E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish

Autoři: Haibo Xie aff001;  Yunsi Kang aff001;  Shuo Wang aff001;  Pengfei Zheng aff001;  Zhe Chen aff001;  Sudipto Roy aff004;  Chengtian Zhao aff001
Působiště autorů: Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao, Shandong, China aff001;  Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, China aff002;  Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China aff003;  Institute of Molecular and Cell Biology, Singapore, Singapore aff004;  Department of Biological Sciences, National University of Singapore, Singapore, Singapore aff005;  Department of Pediatrics, Yong Loo Ling School of Medicine, National University of Singapore, Singapore, Singapore aff006
Vyšlo v časopise: E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish. PLoS Genet 16(3): e32767. doi:10.1371/journal.pgen.1008655
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008655


E2f5 is a member of the E2f family of transcription factors that play essential roles during many cellular processes. E2f5 was initially characterized as a transcriptional repressor in cell proliferation studies through its interaction with the Retinoblastoma (Rb) protein for inhibition of target gene transcription. However, the precise roles of E2f5 during embryonic and post-embryonic development remain incompletely investigated. Here, we report that zebrafish E2f5 plays critical roles during spermatogenesis and multiciliated cell (MCC) differentiation. Zebrafish e2f5 mutants develop exclusively as infertile males. In the mutants, spermatogenesis is arrested at the zygotene stage due to homologous recombination (HR) defects, which finally leads to germ cell apoptosis. Inhibition of cell apoptosis in e2f5;tp53 double mutants rescued ovarian development, although oocytes generated from the double mutants were still abnormal, characterized by aberrant distribution of nucleoli. Using transcriptome analysis, we identified dmc1, which encodes an essential meiotic recombination protein, as the major target gene of E2f5 during spermatogenesis. E2f5 can bind to the promoter of dmc1 to promote HR, and overexpression of dmc1 significantly increased the fertilization rate of e2f5 mutant males. Besides gametogenesis defects, e2f5 mutants failed to develop MCCs in the nose and pronephric ducts during early embryonic stages, but these cells recovered later due to redundancy with E2f4. Moreover, we demonstrate that ion transporting principal cells in the pronephric ducts, which remain intercalated with the MCCs, do not contain motile cilia in wild-type embryos, while they generate single motile cilia in the absence of E2f5 activity. In line with this, we further show that E2f5 activates the Notch pathway gene jagged2b (jag2b) to inhibit the acquisition of MCC fate as well as motile cilia differentiation by the neighboring principal cells. Taken together, our data suggest that E2f5 can function as a versatile transcriptional activator and identify novel roles of the protein in spermatogenesis as well as MCC differentiation during zebrafish development.

Klíčová slova:

Cell differentiation – Cell motility – Cilia – Embryos – Gene expression – Spermatogenesis – Testes – Zebrafish


1. DeGregori J., Johnson D. G., Distinct and Overlapping Roles for E2F Family Members in Transcription, Proliferation and Apoptosis. Current molecular medicine 6, 739–748 (2006). doi: 10.2174/1566524010606070739 17100600

2. Chen H. Z., Tsai S. Y., Leone G., Emerging roles of E2Fs in cancer: an exit from cell cycle control. Nature reviews. Cancer 9, 785–797 (2009). doi: 10.1038/nrc2696 19851314

3. Dick F. A., Goodrich D. W., Sage J., Dyson N. J., Non-canonical functions of the RB protein in cancer. Nature reviews. Cancer 18, 442–451 (2018).

4. Ertosun M. G., Hapil F. Z., Osman Nidai O., E2F1 transcription factor and its impact on growth factor and cytokine signaling. Cytokine & growth factor reviews 31, 17–25 (2016).

5. Attwooll C., Lazzerini Denchi E., Helin K., The E2F family: specific functions and overlapping interests. The EMBO journal 23, 4709–4716 (2004). doi: 10.1038/sj.emboj.7600481 15538380

6. Humbert P. O. et al., E2F4 is essential for normal erythrocyte maturation and neonatal viability. Molecular cell 6, 281–291 (2000). doi: 10.1016/s1097-2765(00)00029-0 10983976

7. Rempel R. E. et al., Loss of E2F4 activity leads to abnormal development of multiple cellular lineages. Molecular cell 6, 293–306 (2000). doi: 10.1016/s1097-2765(00)00030-7 10983977

8. Ruzhynsky V. A. et al., Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon. The Journal of neuroscience: the official journal of the Society for Neuroscience 27, 5926–5935 (2007).

