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DNA cross-link repair safeguards genomic stability during premeiotic germ cell development

Nature Genetics volume 51, pages 1283–1294 (2019)Cite this article

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Abstract

Germline de novo mutations are the basis of evolutionary diversity but also of genetic disease. However, the molecular origin, mechanisms and timing of germline mutagenesis are not fully understood. Here, we define a fundamental role for DNA interstrand cross-link repair in the germline. This repair process is essential for primordial germ cell (PGC) maturation during embryonic development. Inactivation of cross-link repair leads to genetic instability that is restricted to PGCs within the genital ridge during a narrow temporal window. Having successfully activated the PGC transcriptional program, a potent quality control mechanism detects and drives damaged PGCs into apoptosis. Therefore, these findings define a source of DNA damage and the nature of the subsequent DNA repair response in germ cells, which ensures faithful transmission of the genome between generations.

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Fig. 1: Ercc1−/− mice have reduced numbers of germ cells.
Fig. 2: ERCC1 is critical for PGC development.
Fig. 3: ERCC1 acts together with the Fanconi anemia pathway to preserve PGC development.
Fig. 4: Cross-link repair-deficient PGCs undergo normal PGC transcriptional activation and epigenetic reprogramming.
Fig. 5: PGCs accumulate unrepaired DNA breaks.
Fig. 6: Fetal aldehyde catabolism protects developing PGCs.
Fig. 7: Damaged PGCs are eliminated by apoptosis.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We would like to thank the Wellcome Trust Sanger Institute, the Knockout Mouse Project Consortium and the Mouse Biology Program, University of California, Davis for the Ercc1-targeted mESCs. We thank A. Surani for the gift of Stella-GFP mice, F. Hildebrandt for Fan1-deficient mice, G.T. van der Horst for the gift of Xpa- and Csb-deficient mice. We would like to thank K.J. Patel for the ALDH2- and ADH5-deficient mice. We thank the Human Research Tissue Bank (National Institute for Health Research Cambridge Biomedical Research Centre) for processing the histology. We would like to thank R. Pannell, C. Knox, C. Watson, K. Kemp, R. Higginson, J. Clark, the Ares and Biomed staff for assistance with animal procedures and experiments. We thank M. Daly, F. Zhang and the Flow Cytometry Core staff for their technical assistance. We would like to thank G. Oliveira for assistance with the PGCLC assays. We would like to thank V. Sacalean for assistance with apoptosis assays. We would like to thank J. Garaycoechea for his criticism, for screening the Ercc1 founder mice, for collecting samples from NER-deficient mice and for critically reading the manuscript. We would like to thank J. Sale for critically reading the manuscript and for useful discussions. R.J.H. and G.P.C. are supported by the Medical Research Council. This work was supported by the Medical Research Council as part of UK Research and Innovation (file reference no. MC_UP_1201/18). G.P.C. would like to thank K.J. Patel for scientific discussion and support.

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  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Ross J. Hill & Gerry P. Crossan

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G.P.C. and R.J.H. conceived the study, designed experiments and wrote the paper. R.J.H. performed all the experiments.

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Correspondence to Gerry P. Crossan.

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Integrated supplementary information

Supplementary Figure 1 Ercc1 knockout mice recapitulate key features of ERCC1 deficiency.

