX single staining. The activities are normalized to that of region 2B cysts wt. COX activity is significantly reduced in the mycP0 mutant, but is ectopically induced when Myc is over-expressed in region 2A by bam-Gal4. Scale bars, 10 μm. (C) Quantifications of relative COX activity in germarium regions from wt, mycP0, bam >ctrl, and bam >mycOE flies. ETC activities is normalized to that in the wt 2B cysts. n = 10 germaria for each genotype. Error bars represent SEM. *p<0.05. (D) Visualization of mtDNA replication in germaria from wt and mycP0 ovaries with EdU incorporation (Green) and co-staining with anti-1B1 (Red). Arrowheads point to EdU incorporation into mtDNA, while arrows point to EdU incorporation into the nuclear genome. Scale bars, 10 μm. (E) Area of EdU puncta (pixels) normalized to total pixels at indicated germarium stages in the germline cysts from wt and mycP0 ovaries. n = 11 cysts for each column. Error bars represent SEM. *p<0.005. (F) Table of genes functioning in mitochondrial processes with at least 3-fold decreased expression in mycP0 ovaries compared with wt ovaries. (G) Diagrams of decreased genes encoding mitochondrial processes and transcription factors (TFs) in the mycP0 ovaries. A number of genes in either category has a Myc binding site in their regulatory region.

Figure 2—source data 1. Relative ETC activity and mtDNA area in the germarium regions.
elife-49309-fig2-data1.xlsx (12.9KB, xlsx)
DOI: 10.7554/eLife.49309.008

Figure 2.

Figure 2—figure supplement 1. Myc protein pattern in the ovary.

Figure 2—figure supplement 1.

(A) A representative low-magnification image of ovarioles from flies endogenously expressing Myc-GFP stained with anti-GFP, anti-1B1, and DAPI. Note that Myc level is low in early germarium stages, becomes high from germarium region 2B, and reduces from the stage-10 egg chamber. Scale bar, 50 μm.
Figure 2—figure supplement 2. Standards for COX activity staining reveal that staining intensity is linearly correlated with the amount of complexes.

Figure 2—figure supplement 2.

(A) Upper panel: representative images of wt germaria stained for COX activity, respectively. Lower panel: germaria from wt ovaries incubated in COX activity solution supplied with complex IV inhibitor KCN. Note the onset of COX activity from the germarium region 2B (arrowhead). Scale bars, 5 μm. (B) COX activity was visualized in an ovariole from germarium to the stage-10 egg chamber. Scale bar, 50 μm. (C) Standards for COX activity visualized on a nitrocellulose membrane. COX proteins from bovine heart were used for generating COX standards. The amounts of COX proteins for each band were indicated. (D) Intensities of bands from (C) were calculated by the ‘Gels’ function in ImageJ and plotted against corresponding COX protein amounts. COX protein amounts and their corresponding band intensity values fit well a linear relation (R2 = 0.9923).