Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Apoptosis in the fetal testis eliminates developmentally defective germ cell clones

Nature Cell Biology volume 22, pages 1423–1435 (2020)Cite this article

Subjects

Abstract

Many germ cells are eliminated during development, long before oogenesis or spermatogenesis. In mouse fetal testes, the majority of germ cell apoptosis coincides with the onset of male differentiation, suggesting coordination of these processes. We studied fetal germ-cell fates and discovered that both apoptosis and differentiation initiate in clonally related clusters. Lineage tracing confirmed that germ cells die as clones independent of intercellular bridges, suggesting that shared intrinsic properties are apoptotic determinants. We identified transcriptional heterogeneity among fetal germ cells that included an apoptosis-susceptible population characterized by failure to differentiate, whereas successful differentiation to prospermatogonia occurred through the expression of epigenetically regulated genes, including LINE1. Our results indicate that the fetal germ-cell fate is based on discrete cell-heritable identities. Elevated DNA methylation in the apoptosis-susceptible subpopulation supports our hypothesis that earlier errors in germ-cell epigenetic reprogramming derail differentiation in cellular progeny, leading to fetal apoptotic selection that ultimately improves the gamete quality.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Germ-cell apoptosis is spatially clustered in fetal testes.
Fig. 2: Clustered germ-cell apoptosis does not require cytoplasmic sharing.
Fig. 3: Multicolour clonal labelling reveals clonal apoptosis during the fetal apoptotic wave.
Fig. 4: Identification of an apoptotically poised subpopulation by scRNA-seq of male germ cells.
Fig. 5: AP germ cells exhibit aberrant male sex differentiation at E13.5.
Fig. 6: Aberrantly differentiated germ cells are retained in the absence of apoptosis.
Fig. 7: Epigenetic differences distinguish the AP and MD germ-cell subpopulations.
Fig. 8: LINE1 expression is clonally heterogeneous and associated with male differentiation.

Similar content being viewed by others

Data availability

The scRNA-seq and BS-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under the accession codes GSE119045 and GSE155122. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Gene-ontology analysis on MSigDB computed overlaps with GO:BP (https://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=GO:BP). Source data are provided with this paper.

Code availability

Code for the scRNA-seq analysis is available at https://github.com/dnucsf/NatureCellBiology2020.

References

  1. Lehtonen, J. & Parker, G. A. Gamete competition, gamete limitation, and the evolution of the two sexes. Mol. Hum. Reprod. 20, 1161–1168 (2014).

    PubMed  Google Scholar 

  2. Gardner, R. L. & Rossant, J. Investigation of the fate of 4–5 day post-coitum mouse inner cell mass cells by blastocyst injection. J. Embryol. Exp. Morphol. 52, 141–152 (1979).

    CAS  PubMed  Google Scholar 

  3. Anderson, R., Copeland, T. K., Schöler, H., Heasman, J. & Wylie, C. The onset of germ cell migration in the mouse embryo. Mech. Dev. 91, 61–68 (2000).

    CAS  PubMed  Google Scholar 

  4. Cantú, A. V., Altshuler-Keylin, S. & Laird, D. J. Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling. J. Cell. Biol. 214, 215–229 (2016).

    PubMed  PubMed Central  Google Scholar 

  5. Saitou, M., Kagiwada, S. & Kurimoto, K. Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development 139, 15–31 (2012).

    CAS  PubMed  Google Scholar 

  6. Menke, D. B., Koubova, J. & Page, D. C. Sexual differentiation of germ cells in XX mouse gonads occurs in an anterior-to-posterior wave. Dev. Biol. 262, 303–312 (2003).

    CAS  PubMed  Google Scholar 

  7. Ohta, K. et al. Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice. Biol. Reprod. 86, 112 (2012).

    PubMed  PubMed Central  Google Scholar 

  8. Aitken, R. J., Findlay, J. K., Hutt, K. J. & Kerr, J. B. Apoptosis in the germ line. Reproduction 141, 139–150 (2011).

    CAS  PubMed  Google Scholar 

  9. Runyan, C. et al. Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration. Development 133, 4861–4869 (2006).

    CAS  PubMed  Google Scholar 

  10. Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A. & Korsmeyer, S. J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270, 96–99 (1995).