a, Schematic representation of the wildtype (Ercc1+) and disrupted Ercc1tm1a(KOMP)Wtsi allele (Ercc1−), when crossed with Flp-e recombinase this generates the conditional allele (Ercc1f). This can produce a null allele by crossing with Cre-recombinase, deleting the floxed exon 5 (Ercc1Δ). b, Verification of gene targeting in mESCs by long-range PCR. Oligonucleotide pairs were designed so that one oligonucleotide binds within the targeting construct and the other binds within the genomic sequence beyond the homology arm. c, Mice were genotyped using two PCR strategies to allow the discrimination between the four different alleles. Firstly, oligonucleotides ER1F2, ER1R1 and En2A would amplify a 594 bp product from the Ercc1+ allele, a 477 bp product from the Ercc1− allele and a 965 bp product from the Ercc1f allele (top panel). Secondly, oligonucleotides ER1NF and ER1NR would amplify a 776 bp product from the Ercc1+ allele, a 947 bp product from the Ercc1f allele and a 339 bp product from the Ercc1Δ allele (bottom panel). d, Immunoblot showing the absence of the ERCC1 protein in whole cell lysates of MEFs derived from Ercc1-/- embryos (representative data from 2 independent experiments). Uncropped images in Supplementary Fig. 17 e, Ercc1-/- MEFs are hypersensitive to mitomycin c (MMC) and ultraviolet (UV) irradiation (data represent mean and s.e.m.; data represent 3 independent experiments each carried out in triplicate). f, Kaplan-Meier curve showing the survival of cohorts of Ercc1-/- and congenic control mice. g, Ercc1-/- embryos are observed at expected ratios throughout development (E11.5, E12.5 and E18.5). However, Ercc1-/- mice are observed at a significantly reduced frequency in 14-day old (P14) mice, obtained from Ercc1+/- males intercrossed with Ercc1+/- females (p-value calculated by Chi-square test). h, Ercc1-/- mice have a moderate reduction in ovary mass (p-value calculated by 2-tailed Mann-Whitney test; data shown represent mean and s.e.m.; each point represents one ovary, n= 21 and 6, left to right).

Supplementary Figure 2 Ercc1-/- embryos have reduced numbers of germ cells.

a, Quantification of MVH+ cells per seminiferous tubule at E18.5 (p-value calculated by 2-tailed Mann-Whitney test; data shown represent mean and s.e.m.; a total of 150 tubules per genotype were scored, 50 per mouse). b, Distribution of the number of MVH+ cells per tubule in E18.5 wildtype and Ercc1-/- embryos (250 tubules per genotype were scored, 50 per mouse). c, Representative images of E18.5 ovarian sections stained with the marker of apoptosis cleaved-Caspase 3 (CC3) and quantification of the frequency of CC3+ cells (p-value calculated by 2-tailed Mann-Whitney test; data represent mean and s.e.m.; each point represents data from one embryo, n= 4 per genotype). d, Representative images of E18.5 testis sections stained with the marker of apoptosis cleaved-Caspase 3 (CC3) and quantification of the frequency of CC3+ cells (p-value calculated by 2-tailed Mann-Whitney test; data represent mean and s.e.m.; each point represents data from one embryo, n= 5 per genotype).

Supplementary Figure 3 ERCC1 is required for PGC development.

a, Quantification of PGCs (SSEA1+GFP+) per gonad at E12.5 from wildtype and Ercc1-/- embryos carrying either the GOF18-GFP or Stella-GFP reporters show equivalent defects with either reporter (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=11, 7, 10 and 6, left to right). b, Quantification of PGCs (SSEA1+GOF18-GFP+) per gonad by flow cytometry from embryos carrying the GOF18-GFP reporter at E11.5 separated by sex (p-value calculated by Mann-Whitney test; data shown represents mean and s.e.m.; each point represents data from one embryo, n=2, 2, 2 and 2, left to right). c, Quantification of PLZF+ cells per seminiferous tubule in adult Ercc1-/fBlimp1-Cre+ and control mice (p-value calculated by 2-tailed Mann-Whitney test; data represent median and interquartile range ; n=150 tubules per genotype from n=3 mice). d, Frequency of PLZF+ cells per seminiferous tubule excluding tubules with no PLZF+ cells in adult Ercc1-/fBlimp1-Cre+ and control mice (p-value calculated by 2-tailed Mann-Whitney test; data shown represent mean and s.e.m.; n=150 tubules per genotype, 50 per mouse). e-f, Cumulative number of offspring over successive litters from male (e) and female (f) mice carrying the conditional Ercc1 allele and the PGC Cre-recombinase (Ercc1-/fBlimp1-Cre+) and congenic controls, bred with wildtype mates shows that expression of Ercc1 in PGCs is essential for fertility (data shown represent mean and s.e.m.; n=3 mice per genotype.

