sod2 RNA expression pattern and localization of the protein during early embryogenesis. (A, B) RNAscope-based localization of sod2 mRNA and the germ cell marker vasa mRNA at (A) 4 hpf and (B) 7 hpf. (C) Whole-mount in situ hybridization image probing for sod2 mRNA in 24 hpf embryos. Blue arrowhead points at the PGC cluster, yellow arrowhead at hematopoietic precursor cells, and purple arrowhead at the migrating pLLP. (D–D’’) Representative confocal images of Sod2-mCherry protein in germ cells relative to the mitochondrial marker (GrpEL-EGFP). Scale bar 10 µm.

Generation of sod2 maternal zygotic fish lines. (A) The schematic representation of the position of the two Cas9-induced mutations. The mutations resulted in stop codons at amino acid position 31 (sod2^STOP31) and 19 (sod2^STOP19) (marked by asterisks). (B) Western blotting analysis of the Sod2 protein expression in wild type and MZsod2 embryos.

MZsod2 KO embryos exhibit reduction in the germ cell migration speed. (A) The scheme of crosses and genotypes analyzed in this manuscript. (B, C) PGC migration speed was determined by tracking cell migration for 70 min using Imaris software. (D) Normalized PGC cluster lengths at 24 hpf in WT and MZsod2 KO embryos treated with neomycin (enhanced ROS production condition). (E, F) Representative images of embryos analyzed for the PGC cluster length (quantification shown in (D)) ****p < 0.0001, *p < 0.05 was determined by Student’s t-test. n - number of PGCs analyzed in 38 WT and 36 MZsod2 KO embryos (B), 24 MZsod2 KO + control (contr) mRNA and 29 MZsod2 KO + sod2 mRNA embryos (C). N - number of PGC clusters (one cluster per embryo) analyzed.

Defects in protrusion formation and germ cell polarity in sod2 KO and neomycin-treated embryos. Analysis of (A, D) the number of blebs in PGCs per minute and (B, E) the proportion of blebs at the cell back. In graphs A and B, MZsod2^STOP31 embryos were injected with control RNA or with RNA directing Sod2 expression to the germ cells (see Methods); the same cells were analyzed in panels A and (B). In graphs D and E, wild-type embryos were treated with DMSO or with neomycin; the same cells were analyzed in panels D and (E). (C, F) Snapshots from the representative time-lapse movies (movies S1 and S2) analyzed in (A,B,D, E). Yellow asterisks point at blebs at the rear of cells. n - the number of PGCs analyzed. One PGC per embryo was imaged and analyzed. **p < 0.01, *p < 0.05, were calculated by Student’s t-test.

MZsod2 KO germ cells exhibit reduced mitochondria-derived fluorescence signal. Average intensity of the fluorescently-labeled mitochondria in WT and MZsod2 KO germ cells at 8 and 24 hpf. n - number of PGCs analyzed in N–number of embryos. ****p < 0.0001, *p < 0.05 was determined by ANOVA test. Scale bar 10 µm.

Analysis of zebrafish with mosaic gonads: PGC transplantation approach. Embryos with mosaic germ cell clusters (white cells are the transplanted donor PGCs, red cells are the host PGCs in the upper panels) were selected at 24 hpf and raised to adulthood and crossed to wild-type fish. If clutches contained embryos with the dominant marker of the donor line (red eyes), the outcrossed adult fish would be counted as “positive.” Lower graph shows the number of such positive fish relative to the total number of adults tested is presented in percentage for each background. Numbers within the bars represent the number of F0 adults with donor progeny divided by the total number of F0 adults raised for this line.

Generation and analysis of zebrafish with mosaic gonads: Cas9-based approach. (A) Embryos of WT or sod2 KO backgrounds homozygous for Tg(cldnB:lynEGFP) (Tg(GFP)^+/+) were injected at the 8-cell stage into one of the middle blastomeres with Cas9 protein and two guide RNAs targeting egfp and sod2 sequences (sgEGFP and sgSod2). This manipulation affected only a fraction of germ cells per embryo, allowing for a high-throughput generation of animals with mosaic gonads containing WT germ cells (expressing Sod2 and EGFP transgene) and KO germs cells (where both Sod2 and EGFP functions were eliminated). Such embryos were raised to adulthood. Adult males were outcrossed to WT females, and the percentage of GFP-negative embryos (lacking EGFP and Sod2 expression) within the clutch was calculated. Results are presented in graph (B). The results of the negative control experiment with animals injected only with sgGFP (manipulating GFP expression only) are presented in graph (C). n–number of embryos and N - number of adult fish analyzed. ****p < 0.0001, ^n.s.p > 0.05 was determined by Student’s t-test.

The number of telomeric repeats is reduced in sod2 KO fish lines. (A) The relative length of telomeric repeats in wild type and MZsod2 mutants was determined by qPCR (graph). The qPCR values were normalized to the WT condition. (B) The relative number of telomeric repeats was determined by fluorescence in situ hybridization (FISH) using Cy3-conjugated TelC probes. The graph presents intensity measurements of the TelC signal in individual PGC nuclei. n–number of PGCs analyzed in 32 WT and 35 MZsod2 embryos (C, D) Representative confocal images of TelC-FISH staining. PGCs are identified by the expression of EGFP-F′ on their cell membranes (represented by yellow dotted lines). ****p < 0.0001was determined by Student’s t-test. Scale bar 10 µm.

Schematic illustration of changes in mitochondria numbers during zebrafish development. Representation of mitochondria numbers per germ cell at 8 hpf and 24 hpf of zebrafish development in WT (black) and MZsod2 KO (red) animals. The dotted part of the graph is based on (33).