    CAS  PubMed  Google Scholar 

  11. Rodriguez, I., Ody, C., Araki, K., Garcia, I. & Vassalli, P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J. 16, 2262–2270 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Perez, G. I., Knudson, C. M., Leykin, L., Korsmeyer, S. J. & Tilly, J. L. Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction. Nat. Med. 3, 1228–1232 (1997).

    CAS  PubMed  Google Scholar 

  13. Coucouvanis, E. C., Sherwood, S. W., Carswell-Crumpton, C., Spack, E. G. & Jones, P. P. Evidence that the mechanism of prenatal germ cell death in the mouse is apoptosis. Exp. Cell Res. 209, 238–247 (1993).

    CAS  PubMed  Google Scholar 

  14. Wang, R. A., Nakane, P. K. & Koji, T. Autonomous cell death of mouse male germ cells during fetal and postnatal period. Biol. Reprod. 58, 1250–1256 (1998).

    CAS  PubMed  Google Scholar 

  15. Goriely, A., McVean, G. A. T., Röjmyr, M., Ingemarsson, B. & Wilkie, A. O. M. Evidence for selective advantage of pathogenic FGFR2 mutations in the male germ line. Science 301, 643–646 (2003).

    CAS  PubMed  Google Scholar 

  16. Ueno, H., Turnbull, B. B. & Weissman, I. L. Two-step oligoclonal development of male germ cells. Proc. Natl Acad. Sci. USA 106, 175–180 (2009).

    CAS  PubMed  Google Scholar 

  17. Cook, M. S., Munger, S. C., Nadeau, J. H. & Capel, B. Regulation of male germ cell cycle arrest and differentiation by DND1 is modulated by genetic background. Development 138, 23–32 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Foley, J. G. D. & Bard, J. B. L. Apoptosis in the cortex of the developing mouse kidney. J. Anat. 201, 477–484 (2002).

    PubMed  PubMed Central  Google Scholar 

  19. Laird, D. J., Altshuler-Keylin, S., Kissner, M. D., Zhou, X. & Anderson, K. V. Ror2 enhances polarity and directional migration of primordial germ cells. PLOS Genet. 7, e1002428 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Mendis, S. H. S., Meachem, S. J., Sarraj, M. A. & Loveland, K. L. Activin A balances Sertoli and germ cell proliferation in the fetal mouse testis. Biol. Reprod. 84, 379–391 (2011).

    CAS  PubMed  Google Scholar 

  21. Lei, L. & Spradling, A. C. Mouse primordial germ cells produce cysts that partially fragment prior to meiosis. Development 140, 2075–2081 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ventelä, S., Toppari, J. & Parvinen, M. Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing. Mol. Biol. Cell 14, 2768–2780 (2003).

    PubMed  PubMed Central  Google Scholar 

  23. Greenbaum, M. P. et al. TEX14 is essential for intercellular bridges and fertility in male mice. Proc. Natl Acad. Sci. USA 103, 4982–4987 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lu, K. L. & Yamashita, Y. M. Germ cell connectivity enhances cell death in response to DNA damage in the Drosophila testis. eLife 6, e27960 (2017).

    PubMed  PubMed Central  Google Scholar 

  25. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

  26. Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. T. & Weissman, I. L. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. Nature 476, 409–413 (2011).

    CAS  PubMed  Google Scholar 

  27. Greder, L. V. et al. Analysis of endogenous Oct4 activation during induced pluripotent stem cell reprogramming using an inducible Oct4 lineage label. Stem Cells 30, 2596–2601 (2012).

    PubMed  PubMed Central  Google Scholar 

  28. Western, P. S., Miles, D. C., van den Bergen, J. A., Burton, M. & Sinclair, A. H. Dynamic regulation of mitotic arrest in fetal male germ cells. Stem Cells 26, 339–347 (2008).

    CAS  PubMed  Google Scholar 

  29. Bondar, T. & Medzhitov, R. p53-Mediated hematopoietic stem and progenitor cell competition. Cell Stem Cell 6, 309–322 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Bowling, S. et al. P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development. Nat. Commun. 9, 1763 (2018).

    PubMed  PubMed Central  Google Scholar 

  31. Haupt, S., Berger, M., Goldberg, Z. & Haupt, Y. Apoptosis—the p53 network. J. Cell Sci. 116, 4077–4085 (2003).

    CAS  PubMed  Google Scholar 

  32. de Felici, M. D. et al. Bcl-2 and Bax regulation of apoptosis in germ cells during prenatal oogenesis in the mouse embryo. Cell Death Differ. 6, 908–915 (1999).