Supplementary Figure 4 Fanconi-mediated DNA crosslink repair is essential for fertility in mice.

a, Microscopic analysis of H&E-stained ovaries, testes and epididymis of 8-12-week old mice with constitutive deletions of Xpa, Csb, Fanca and mice that lack Ercc1 in PGCs (Ercc1-/fBlimp1-Cre+) (representative data, 3 independent animals per assessed per genotype and gender). b, Cumulative number of offspring over successive litters from male and female mice with constitutive deletions of Xpa, Csb, Fanca and mice that lack Ercc1 in PGCs (Ercc1-/fBlimp1-Cre+) when bred with wildtype mates (data shown represent mean and s.e.m.; n=3 mice per genotype and gender, mice were checked for evidence of copulation). c, Quantification of PLZF+ cells per tubule in 8-12-week old wildtype and Fanca-/- mice (p-value calculated by 2-tailed Mann-Whitney test; data represent median and the interquartile range; n=150 tubules per genotype, 50 per mouse). d, Distribution of the number of PLZF+ cells per tubule in 8-12-week old wildtype and Fanca-/- mice (n=150 tubules per genotype, 50 per mouse). e, Frequency of PLZF+ cells per seminiferous tubule excluding tubules with no PLZF+ cells in 8- to 12-week old mice (p-value calculated by 2-tailed Mann-Whitney test; data shown represent mean and s.e.m.; n=150 tubules per genotype, 50 per mouse).

Supplementary Figure 5 ERCC1 and FANCA are dispensable for entry and progression through meiosis.

a, Frequency of meiotic intermediates observed in wildtype and constitutive Ercc1-/- and Fanca-/- females at E16.5 and E18.5, and male mice at P21 and P50 (as Ercc1-/- mice die before P50 Ercc1-/fStra8-iCre+ males were employed) (data represent mean and s.e.m.; n=3 embryos or mice per genotype and gender). b, Representative immunofluorescence images of meiotic cells from P21 mice (representative data). c, Generation of offspring using a conditional Ercc1 or Fanca allele with Stra8-iCre. d, Flow cytometry plot showing the spermatogonia (Sp) used for isolation of cells from the testes of adult mice (all samples that were sorted were similar to this plot). Gating strategy shown in Supplementary Fig. 18. e, qRT-PCR expression analysis of the spermatogonial stem cell marker Plzf (data represent mean and s.e.m.; each point represents one mouse, n=4 biologically independent samples per genotype, expression was normalized to Gapdh and made relative to the sorted spermatogonia population). f, Immunoblot for PLZF on equivalent cell numbers of whole testis lysate and sorted populations. g,h, Immunoblot for ERCC1 and FANCA in whole cell spermatogonia lysates shows successful Stra8-iCre-mediated loss of ERCC1 or FANCA, respectively. Uncropped images in Supplementary Fig. 17 These blots are representative of 3 independent experiments i, Propidium iodide DNA content analysis of whole testis and sorted spermatogonia. This experiment was repeated on three separate occasions. Gating strategy shown in Supplementary Fig. 19.

Supplementary Figure 6 Generation of functional gametes in the absence of ERCC1 or FANCA.

a, Cumulative number of offspring over successive months from male mice carrying the conditional Ercc1 allele and the Cre-recombinase Stra8-iCre and congenic controls bred with wildtype mates (data shown represent mean and s.e.m.; n=3 mice per genotype). b, Representative genotyping gels from the offspring of Ercc1-/fStra8-iCre+ and congenic control showing the inheritance of recombined alleles in offspring when mated with wildtype mates. c, The frequency of Stra8-iCre-mediated recombination as determined by the inheritance of recombined alleles by offspring of Ercc1-/fStra8-iCre+ and control mice when mated with wildtype mates (p-value calculated by 2-tailed Mann-Whitney test; data shown represent mean and s.e.m.; each point represents the total offspring of one animal over 3 litters, n=4 and 6, left to right). d, Cumulative number of offspring from male mice carrying the conditional Fanca allele and Stra8-iCre and congenic controls bred with wildtype mates (data shown represent mean and s.e.m.; n=3 mice per genotype). e, Representative genotyping gels from the offspring of Fanca-/fStra8-iCre+ and congenic control showing the inheritance of recombined alleles in offspring when mated with wildtype mates. f, The frequency of Stra8-iCre-mediated recombination as determined by the inheritance of recombined alleles by offspring of Fanca-/fStra8-iCre+ and control mice when mated with wildtype mates (p-value calculated by 2-tailed Mann-Whitney test; data shown represent mean and s.e.m.; each point represents the total offspring of one animal over 3 litters, n=3 and 4, left to).

