DOI QR코드

DOI QR Code

NANOG expression in parthenogenetic porcine blastocysts is required for intact lineage specification and pluripotency

  • Mingyun Lee (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Jong-Nam Oh (Department of Cellular and Molecular Physiology, Yale School of Medicine) ;
  • Gyung Cheol Choe (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Kwang-Hwan Choi (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Dong-Kyung Lee (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Seung-Hun Kim (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Jinsol Jeong (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Yelim Ahn (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University) ;
  • Chang-Kyu Lee (Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Sciences, Seoul National University)
  • Received : 2023.06.09
  • Accepted : 2023.08.08
  • Published : 2023.12.01

Abstract

Objective: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. Methods: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. Results: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Conclusion: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

Keywords

Acknowledgement

This work was supported by the BK21 Four program, Alchemist project funded by the MOTIE (20012411), and a National Research Foundation of Korea (NRF) grant (2021R1A2C4001837), (2021R1A6A3A13038516).

References

  1. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448:313-7. https://doi.org/10.1038/nature05934
  2. Mitsui K, Tokuzawa Y, Itoh H, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631-42. https://doi.org/10.1016/S0092-8674(03)00393-3
  3. Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643-55. https://doi.org/10.1016/S0092-8674(03)00392-1
  4. Boyer LA, Lee TI, Cole MF, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005;122:947-56. https://doi.org/10.1016/j.cell.2005.08.020
  5. Darr H, Mayshar Y, Benvenisty N. Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features. Development 2006;133:1193-201. https://doi.org/10.1242/dev.02286
  6. Yang F, Zhang J, Liu Y, Cheng D, Wang H. Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes. Int J Biochem Cell Biol 2015;59:142-52. https://doi.org/10.1016/j.biocel.2014.12.009
  7. Chen L, Yabuuchi A, Eminli S, et al. Cross-regulation of the Nanog and Cdx2 promoters. Cell Res 2009;19:1052-61. https://doi.org/10.1038/cr.2009.79
  8. Mendjan S, Mascetti VL, Ortmann D, et al. NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency. Cell Stem Cell 2014;15:310-25. https://doi.org/10.1016/j.stem.2014.06.006
  9. Zhang X, Neganova I, Przyborski S, et al. A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and CDC25A. J Cell Biol 2009;184:67-82. https://doi.org/10.1083/jcb.200801009
  10. Chazaud C, Yamanaka Y. Lineage specification in the mouse preimplantation embryo. Development 2016;143:1063-74. https://doi.org/10.1242/dev.128314
  11. Allegre N, Chauveau S, Dennis C, et al. NANOG initiates epiblast fate through the coordination of pluripotency genes expression. Nat Commun 2022;13:3550. https://doi.org/10.1038/s41467-022-30858-8
  12. Frankenberg S, Gerbe F, Bessonnard S, et al. Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling. Dev Cell 2011;21:1005-13. https://doi.org/10.1016/j.devcel.2011.10.019
  13. Ortega MS, Kelleher AM, O'Neil E, et al. NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo. Mol Reprod Dev 2020;87:152-60. https://doi.org/10.1002/mrd.23304
  14. Levasseur DN, Wang J, Dorschner MO, Stamatoyannopoulos JA, Orkin SH. Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells. Genes Dev 2008;22:575-80. https://doi.org/10.1101/gad.1606308
  15. Lee M, Choi KH, Oh JN, et al. SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development. Cell Prolif 2021;54:e13097. https://doi.org/10.1111/cpr.13097
  16. Cauffman G, De Rycke M, Sermon K, Liebaers I, Van De Velde H. Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos. Hum Reprod 2009;24:63-70. https://doi.org/10.1093/humrep/den351
  17. Canizo JR, Ynsaurralde Rivolta AE, Vazquez Echegaray C, et al. A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos. BMC Dev Biol 2019;19:13. https://doi.org/10.1186/s12861-019-0193-9
  18. Liu J, An L, Wang J, et al. Dynamic patterns of H3K4me3, H3K27me3, and Nanog during rabbit embryo development. Am J Transl Res 2019;11:430-41.
  19. Ghimire S, Heindryckx B, Van der Jeught M, et al. Inhibition of transforming growth factor β signaling promotes epiblast formation in mouse embryos. Stem Cells Dev 2015;24:497-506. https://doi.org/10.1089/scd.2014.0206
  20. Heng JC, Orlov Y, Ng HH. Transcription factors for the modulation of pluripotency and reprogramming. Cold Spring Harb Symp Quant Biol 2010;75:237-44. https://doi.org/10.1101/sqb.2010.75.003
  21. Springer C, Zakhartchenko V, Wolf E, Simmet K. Hypoblast formation in bovine embryos does not depend on NANOG. Cells 2021;10:2232. https://doi.org/10.3390/cells10092232
  22. Kim SH, Lee M, Choi KH, et al. Species-specific enhancer activity of OCT4 in porcine pluripotency: the porcine OCT4 reporter system could monitor pluripotency in porcine embryo development and embryonic stem cells. Stem Cells Int 2022;2022:6337532. https://doi.org/10.1155/2022/6337532
  23. Stamatiadis P, Boel A, Cosemans G, et al. Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences. Hum Reprod 2021;36:1242-52. https://doi.org/10.1093/humrep/deab027
  24. Oh JN, Lee M, Choe GC, et al. Identification of the lineage markers and inhibition of DAB2 in in vitro fertilized porcine embryos. Int J Mol Sci 2020;21:7275. https://doi.org/10.3390/ijms21197275
  25. Choi KH, Lee CK. Pig pluripotent stem cells as a candidate for biomedical application. J Anim Reprod Biotechnol 2019;34:139-47. https://doi.org/10.12750/JARB.34.3.139
  26. Park JK, Kim HS, Uh KJ, et al. Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig. PloS One 2013;8:e52481. https://doi.org/10.1371/journal.pone.0052481
  27. Mashiko D, Fujihara Y, Satouh Y, et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 2013;3:3355. https://doi.org/10.1038/srep03355
  28. Lee DK, Choi KH, Hwang JY, Oh JN, Kim SH, Lee CK. Stearoyl-coenzyme A desaturase 1 is required for lipid droplet formation in pig embryo. Reproduction 2019;157:235-43. https://doi.org/10.1530/REP-18-0556
  29. Lee M, Oh JN, Choe GC, et al. Changes in OCT4 expression play a crucial role in the lineage specification and proliferation of preimplantation porcine blastocysts. Cell Prolif 2022;55:e13313. https://doi.org/10.1111/cpr.13313
  30. Silva J, Nichols J, Theunissen TW, et al. Nanog is the gateway to the pluripotent ground state. Cell 2009;138:722-37. https://doi.org/10.1016/j.cell.2009.07.039
  31. Bou G, Guo S, Guo J, et al. Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro. Zygote 2022;30:324-9. https://doi.org/10.1017/S0967199421000678
  32. Bessonnard S, De Mot L, Gonze D, et al. Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network. Development 2014;141:3637-48. https://doi.org/10.1242/dev.109678
  33. Messerschmidt DM, Kemler R. Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism. Dev Biol 2010;344:129-37. https://doi.org/10.1016/j.ydbio.2010.04.020
  34. Kirchhof N, Carnwath JW, Lemme E, Anastassiadis K, Schöler H, Niemann H. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol Reprod 2000;63:1698-705. https://doi.org/10.1095/biolreprod63.6.1698