9. Danielian P. S., Hess R. A., Lees J. A., E2f4 and E2f5 are essential for the development of the male reproductive system. Cell cycle 15, 250–260 (2016). doi: 10.1080/15384101.2015.1121350 26825228

10. Lindeman G. J. et al., A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting. Genes & development 12, 1092–1098 (1998).

11. Spassky N., Meunier A., The development and functions of multiciliated epithelia. Nature reviews. Molecular cell biology 18, 423–436 (2017). doi: 10.1038/nrm.2017.21 28400610

12. Brooks E. R., Wallingford J. B., Multiciliated cells. Current biology: CB 24, R973–982 (2014). doi: 10.1016/j.cub.2014.08.047 25291643

13. Liu Y., Pathak N., Kramer-Zucker A., Drummond I. A., Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros. Development 134, 1111–1122 (2007). doi: 10.1242/dev.02806 17287248

14. Mori M. et al., Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors. Development 142, 258–267 (2015). doi: 10.1242/dev.116855 25564622

15. Tsao P. N. et al., Notch signaling controls the balance of ciliated and secretory cell fates in developing airways. Development 136, 2297–2307 (2009). doi: 10.1242/dev.034884 19502490

16. Marcet B. et al., Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway. Nature cell biology 13, 693–699 (2011). doi: 10.1038/ncb2241 21602795

17. Deblandre G. A., Wettstein D. A., Koyano-Nakagawa N., Kintner C., A two-step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos. Development 126, 4715–4728 (1999). 10518489

18. Ma M., Jiang Y. J., Jagged2a-notch signaling mediates cell fate choice in the zebrafish pronephric duct. PLoS genetics 3, e18 (2007). doi: 10.1371/journal.pgen.0030018 17257056

19. Zhou F. et al., Gmnc Is a Master Regulator of the Multiciliated Cell Differentiation Program. Current biology: CB 25, 3267–3273 (2015). doi: 10.1016/j.cub.2015.10.062 26778655

20. Terre B. et al., GEMC1 is a critical regulator of multiciliated cell differentiation. The EMBO journal 35, 942–960 (2016). doi: 10.15252/embj.201592821 26933123

21. Arbi M. et al., GemC1 controls multiciliogenesis in the airway epithelium. EMBO reports 17, 400–413 (2016). doi: 10.15252/embr.201540882 26882546

22. Stubbs J. L., Vladar E. K., Axelrod J. D., Kintner C., Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation. Nature cell biology 14, 140–147 (2012). doi: 10.1038/ncb2406 22231168

23. Chong Y. L., Zhang Y., Zhou F., Roy S., Distinct requirements of E2f4 versus E2f5 activity for multiciliated cell development in the zebrafish embryo. Developmental biology 443, 165–172 (2018). doi: 10.1016/j.ydbio.2018.09.013 30218642

24. Ma L., Quigley I., Omran H., Kintner C., Multicilin drives centriole biogenesis via E2f proteins. Genes & development 28, 1461–1471 (2014).

25. Mori M. et al., Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis. Nature communications 8, 15857 (2017). doi: 10.1038/ncomms15857 28675157

26. Zhao H. et al., The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis. Nature cell biology 15, 1434–1444 (2013). doi: 10.1038/ncb2880 24240477

27. Wang Y., Copenhaver G. P., Meiotic Recombination: Mixing It Up in Plants. Annu Rev Plant Biol 69, 577–609 (2018). doi: 10.1146/annurev-arplant-042817-040431 29489392

28. Neale M. J., Keeney S., Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158 (2006). doi: 10.1038/nature04885 16838012

29. Sehorn M. G., Sigurdsson S., Bussen W., Unger V. M., Sung P., Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature 429, 433–437 (2004). doi: 10.1038/nature02563 15164066