Supplementary Figure 7 FANCA and ERCC1 are required for PGC-like cell (PGCLC) development in vitro.

a, Immunoblot for ERCC1 and FANCA in whole cell lysates shows successful loss of ERCC1 and FANCA, respectively, in targeted mouse embryonic stem cells (mESCs) (ES clones were screened by immunoblot and the blot repeated for the clones used in the assays below, the blot was repeated on 3 occasions). Uncropped images in Supplementary Fig. 17 b, Ercc1-/- mESCs are hypersensitive to ultraviolet (UV) irradiation and mitomycin c (MMC) and Fanca-/- mESCs are hypersensitive to just mitomycin c (MMC) (data represent mean and s.e.m.; data represent 3 independent experiments, each carried out in triplicate). c,d, In vitro differentiation of mESCs carrying the PGC-specific GOF18-GFP reporter into primordial germ cell-like cells (PGCLCs). (c) Representative images of GOF18-GFP fluorescence and flow cytometry quantification of PGCLCs (SSEA1+GOF18-GFP+) formed from parental and ERCC1-deficient mESCs (p-value calculated by 2-tailed Mann-Whitney test; data represent mean and s.e.m.; each point represents data from one independent experiment, n= 9 and 5, left to right). (d) Representative images of GOF18-GFP fluorescence and flow cytometry quantification of PGCLCs (SSEA1+GOF18-GFP+) formed from FANCA-deficient and congenic control mESCs (p-value calculated by 2-tailed Mann-Whitney test; data represent mean and s.e.m.; each point represents data from one independent experiment, n= 9 and 8, left to right).

Supplementary Figure 8 FANCA is required for both male and female PGC development.

a, Quantification of PGCs (SSEA1+GOF18-GFP+) per gonad at E11.5, E12.5 and E13.5 from wildtype and Fanca-/- embryos (p-value calculated by 2-tailed Mann-Whitney test; data represent mean and s.e.m.; each point represents data from one embryo, E11.5 n=9 and 13, E12.5 n=13 and 22, E13.5 n=5 and 9, left to right). b, Quantification of PGCs (SSEA1+GOF18-GFP+) per gonad at E11.5, E12.5 and E13.5 from wildtype and Fanca-/- embryos separated by sex (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, E11.5 n=6, 2, 6 and 5, E12.5 n=9, 4, 7 and 4, E13.5 n=2, 2, 14 and 7, left to right).

Supplementary Figure 9 Fanconi-mediate crosslink repair is essential for normal fertility in mice and crosslink repair-deficient PGCs express germ cell markers.

a, H&E staining of ovaries, testes and epididymis of 8-12-week old mice with constitutive deletions of Fan1 and Slx4 shows that SLX4 but not FAN1 is required for gamete production in mice (representative data from 3 independent animals per genotype and gender). b, Cumulative number of offspring over successive litters from male and female mice with constitutive deletions of Fan1 or Slx4 when mated with wildtype mates (data represent mean and s.e.m.; n=3 mice per genotype and gender, mice were checked for evidence of copulation). c, Quantification of the germ cell compartment by flow cytometry of gonads from wildtype, Fanca-/-, Ercc1-/- and Fan1-/- E12.5 embryos shows that Fanconi-mediated crosslink repair is required for germ cell development (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=14, 10, 7 and 4, left to right). d, qRT-PCR expression analysis of early genital ridge germ cell markers (Ap2γ, Prdm14, Blimp-1 and Stella) in PGCs (SSEA1+GOF18-GFP+) and somatic cells (SSEA1-GOF18-GFP-) purified from E10.5 wildtype embryos (data represent mean and s.e.m.; each point represents data from one embryo, Ap2γ n=6, 4, 2, and 2, Prdm14 n=6, 2, 2 and 2, Blimp-1 n=4, 3, 3, and 3, Stella n=6, 3, 3, and 3, left to right). For each marker, the expression of each sample was normalized to Gapdh and made relative to the somatic cell expression.