    CAS  PubMed  Google Scholar 

  33. Rucker, E. B. et al. Bcl-x and Bax regulate mouse primordial germ cell survival and apoptosis during embryogenesis. Mol. Endocrinol. 14, 1038–1052 (2000).

    CAS  PubMed  Google Scholar 

  34. Maclean, J. A. et al. Rhox: a new homeobox gene cluster. Cell 120, 369–382 (2005).

    CAS  PubMed  Google Scholar 

  35. Spiller, C. M. et al. Endogenous Nodal signaling regulates germ cell potency during mammalian testis development. Development 139, 4123–4132 (2012).

    CAS  PubMed  Google Scholar 

  36. Saba, R., Kato, Y. & Saga, Y. NANOS2 promotes male germ cell development independent of meiosis suppression. Dev. Biol. 385, 32–40 (2014).

    CAS  PubMed  Google Scholar 

  37. Molaro, A. et al. Two waves of de novo methylation during mouse germ cell development. Genes Dev. 28, 1544–1549 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Suzuki, A. & Saga, Y. Nanos2 suppresses meiosis and promotes male germ cell differentiation. Genes Dev. 22, 430–435 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Milholland, B. et al. Differences between germline and somatic mutation rates in humans and mice. Nat. Commun. 8, 15183 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Seisenberger, S. et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol. Cell 48, 849–862 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Hill, P. W. S. et al. Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte. Nature 555, 392–396 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Hargan-Calvopina, J. et al. Stage-specific demethylation in primordial germ cells safeguards against precocious differentiation. Dev. Cell 39, 75–86 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hajkova, P. et al. Epigenetic reprogramming in mouse primordial germ cells. Mech. Dev. 117, 15–23 (2002).

    CAS  PubMed  Google Scholar 

  44. Malki, S., van der Heijden, G. W., O’Donnell, K. A., Martin, S. L. & Bortvin, A. A role for retrotransposon LINE-1 in fetal oocyte attrition in mice. Dev. Cell 29, 521–533 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Reznik, B. et al. Heterogeneity of transposon expression and activation of the repressive network in human fetal germ cells. Development 146, dev.171157 (2019).

  46. Brockway, N. L. et al. Multicolour lineage tracing using in vivo time-lapse imaging reveals coordinated death of clonally related cells in the developing vertebrate brain. Dev. Biol. 453, 130–140 (2019).

    CAS  PubMed  Google Scholar 

  47. Watanabe, K. et al. Cripto-1 is a cell surface marker for a tumorigenic, undifferentiated subpopulation in human embryonal carcinoma cells. Stem Cells 28, 1303–1314 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Spiller, C. M., Bowles, J. & Koopman, P. Nodal/Cripto signaling in fetal male germ cell development: implications for testicular germ cell tumors. Int. J. Dev. Biol. 57, 211–219 (2013).

    CAS  PubMed  Google Scholar 

  49. Dawson, E. P. et al. Delayed male germ cell sex-specification permits transition into embryonal carcinoma cells with features of primed pluripotency. Development 145, dev156612 (2018).

    PubMed  PubMed Central  Google Scholar 

  50. Aravin, A. A. et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 31, 785–799 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Hansson K. et al. RipleyGUI: software for analyzing spatial patterns in 3D cell distributions. Front. Neuroinform. 7, 5 (2013).

  52. Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    CAS  PubMed  Google Scholar 

  53. Jin, Y. et al. TEtranscripts: a package for including transposable elements in differential expression analysis of RNA-seq datasets. Bioinformatics 31, 3593–3599 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Liberzon, A. et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 1, 417–425 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Subramanaian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    Google Scholar 

Download references

Acknowledgements

Mice were provided by I.L. Weissman, M. Matzuk, and M. Bartolomei. ORF1p antibody was provided by A. Wilkin, A. Bortvin, and A. Wiblin. J. Sneddon, M. Conti, T. Nystul, A.W. De Tomaso, L. Byrnes, D. Wong, R.G. Jaszczak, S. Cincotta, and B. Reznik provided feedback. J. Daza, G. Zhang, A. Villanueva, D. Palomera, I. Vainchtein, C. Bispo, R. Prasasva, and B. Dreskin assisted with experiments. Funding included an NSF Predoctoral Fellowship to D.H.N. and NIH 1DP2OD007420, R01GM122902, R01ES028212, and Cancer Research Coordinating Committee grants to D.J.L.

