Abstract
Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.
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Abbreviations
- bFGF:
-
Basic fibroblast growth factor
- BMP4:
-
Bone morphogenetic protein 4
- dpc or hpc:
-
Day(s) or hour(s) post culture
- dpf or hpf:
-
Day(s) or hour(s) post fertilization
- ES cell:
-
Embryonic stem cell
- FBS:
-
Fetal bovine serum
- FS:
-
Fish serum from rainbow trout
- GFP:
-
Green fluorescent protein
- MBE:
-
Midblastula embryo
- MEE:
-
Medaka embryo extract
- PR:
-
Fusion between puromycin acetyltransferase and red fluorescent protein
- PBS:
-
Phosphate-buffered saline
- PBST:
-
Tween-20 (0.1%) in PBS
- pPGC:
-
Presumptive primordial germ cell
- RT-PCR:
-
Reverse transcription plus polymerase chain reaction
- 3′-UTR:
-
3′ Untranslated region
References
Aizawa K, Shimada A, Naruse K, Mitani H, Shima A (2003) The medaka midblastula transition as revealed by the expression of the paternal genome. Gene Expr Patterns 3:43–47
Chourrout D (1982) Tetraploidy induced by heat shocks in the rainbow trout (Salmo gairdneri R.). Reprod Nutr Dev 22:569–574
Clark A, Bodnar M, Fox M, Rodriquez R, Abeyta M, Firpo M, Pera R (2004) Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Stem Cells 22:169–179
Ephrussi A, Lehmann R (1992) Induction of germ cell formation by oskar. Nature 358:387–392
Extavour C, Akam M (2003) Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 130:5869–5884
Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ (2004) Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427:148–154
Guan G, Yan Y, Chen T, Yi M, Ni H, Naruse K, Nagahama Y, Hong Y (2013) Nanos3 gene targeting in medaka ES cells. Int J Biol Sci 9:444–454
Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M (2011) Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146:519–532
Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M (2012) Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338:971–975
Herpin A, Rohr S, Riedel D, Kluever N, Raz E, Schartl M (2007) Specification of primordial germ cells in medaka (Oryzias latipes). BMC Dev Biol 7:3
Hong N, Li M, Zeng Z, Yi M, Deng J, Gui J, Winkler C, Schartl M, Hong Y (2010) Accessibility of host cell lineages to medaka stem cells depends on genetic background and irradiation of recipient embryos. Cell Mol Life Sci 67:1189–1202
Hong N, Chen S, Ge R, Song J, Yi M, Hong Y (2012) Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation. Stem Cells Dev 21:2333–2341
Hong Y, Schartl M (1996) Establishment and growth responses of early medaka (Oryzias latipes) embryonic cells in feeder layer-free culture. Mol Mar Biol Biotechnol 5:93–104
Hong Y, Winkler C, Schartl M (1996) Pluripotency and differentiation of embryonic stem cell lines from the medakafish (Oryzias latipes). Mech Dev 60:33–44
Hong Y, Chen S, Gui J, Schartl M (2004a) Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish. Transgenic Res 13:41–50
Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, Chen T, Deng J, Gui J (2004b) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci U S A 101:8011–8016
Houston DW, Zhang J, Maines JZ, Wasserman SA, King ML (1998) A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule. Development 125:171–180
Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Scholer HR (2003) Derivation of oocytes from mouse embryonic stem cells. Science 300:1251–1256
Iwamatsu T (2004) Stages of normal development in the medaka Oryzias latipes. Mech Dev 121:605–618
Johnson AD, Bachvarova RF, Drum M, Masi T (2001) Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad. Dev Biol 234:402–415
Kawakami Y, Goto-Kazeto R, Saito T, Fujimoto T, Higaki S, Takahashi Y, Arai K, Yamaha E (2011) Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids. Int J Dev Biol 54:1493–1501
Kawasaki T, Saito K, Sakai C, Shinya M, Sakai N (2012) Production of zebrafish offspring from cultured spermatogonial stem cells. Genes Cells 17:316–325
Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA (2009) Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462:222–225
Knaut H, Pelegri F, Bohmann K, Schwarz H, Nusslein-Volhard C (2000) Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification. J Cell Biol 149:875–888
Koprunner M, Thisse C, Thisse B, Raz E (2001) A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev 15:2877–2885
Krovel AV, Olsen LC (2002) Expression of a vas::EGFP transgene in primordial germ cells of the zebrafish. Mech Dev 116:141–150
Kurokawa H, Aoki Y, Nakamura S, Ebe Y, Kobayashi D, Tanaka M (2006) Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes. Dev Growth Differ 48:209–221
Lavoir MC van de, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ (2006) Germline transmission of genetically modified primordial germ cells. Nature 441:766–769
Lavoir MC van de, Collarini EJ, Leighton PA, Fesler J, Lu DR, Harriman WD, Thiyagasundaram TS, Etches RJ (2012) Interspecific germline transmission of cultured primordial germ cells. PloS ONE 7:e35664
Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL (1999) Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 13:424–436
Li M, Hong N, Xu H, Yi M, Li C, Gui J, Hong Y (2009) Medaka vasa is required for migration but not survival of primordial germ cells. Mech Dev 126:366–381
Li M, Hong N, Gui J, Hong Y (2012) Medaka piwi is essential for primordial germ cell migration. Curr Mol Med 12:1040–1049
Li Z (2009) Nanog in the twin fish models medaka and zebrafish: functional divergence or pleiotropy of vertebrate pluripotency gene. Doctoral thesis, National University of Singapore.