Supplementary Figure 10 Crosslink repair-deficient PGCs migrate to the genital ridge.

a, Representative GOF18-GFP fluorescence images of whole-mount E9.5 wildtype and Ercc1-/- embryos. b, Microscopic quantification of the number of PGCs (GFP+ for GOF18-GFP transgene) in E9.5 wildtype and Ercc1-/- embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=9 and 3, left to right). c, Quantification of the distribution of PGCs (GFP+ for GOF18-GFP transgene) within the developing E9.5 embryo using somites as landmarks (data represent mean and s.e.m.; n=3 embryos per genotype). d, Representative GOF18-GFP fluorescence images of whole-mount E10.5 wildtype and Ercc1-/- embryos, the dashed line marks the outline of the embryo. e, Microscopic quantification of the number of PGCs (GFP+ for GOF18-GFP transgene) in E10.5 wildtype and Ercc1-/- embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=6 per genotype). f, Quantification of the number of ectopically located PGCs (GFP+ for GOF18-GFP transgene) in E10.5 wildtype and Ercc1-/- embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=4 and 5, left to right). g, Quantification of the distribution of PGCs (GFP+ for GOF18-GFP transgene) within the developing E10.5 embryo using somites as physiological landmarks (data represent mean and s.e.m.; n=4 embryos per genotype).

Supplementary Figure 11 Crosslink repair-deficient PGCs are reduced in number but localize to the genital ridges.

a, Representative GOF18-GFP fluorescence images of whole-mount E11.5 wildtype and Ercc1-/- embryos, the dashed line marks the outline of the two gonads. b, Quantification of the number of PGCs (GFP+ for GOF18-GFP transgene) in E11.5 wildtype and Ercc1-/- embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=9 and 5, left to right). c, Microscopic quantification of the number of PGCs (GFP+ for GOF18-GFP transgene) confined within the genital ridges of E11.5 wildtype and Ercc1-/- embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=4 and 3, left to right).

Supplementary Figure 12 Ercc1-/- and Fanca-/- PGCs successfully undergo DNA demethylation at Tex19.1 and Mili.

a, Genomic bisulfite sequencing analysis demonstrates promoter demethylation at Tex19.1 in FACS-purified PGCs (SSEA1+GOF18-GFP+) compared to surrounding somatic cells (SSEA1-GOF18-GFP-) in E12.5 Wildtype, Ercc1-/- and Fanca-/- embryos. b, Genomic bisulfite sequencing analysis demonstrates promoter demethylation at Mili in FACS-purified PGCs (SSEA1+GOF18-GFP+) compared to surrounding somatic cells (SSEA1-GOF18-GFP-) in E12.5 Wildtype, Ercc1-/- and Fanca-/- embryos. All data shown represent individual sequencing reads (filled = methylated CpG, open = unmethylated CpG, n=3 independent embryos per genotype).

Supplementary Figure 13 Ercc1-/- and Fanca-/- PGCs successfully undergo DNA demethylation at H19 DMR and Line1.

a, Bisulfite analysis of the H19 DMR in FACS-purified PGCs (SSEA1+GOF18-GFP+) compared to surrounding somatic cells (SSEA1-GOF18-GFP-) in E12.5 Wildtype, Ercc1-/- and Fanca-/- embryos. b, Bisulfite analysis of the CpG-rich region of the transposable element Line1 in FACS-purified PGCs (SSEA1+GOF18-GFP+) compared to surrounding somatic cells (SSEA1-GOF18-GFP-) in E12.5 Wildtype, Ercc1-/- and Fanca-/- embryos. All data shown represent individual sequencing reads (filled = methylated CpG, open = unmethylated CpG, n=3 independent embryos per genotype).