Author information

Authors and Affiliations

  1. Department of Obstetrics, Gynecology and Reproductive Science, Center for Reproductive Sciences, Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA, USA

    Daniel H. Nguyen, Bikem Soygur, Su-Ping Peng & Diana J. Laird

  2. Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Durham, NC, USA

    Safia Malki & Guang Hu

Authors
  1. Daniel H. Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bikem Soygur
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Su-Ping Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Safia Malki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guang Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Diana J. Laird
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.H.N. and D.J.L. conceived of the study, analysed the data and wrote the manuscript. D.H.N., B.S., S.P., S.M. and G.H. conducted experiments and analysed results. All authors contributed comments.

Corresponding author

Correspondence to Diana J. Laird.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Apoptotic clusters are randomly distributed throughout fetal testes.

a, Percent apoptotic cleaved-PARP+ germ cells in whole-mount testes on a mixed genetic background during the apoptotic wave. n=4 animals. Data presented as mean values ± SD. Dashed line denotes reported average apoptotic index in developing fetal tissues18. b, Percent cleaved-PARP+ cells on apoptosis-sensitive C57/Bl6 background20 at peak apoptosis (E13.5). c, Distribution of apoptotic clusters measured for spatial bias. 3D coordinates of each cluster’s centroid were evaluated by Ripley’s K function. These were compared against simulated distributions for an equivalent number of points. Representative plots depict each simulated distribution type (right). Cluster coordinates from n=5 animals.**: p=0, †: p=0.10622, between-treatments sum of squares. d, E13.5 testis section stained for Sertoli cells (AMH), germ cells (TRA98), and apoptosis (cleaved-PARP). Apoptotic cords are defined as cords containing any apoptotic germ cells (red outline) in contrast to non-apoptotic cords (white outline). Scale bar = 20µm. e, Ratio of total germ cells to Sertoli cells in apoptotic and non-apoptotic cords, p=0.584, two-tailed Student’s t-test. Data points represent individual cords pooled from sections from 4 animals. n =15 non-apoptotic cords and n = 11 apoptotic cords. Data presented as mean values ± SD. f, Average numbers of Sertoli cells in direct contact with each germ cell in apoptotic and non-apoptotic cords. Within apoptotic cords, apoptotic (PARP+) and non-apoptotic (PARP-) germ cells were separately considered. p=0.737154, one-way ANOVA. Data points represent individual germ cells (n) analysed in sections from 4 animals. Total cell counts per category are detailed in source data. Data presented as mean values ± SD. g, Quantification of apoptotic germ cells in E13.5 Tex14 mutants. p=0.4491, two-tailed Student’s t-test. n=4 animals per genotype. Data presented as mean values ± SD. h, Apoptotic cluster size by cell number in Tex14 E13.5. p=0.0073, two-tailed Student’s t-test. Data points (n) are available in source data and represent individual apoptotic clusters analysed from 2 animals (Tex14+/+) and 3 animals (Tex14-/-). Data presented as mean values ± SD.

Source data

Extended Data Fig. 2 Clonal boundary determination for fragmented apoptotic clusters.

a, Detection of clonal apoptosis in Confetti-labelled germ cells at E13.5. Apoptotic germ cells identified by cleaved-PARP staining (white outlines). Scale bar = 20um. 5 animals. Inset: Hoechst staining. b, Confetti -labelled germ cells imaged in whole-mount and segmented using the Imaris Cell module displayed fragmentation by E13.5, in accordance with published findings21. We identified three-dimensional coordinates for centroids (grey dots) of each cell (represented by yellow spheres) within a clone to measure the centroid-to-centroid distances between nearest-neighbour germ cells belonging to different fragments. In depicted clone, \(\overline {BC}\) spans nearest-neighbour cells between Fragments 1 and 2 while \(\overline {DE}\) spans nearest-neighbour cells from Fragments 3 and 2. We took the largest of these nearest-neighbour distances from all fragments pairs to represent the maximum potential dispersion between fragments of a single clone. Scale bar = 5um.