30. Cloud V., Chan Y. L., Grubb J., Budke B., Bishop D. K., Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science 337, 1222–1225 (2012). doi: 10.1126/science.1219379 22955832

31. Da Ines O. et al., Meiotic recombination in Arabidopsis is catalysed by DMC1, with RAD51 playing a supporting role. PLoS genetics 9, e1003787 (2013). doi: 10.1371/journal.pgen.1003787 24086145

32. Bisig C. G. et al., Synaptonemal complex components persist at centromeres and are required for homologous centromere pairing in mouse spermatocytes. PLoS genetics 8, e1002701 (2012). doi: 10.1371/journal.pgen.1002701 22761579

33. Saito K., Siegfried K. R., Nusslein-Volhard C., Sakai N., Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants. Dev Dyn 240, 1779–1792 (2011). doi: 10.1002/dvdy.22661 21594953

34. Mahadevaiah S. K. et al., Recombinational DNA double-strand breaks in mice precede synapsis. Nat Genet 27, 271–276 (2001). doi: 10.1038/85830 11242108

35. Rodriguez-Mari A. et al., Roles of brca2 (fancd1) in oocyte nuclear architecture, gametogenesis, gonad tumors, and genome stability in zebrafish. PLoS genetics 7, e1001357 (2011). doi: 10.1371/journal.pgen.1001357 21483806

36. Shive H. R. et al., brca2 in zebrafish ovarian development, spermatogenesis, and tumorigenesis. Proc Natl Acad Sci U S A 107, 19350–19355 (2010). doi: 10.1073/pnas.1011630107 20974951

37. Zhao W., Wiese C., Kwon Y., Hromas R., Sung P., The BRCA Tumor Suppressor Network in Chromosome Damage Repair by Homologous Recombination. Annu Rev Biochem 88, 221–245 (2019). doi: 10.1146/annurev-biochem-013118-111058 30917004

38. Sun Y., McCorvie T. J., Yates L. A., Zhang X., Structural basis of homologous recombination. Cell Mol Life Sci 77, 3–18 (2020). doi: 10.1007/s00018-019-03365-1 31748913

39. Kagawa W., Kurumizaka H., From meiosis to postmeiotic events: uncovering the molecular roles of the meiosis-specific recombinase Dmc1. FEBS J 277, 590–598 (2010). doi: 10.1111/j.1742-4658.2009.07503.x 20015079

40. Chen J. et al., Disruption of dmc1 Produces Abnormal Sperm in Medaka (Oryzias latipes). Sci Rep 6, 30912 (2016). doi: 10.1038/srep30912 27480068

41. Drummond I. A., Davidson A. J., Zebrafish kidney development. Methods Cell Biol 100, 233–260 (2010). doi: 10.1016/B978-0-12-384892-5.00009-8 21111220

42. Zhao C., Malicki J., Genetic defects of pronephric cilia in zebrafish. Mech Dev 124, 605–616 (2007). doi: 10.1016/j.mod.2007.04.004 17576052

43. Nguyen A. T. et al., An inducible kras(V12) transgenic zebrafish model for liver tumorigenesis and chemical drug screening. Disease models & mechanisms 5, 63–72 (2012).

44. Kang Y., Xie H., Zhao C., Ankrd45 Is a Novel Ankyrin Repeat Protein Required for Cell Proliferation. Genes (Basel) 10 (2019).

45. Kossack M. E., Draper B. W., Genetic regulation of sex determination and maintenance in zebrafish (Danio rerio). Curr Top Dev Biol 134, 119–149 (2019). doi: 10.1016/bs.ctdb.2019.02.004 30999973

46. Rodriguez-Mari A. et al., Sex reversal in zebrafish fancl mutants is caused by Tp53-mediated germ cell apoptosis. PLoS genetics 6, e1001034 (2010). doi: 10.1371/journal.pgen.1001034 20661450

47. Lu H. et al., Mcidas mutant mice reveal a two-step process for the specification and differentiation of multiciliated cells in mammals. Development 146 (2019).

Článek vyšel v časopise

PLOS Genetics

2020 Číslo 3
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy Podcasty Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se