Supplementary Figure 14 Mutant PGCs replicate and accumulate markers of DNA damage, but undergo normal changes in histone modifications.

a, Representative flow cytometry plot and quantification of EdU+ PGCs (SSEA1+GOF18-GFP+) from E11.5 embryos following a single intraperitoneal (IP) injection of EdU. This shows that equivalent numbers of Ercc1-/- and Fanca-/- PGCs incorporate EdU compared to controls (p-value calculated by unpaired t-test; data shown as mean and SD, each point represents data from one embryo, n=2 per genotype). Gating strategy shown in Supplementary Figure 19. b-c, Quantification of the number of PGCs that stain positively for H3K27me3 from wildtype and Fanca-/- E11.5 embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=5, 3 and 3, left to right) and (c) quantification of the number of γ−H2A.X foci per nucleus. Left bar illustrates the percentage of PGCs with <10 γ−H2A.X foci per nucleus, which stain positively (red) or negatively (grey) for H3K27me3. The central pie chart illustrates the percentage of PGCs with <10 γ−H2A.X foci (blue) or >10 γ−H2A.X foci per nucleus (orange). The right bar illustrates the percentage of PGCs with >10 γ−H2A.X foci per nucleus, which either stain positively (red) or negatively (gray) for H3K27me3 (representative data from 3 independent animals per genotype). d-e, Quantification of the number of PGCs that stain positively for H3K9me2 from wildtype and Fanca-/- E11.5 embryos (p-value calculated by 2-tailed Mann-Whitney test; data shown as mean and s.e.m.; each point represents data from one embryo, n=5, 3 and 2, left to right) and (e) quantification of the number of γ−H2A.X foci per nucleus. Left bar illustrates the percentage of PGCs with <10 γ−H2A.X foci per nucleus, which stain positively (red) or negatively (gray) for H3K9me2. The central pie chart illustrates the percentage of PGCs with <10 γ−H2A.X foci (blue) or >10 γ−H2A.X foci per nucleus (orange). The right bar illustrates the percentage of PGCs with >10 γ−H2A.X foci per nucleus, which either stain positively (red) or negatively (gray) for H3K9me2 (representative data from 3 independent animals per genotype).

Supplementary Figure 15 PGCs with markers of DNA damage have phosphorylated p53.

a, Quantification of the number of PGCs that stain positively for phosphorylated p53 (pSer15-P53) and the number of γ−H2A.X foci per nucleus from E11.5 wildtype, Ercc1-/- and Fanca-/- embryos. Left bar illustrates the percentage of PGCs with <10 γ−H2A.X foci per nucleus, which either stain positively (red) or negatively (gray) for pSer15-P53. The central pie chart illustrates the percentage of PGCs with <10 γ−H2A.X foci (blue) or >10 γ−H2A.X foci per nucleus (orange). The right bar illustrates the percentage of PGCs with >10 γ−H2A.X foci per nucleus, which either stain positively (red) or negatively (gray) for pSer15-P53 (representative data from 3 independent animals per genotype).

Supplementary Figure 16 FA Crosslink repair is crucial during a narrow temporal window of PGC development.

DNA crosslink repair plays a critical role during a narrow window of embryonic PGC development. Crosslink repair is critical to prevent the accumulation of DNA double strand breaks. Surprisingly, this accumulation of damaged DNA is specific to the PGCs and does not occur in surrounding somatic cells. Damaged PGCs accumulated phosphorylated p53 and enter into apoptosis, providing a mechanism through which corrupted PGCs are eliminated, preventing their mutated genome being passed on to the next generation. (Upper panel) schema showing formation of an early embryo following fertilisation, which gives rise to somatic tissue from which the initial primordial germ cell (PGC) population is specified. The PGCs migrate towards and seed the genital ridge where they undergo epigenetic reprogramming prior to entering meiosis (at E13.5 in females and postnatally in males) to generate mature gametes. (Middle panel) FA crosslink repair-deficient PGCs migrate towards the genital ridge but are subsequently lost. (Lower panel) Crosslink repair-deficient PGCs accumulate unrepaired DNA DSBs within the developing gonad, stabilize p53 and are lost by apoptosis whilst the surrounding tissue is preserved.

Supplementary Figure 17

Uncropped western blots images.

Supplementary Figure 18

Flow cytometry gating strategy of PGCs and Spermatogonia

Supplementary Figure 19

Flow cytometry gating strategy of DNA content analysis and EdU incorporation.

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Supplementary Information

Supplementary Figs. 1–19, Supplementary Table 1 and Supplementary Note

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Hill, R.J., Crossan, G.P. DNA cross-link repair safeguards genomic stability during premeiotic germ cell development. Nat Genet 51, 1283–1294 (2019). https://doi.org/10.1038/s41588-019-0471-2

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