Extended Data Fig. 3 Clonal apoptotic clusters are maintained in the absence of intercellular bridges.

a, Cleaved-PARP staining for apoptosis in whole-mount E13.5 Tex14-/- testes on a R26R Rainbow clonally labelled background reveals mCherry apoptotic clone (outlined in yellow). Scale bar = 10µm. Rotated view of same clones confirming that constituent cells of mCherry clone are disconnected. Nearby mCerulean clone is negative for apoptosis. b, Extended view of entire whole-mount E13.5 testis. Scale bar = 100µm.

Extended Data Fig. 4 Single-cell-RNAseq analysis of E13.5 male germ cells.

a, Average expression of p53-activated genes by germ cell subpopulation. Expression from n=2,556 single cells. b, Subpopulation size in cell number and as a percentage of total germ cells analysed for scRNA-seq at E13.5. c, Average pro-survival gene Nanos3 expression by germ cell subpopulation. Expression from n=2,556 single cells. d, Average expression of prosurvival genes Kit, adhesion factor Epcam, and PGC identity gene Ifitm3 by germ cell subpopulation. Expression from n=2,556 single cells.

Source data

Extended Data Fig. 5 LEFTY1/2+ cells are increased in p53-/- testes by E13.5 compared to p53+/-.

a, Immunofluorescence detection of LEFTY1/2 in sections from E13.5 p53+/- and p53-/- testes. Scale bar = 50µm. 2 animals per genotype. b, Quantification of percent LEFTY1/2+ germ cells from E13.5 p53+/- and p53-/- testes. n=2 animals per genotype.

Source data

Extended Data Fig. 6 E12.5 and E13.5 germ cells remain transcriptionally distinct in merged analysis.

a, Merged dataset with E12.5 and E13.5 germ cells after transcriptional clustering. E13.5 AP-germ cell identities coloured while E12.5 boundary is denoted by dashed black line. t-SNE represents 5,106 cells. b, t-SNE plots showing expression of apoptotic genes (Bad, Bax), male differentiation genes (Nanos2, Mael), and genes associated with a pre-male differentiation population (Rhox6, Lefty2). Cell shading is derived from single-cell expression values normalized to global expression for all genes and log-transformed via Seurat.

Extended Data Fig. 7 Model for coordination of clonal apoptosis with male differentiation.

Clonal variation in differentiation potential results in divergent clonal fates: germ cells normally activate male differentiation programs marked by Nanos2 but a subset of clones aberrantly maintain an undifferentiated state characterized by expression of pro-apoptotic genes that ultimately results in clonal death.

Extended Data Fig. 8 LEFTY expression is clonally heterogeneous.

LEFTY expression in E13.5 Confetti testis, LEFTY-high cells (open arrowheads) and LEFTY-low cells (white arrowheads) with clone boundaries indicated by dashed outlines. Scale bar = 20µm.

Extended Data Fig. 9 Population-specific expression of epigenetically regulated genes including LINE1 during male differentiation.

a, Whole-genome bisulfite sequencing comparison of sorted AP and MD populations for relative methylation percentage. Data are presented as median values with first and third quartiles. Whiskers represent minimum and maximums. Sequencing was performed on 2 separate runs of n=2,000 and n=6,000 cells each for both populations. b, ORF1p expression during the apoptotic wave. ORF1p is expressed in small clusters of germ cells at E13.5 (white arrowhead) and progresses toward homogenous expression by E15.5. Scale bar = 100µm. c, ORF1p and cleaved PARP staining in E13.5 wild-type sections. Scale bar = 25µm. d, ORF1p-negative germ cell clusters in E15.5 Bax-/- compared to Bax+/-. Arrowheads denote ORF1p-negative cells. Insets: Higher-magnification image of ORF1p-negative cluster compared to ORF1p-positive neighbouring germ cells. Scale bar = 50µm.

