Figure 3. Primordial germ cell (PGC) lobe cannibalism and autophagy generate a mitochondrial DNA (mtDNA) low point and set point.

(A–F) Germline mitochondria and mitochondrial transcription factor-A (TFAM)-GFP₁₁ in live embryos and larvae at the indicated stage. Dashed lines outline the PGCs or germline stem cells (GSCs). (G–H) Quantification of TFAM-GFP₁₁ foci per germ line (G) and per germ cell (H) in embryos and larvae. (I–J) Quantification of mtDNAs per germ line (I) or per germ cell (J) in embryos and larvae; data shown for PGC mtDNA copy number in embryos and starved L1s are provided for comparison and originate from Figure 2B. (K–L) Quantification of mtDNAs per germ line (K) or per germ cell (L) in nop-1 mutant embryos and larvae; data shown for PGC mtDNA copy number in nop-1 mutant embryos and starved L1s are provided for comparison and originate from Figure 2E. Data in graphs: small dots are individual animals (TFAM-GFP₁₁ measurements) or technical replicates (droplet digital PCR [ddPCR] experiments) from three color-coded biological replicates; the mean from each experiment is shown as a larger circle, the mean of means as a horizontal line, and the SEM as error bars. n.s., not significant (p>0.05), *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001 unpaired two-tailed Student’s t-test. Scale bars, 5 µm.

Figure 3—figure supplement 1. TFAM-GFP₁₁ visualization and effect on mitochondrial DNA (mtDNA) copy number.

(A-B’’) Germline mitochondria and GFP_1-10 in L2 larvae, with (A-A’’) or without (B-B’’) endogenously tagged TFAM-GFP₁₁. Dashed line, the outline of the gonad. (C) Quantification of mtDNA copy number in whole L4 larvae assayed by quantitative PCR (qPCR) in wild-type, TFAM-GFP, and TFAM-GFP₁₁; Mito-GFP_1-10^(PGC) genetic backgrounds. Small dots are data points from individual L4 worms from each of three color-coded biological replicates; the mean from each replicate is shown as a larger circle, the mean of means as a horizontal line, and the SEM as error bars. n.s., not significant (p>0.05), **p≤0.01, ***p≤0.001, unpaired two-tailed Student’s t-test. Scale bars, 10 µm.

Figure 3—figure supplement 2. Fluorescence activated cell sorting (FACS), ploidy, and purity of sorted larval germline stem cells (GSCs).

(A–D) Full gating strategies for isolating mid-L1 and L2 GSCs from dissociations. Abbreviations: FSC-A, forward scatter area; FSC-W, forward scatter width; SSC-A, side scatter area; SSC-W, side scatter width; GFP-A, green fluorescent protein area; mCherry-A, mCherry area; DAPI-A, 4′,6-diamidino-2-phenylindole area. Size exclusion gates (SSC-A × FSC-A) containing GSCs were determined by backgating on all GFP⁺mCherry^– events in mid-L1 (A) and L2 (B). (C–D) Following size exclusion, two doublet discrimination gates (FSC-A × FSC-W and SSC-A × SSC-W) were applied to select singlet cells, DAPI-negative cells were selected for viability, and pure GFP⁺mCherry^– mid-L1 (C) and L2 (D) GSCs were sorted. (E) Ploidy of sorted mid-L1 and L2 GSCs (see Methods). Small dots are three technical replicates of droplet digital PCR (ddPCR) quantification from each of 3–9 color-coded biological replicates; the technical replicate mean from each experiment is shown as a larger circle, the mean of means as a horizontal line, and the SEM as error bars. *p≤0.05, unpaired two-tailed Student’s t-test. (F) Representative images of mid-L1 and L2 GSCs post-FACS. Scale bars, 5 µm.