Li Z, Bhat N, Manali D, Wang D, Hong N, Yi M, Ge R, Hong Y (2011) Medaka cleavage embryos are capable of generating ES-like cell cultures. Int J Biol Sci 7:418–425
Nakaki F, Hayashi K, Ohta H, Kurimoto K, Yabuta Y, Saitou M (2013) Induction of mouse germ-cell fate by transcription factors in vitro. Nature 501:222–226
Nieuwkoop PD, Sutasurya LA (1981) Primordial germ cells in the invertebrates: from epigenesis to preformation. Cambridge University Press, Cambridge
Olsen LC, Aasland R, Fjose A (1997) A vasa-like gene in zebrafish identifies putative primordial germ cells. Mech Dev 66:95–105
Panda RP, Barman HK, Mohapatra C (2011) Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation. Theriogenology 76:241–251
Rao F, Wang T, Li M, Li Z, Hong N, Zhao H, Yan Y, Lu W, Chen T, Wang W, Lim M, Yuan Y, Liu L, Zeng L, Wei Q, Guan G, Li C, Hong Y (2011) Medaka tert produces multiple variants with differential expression during differentiation in vitro and in vivo. Int J Biol Sci 7:426–439
Raz E (2003) Primordial germ-cell development: the zebrafish perspective. Nat Rev Genet 4:690–700
Sanchez-Sanchez AV, Camp E, Leal-Tassias A, Atkinson SP, Armstrong L, Diaz-Llopis M, Mullor JL (2010) Nanog regulates primordial germ cell migration through Cxcr4b. Stem Cells 28:1457–1464
Sawatari E, Shikina S, Takeuchi T, Yoshizaki G (2007) A novel transforming growth factor-beta superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss). Dev Biol 301:266–275
Seydoux G, Strome S (1999) Launching the germline in Caenorhabditis elegans: regulation of gene expression in early germ cells. Development 126:3275–3283
Shikina S, Yoshizaki G (2010) Improved in vitro culture conditions to enhance the survival, mitotic activity, and transplantability of rainbow trout type A spermatogonia. Biol Reprod 83:268–276
Shinomiya A, Tanaka M, Kobayashi T, Nagahama Y, Hamaguchi S (2000) The vasa-like gene, olvas, identifies the migration path of primordial germ cells during embryonic body formation stage in the medaka, Oryzias latipes. Develop Growth Differ 42:317–326
Tam PP, Zhou SX (1996) The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo. Dev Biol 178:124–132
Tanaka M, Kinoshita M, Kobayashi D, Nagahama Y (2001) Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate. Proc Natl Acad Sci U S A 98:2544–2549
Tilgner K, Atkinson SP, Golebiewska A, Stojkovic M, Lako M, Armstrong L (2008) Isolation of primordial germ cells from differentiating human embryonic stem cells. Stem Cells 26:3075–3085
Wang D, Manali D, Wang T, Bhat N, Hong N, Li Z, Wang L, Yan Y, Liu R, Hong Y (2011) Identification of pluripotency genes in the fish medaka. Int J Biol Sci 7:440–451
Weidinger G, Stebler J, Slanchev K, Dumstrei K, Wise C, Lovell-Badge R, Thisse C, Thisse B, Raz E (2003) Dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Curr Biol 13:1429–1434
Wong TT, Tesfamichael A, Collodi P (2013) Production of zebrafish offspring from cultured female germline stem cells. PloS One 8:e62660
Wylie C (1999) Germ cells. Cell 96:165–174
Xu H, Gui J, Hong Y (2005) Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate. Dev Dyn 233:872–882
Xu H, Li M, Gui J, Hong Y (2010) Fish germ cells. Sci China Life Sci 53:435–446
Xu H, Li Z, Li M, Wang L, Hong Y (2009) Boule is present in fish and bisexually expressed in adult and embryonic germ cells of medaka. PloS One 4:e6097
Yan Y, Hong N, Chen T, Li M, Wang T, Guan G, Qiao Y, Chen S, Schartl M, Li CM, Hong Y (2013) p53 gene targeting by homologous recombination in fish ES cells. PLoS One 8:e59400
Yi M, Hong N, Hong Y (2009) Generation of medaka fish haploid embryonic stem cells. Science 326:430–433
Yi M, Hong N, Hong Y (2010) Derivation and characterization of haploid embryonic stem cell cultures in medaka fish. Nat Protoc 5:1418–1430
Ying Y, Qi X, Zhao GQ (2001) Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways. Proc Natl Acad Sci U S A 98:7858–7862
Yoon C, Kawakami K, Hopkins N (1997) Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124:3157–3165
Zhao H, Hong N, Lu W, Zeng H, Song J, Hong Y (2012) Fusion gene vectors allowing for simultaneous drug selection, cell labeling, and reporter assay in vitro and in vivo. Anal Chem 84:987–993
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We thank J. Deng for fish breeding.
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This work was supported by grants to Y.H. from the National Research Foundation Singapore (grant NRF-CRP7-2010-03) and to M.Y. from the National Natural Science Foundation of China (grant 31271576).
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Fig. S1
PGC formation in vivo is independent of somatic development. Vg embryos at the 1-cell stage were non-injected or injected with MOpw (an antisense morpholino oligo) and monitored for somatic and PGC development at 4 dpf. a, b Control embryo. c-g MOpw-injected embryos, showing PGC formation in the absence of somatic structures (red fluorescence attributable to the co-injected dye, ey eye, od oil droplet, pc pigment cell, gr gonad region). Bars 100 μm. (TIFF 6795 kb)
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Li, Z., Li, M., Hong, N. et al. Formation and cultivation of medaka primordial germ cells. Cell Tissue Res 357, 71–81 (2014). https://doi.org/10.1007/s00441-014-1867-z
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DOI: https://doi.org/10.1007/s00441-014-1867-z