Source data

Extended Data Fig. 10 Differentially expressed repeat elements identify distinct subpopulations of E13.5 germ cells.

a, Abridged table of repeat element markers for subpopulations defined by scRNA-seq clustering based on expression of non-repetitive elements. p value calculated by two-sided Wilcoxon rank-sum test. Expression from n=2,043 cells. Full table of markers available in source data. b, Expression of MD marker Nanos2, AP marker Rhox6, and LINE1 members across clusters identified by expression of non-repetitive elements. Predominant expression of Nanos2 in cluster 4 is characteristic of MD population. Predominant expression of AP marker Rhox6 in cluster 5 is characteristic of AP population. Violin plots display cell densities as shape and individual cells as dots. The number of cells (n) for each cluster are detailed in source data. c, Abridged table of repeat element markers for subpopulations defined by scRNA-seq based only on expression of repetitive elements. P value calculated by two-sided Wilcoxon rank-sum test. Expression from n=2,043 cells. Full table of markers available in source data. d, Expression of MD marker Nanos2, AP marker Rhox6, and LINE1 members across clusters identified only by expression of repeat elements. Violin plots display cell densities as shape and individual cells as dots. The number of cells (n) for each cluster are detailed in source data.

Source data

Supplementary information

Reporting Summary

Supplementary Tables 1 and 2

Supplementary Table 1. Transcriptional markers that distinguish each germ-cell state compared with all others were identified among differentially expressed genes in an E13.5 germ-cell population using the two-tailed Wilcoxon rank-sum test-based FindMarkers operation in Seurat, which provides a P value that was further adjusted using Bonferroni’s correction using all genes in the dataset. Supplementary Table 2. Comparison of the markers for MD germ cells with GRRs and genes upregulated in Dnmt1-cKO male germ cells42. Comparison of markers for AP germ cells with genes upregulated in Tet1-KO41 male germ cells and genes downregulated in Dnmt1 cKO. Adjusted P values for the MD and AP markers were generated using the Wilcoxon rank-sum test with Bonferroni’s correction. Statistics for markers from Dnmt1 cKO and Tet1-KO were obtained from referenced studies.

41556_2020_603_MOESM3_ESM.mp4

Supplementary Video 1. Whole-mount visualization of apoptotic clusters of PGCs at E13.5. Whole E13.5 testis with cell nuclei identified by Hoechst and PGCs by TRA98. Apoptosis was identified with cPARP and rotated views highlight the spatial distribution of apoptotic PGCs. The magnified views show a representative apoptotic germ cell cluster.

41556_2020_603_MOESM4_ESM.mp4

Supplementary Video 2. PGC proliferation is clonal. E13.5 PGCs were clonally labelled using Rainbow and cells in the late G2/M phase were marked by phospho-histone H3. The magnified view shows two separate groups of phospho-histone H3+ cells that are clonal (mOrange clone and a GFP− clone).

41556_2020_603_MOESM5_ESM.mov

Supplementary Video 3. Multicolour clone labelling reveals shared fluorophores within germ-cell cysts. Three-dimensional perspective of whole E13.5 Rainbow; Tex14+/+; Pou5f1-Cre-ER testis with a bicoloured clone (mCerulean and mCherry) outlined as a surface, followed by individual channels confirming bicoloured labelling.

41556_2020_603_MOESM6_ESM.mp4

Supplementary Video 4. Tex14-mutant testes differ in clone structure and do not form bicoloured clones. Three-dimensional perspective of whole E13.5 Rainbow; Tex14−/−; Pou5f1-Cre-ER testis with strictly single-coloured clones as a consequence of the absence of intercellular bridges.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1b

Source Data Fig. 2

Statistical source data for Fig. 2b

Source Data Fig. 3

Statistical source data for Fig. 3d,e

Source Data Fig. 4

Statistical source data for Fig. 4b,d

Source Data Fig. 6

Statistical source data for Fig. 6a–c

Source Data Fig. 7

Statistical source data for Fig. 7c–e

Source Data Fig. 8

Statistical source data for Fig. 8b,d,e

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1c,e–h

Source Data Extended Data Fig. 4

Statistical source data for Extended Data Fig. 4a,c,d

Source Data Extended Data Fig. 5

Statistical source data for Extended Data Fig. 5b

Source Data Extended Data Fig. 9

Statistical source data for Extended Data Fig. 9a

Source Data Extended Data Fig. 10

Statistical source data for Extended Data Fig. 10a–d

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nguyen, D.H., Soygur, B., Peng, SP. et al. Apoptosis in the fetal testis eliminates developmentally defective germ cell clones. Nat Cell Biol 22, 1423–1435 (2020). https://doi.org/10.1038/s41556-020-00603-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-020-00603-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing