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Genetic, Immunofluorescence Labeling, and In Situ Hybridization Techniques in Identification of Stem Cells in Male and Female Germline Niches

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Stem Cell Niche

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1035))

Abstract

Stem cells have an enormous capacity of self-renewal, as well as the ability to differentiate into specialized cell types. Proper control of these two properties of stem cells is crucial for animal development, growth control, and reproduction. Germline stem cells (GSCs) are a self-renewing population of germ cells, which generate haploid gametes (sperms or oocyte) that transmit genetic information from generation to generation. In Drosophila testis and ovary, GSCs are anchored around the niche cells. The cap cells cluster in females and hub cells in males act as a niche to control GSC behavior. With highly sophisticated genetic techniques in Drosophila, tremendous progress has been made in understanding the interactions between stem cells and niches at cellular and molecular levels. Here, we provide details of genetic, immunofluorescence labeling, and in situ hybridization techniques in identification and characterization of stem cells in Drosophila male and female germline niches.

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References

  1. Morrison SJ, Spradling AC (2008) Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132:598–611

    Article  PubMed  CAS  Google Scholar 

  2. Singh SR, Chen X, Hou SX (2005) JAK/STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila. Cell Res 15:1–5

    Article  PubMed  CAS  Google Scholar 

  3. Singh SR, Zhen W, Zheng Z, Wang H, Oh SW, Liu W, Zbar B, Schmidt LS, Hou SX (2006) The Drosophila homolog of the human tumor suppressor gene BHD interacts with the JAK-STAT and Dpp signaling pathways in regulating male germline stem cell maintenance. Oncogene 25:5933–5941

    Article  PubMed  CAS  Google Scholar 

  4. Singh SR, Liu W, Hou SX (2007) The adult Drosophila malpighian tubules are maintained by multipotent stem cells. Cell Stem Cell 1:191–203

    Article  PubMed  CAS  Google Scholar 

  5. Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111

    Article  PubMed  CAS  Google Scholar 

  6. Wang H, Singh SR, Zheng Z, Oh SW, Chen X, Edwards K, Hou SX (2006) Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis. Dev Cell 10:117–126

    Article  PubMed  CAS  Google Scholar 

  7. Wilson E (1896) The cell in development and inheritance. Macmillan, New York

    Book  Google Scholar 

  8. Nishimiya-Fujisawa C, Sugiyama T (1993) Genetic analysis of developmental mechanisms in hydra. XX. Cloning of interstitial stem cells restricted to the sperm differentiation pathway in Hydra magnipapillata. Dev Biol 157:1–9

    Article  PubMed  CAS  Google Scholar 

  9. Lin H, Spradling AC (1993) Germline stem cell division and egg chamber development in transplanted Drosophila germaria. Dev Biol 159:140–152

    Article  PubMed  CAS  Google Scholar 

  10. Hou SX, Singh SR (2008) Germline stem cells. Methods Mol Biol, 450, Humana Press, Totowa, NJ

    Google Scholar 

  11. Gilboa L, Lehmann R (2004) How different is Venus from Mars? The genetics of germ-line stem cells in Drosophila females and males. Development 131:4895–4905

    Article  PubMed  CAS  Google Scholar 

  12. Yamashita YM, Mahowald AP, Perlin JR, Fuller MT (2007) Asymmetric inheritance of mother versus daughter centrosome in stem cell division. Science 315:518–521

    Article  PubMed  CAS  Google Scholar 

  13. Cheng J, Türkel N, Hemati N, Fuller MT, Hunt AJ, Yamashita YM (2008) Centrosome misorientation reduces stem cell division during ageing. Nature 456:599–604

    Article  PubMed  CAS  Google Scholar 

  14. Sheng XR, Brawley CM, Matunis EL (2009) Dedifferentiating spermatogonia outcompete somatic stem cells for niche occupancy in the Drosophila testis. Cell Stem Cell 5:191–203

    Article  PubMed  CAS  Google Scholar 

  15. Gönczy P, DiNardo S (1996) The germ line regulates somatic cyst cell proliferation and fate during Drosophila spermatogenesis. Development 122:2437–2447

    PubMed  Google Scholar 

  16. Hardy RW, Tokuyasu KT, Lindsley DL, Garavito M (1979) The germinal proliferation center in the testis of Drosophila melanogaster. J Ultrastruct Res 69:180–190

    Article  PubMed  CAS  Google Scholar 

  17. Fuller MT, Spradling AC (2007) Male and female Drosophila germline stem cells: two versions of immortality. Science 316:402–404

    Article  PubMed  CAS  Google Scholar 

  18. Yamashita YM, Jones DL, Fuller MT (2003) Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301:1547–1550

    Article  PubMed  CAS  Google Scholar 

  19. Yamashita YM, Fuller MT (2008) Asymmetric centrosome behavior and the mechanisms of stem cell division. J Cell Biol 180:61–66

    Article  Google Scholar 

  20. Voog J, D'Alterio C, Jones DL (2008) Multipotent somatic stem cells contribute to the stem cell niche in the Drosophila testis. Nature 454:1132–1136

    Article  PubMed  CAS  Google Scholar 

  21. Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT (2001) Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science 294:2542–2545

    Article  PubMed  CAS  Google Scholar 

  22. Tulina N, Matunis E (2001) Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science 294:2546–2549

    Article  PubMed  CAS  Google Scholar 

  23. Brawley C, Matunis E (2004) Regeneration of male germline stem cells by spermatogonial dedifferentiation in vivo. Science 304:1331–1334

    Article  PubMed  CAS  Google Scholar 

  24. Schulz C, Kiger AA, Tazuke SI, Yamashita YM, Pantalena-Filho LC, Jones DL, Wood CG, Fuller MT (2004) A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage. Genetics 167:707–723

    Article  PubMed  CAS  Google Scholar 

  25. Sarkar A, Parikh N, Hearn SA, Fuller MT, Tazuke SI, Schulz C (2007) Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment. Curr Biol 17:1253–1258

    Article  PubMed  CAS  Google Scholar 

  26. Kiger AA, White-Cooper H, Fuller MT (2000) Somatic support cells restrict germline stem cell self-renewal and promote differentiation. Nature 407:750–754

    Article  PubMed  CAS  Google Scholar 

  27. Tran J, Brenner TJ, DiNardo S (2000) Somatic control over the germline stem cell lineage during Drosophila spermatogenesis. Nature 407:754–757

    Article  PubMed  CAS  Google Scholar 

  28. Shivdasani AA, Ingham PW (2003) Regulation of stem cell maintenance and transit amplifying cell proliferation by TGF-beta signaling in Drosophila spermatogenesis. Curr Biol 13:2065–2072

    Article  PubMed  CAS  Google Scholar 

  29. Bunt SM, Hime GR (2004) Ectopic activation of Dpp signalling in the male Drosophila germline inhibits germ cell differentiation. Genesis 39:84–93

    Article  PubMed  CAS  Google Scholar 

  30. Kawase E, Wong MD, Ding BC, Xie T (2004) Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis. Development 131:1365–1375

    Article  PubMed  CAS  Google Scholar 

  31. Schulz C, Wood CG, Jones DL, Tazuke SI, Fuller MT (2002) Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells. Development 129:4523–4534

    PubMed  CAS  Google Scholar 

  32. Leatherman JL, Dinardo S (2008) Zfh-1 controls somatic stem cell self-renewal in the Drosophila testis and nonautonomously influences germline stem cell self-renewal. Cell Stem Cell 3:44–54

    Article  PubMed  CAS  Google Scholar 

  33. Singh SR, Zheng Z, Wang H, Oh SW, Chen X, Hou SX (2010) Competitiveness for the niche and mutual dependence of the germline and somatic stem cells in the Drosophila testis are regulated by the JAK/STAT signaling. J Cell Physiol 223:500–510

    PubMed  CAS  Google Scholar 

  34. Toledano H, D’Alterio C, Czech B, Levine E, Jones DL (2012) The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche. Nature 485:605–610

    Article  PubMed  CAS  Google Scholar 

  35. Srinivasan S, Mahowald AP, Fuller MT (2012) The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche. Development 139:1381–1390

    Article  PubMed  CAS  Google Scholar 

  36. Monk AC, Siddall NA, Fraser B, McLaughlin EA, Hime GR (2011) Differential roles of HOW in male and female Drosophila germline differentiation. PLoS One 6(12):e28508

    Article  PubMed  CAS  Google Scholar 

  37. Inaba M, Yuan H, Yamashita YM (2011) String (Cdc25) regulates stem cell maintenance, proliferation and aging in Drosophila testis. Development 138:5079–5086

    Article  PubMed  CAS  Google Scholar 

  38. Casper AL, Baxter K, Van Doren M (2011) No child left behind encodes a novel chromatin factor required for germline stem cell maintenance in males but not females. Development 138:3357–3366

    Article  PubMed  CAS  Google Scholar 

  39. Zheng Q, Wang Y, Vargas E, DiNardo S (2011) magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis. Dev Biol 357:202–210

    Article  PubMed  CAS  Google Scholar 

  40. Dinardo S, Okegbe T, Wingert L, Freilich S, Terry N (2011) Lines and bowl affect the specification of cyst stem cells and niche cells in the Drosophila testis. Development 138:1687–1696

    Article  PubMed  CAS  Google Scholar 

  41. Leatherman JL, Dinardo S (2010) Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes. Nat Cell Biol 12:806–811

    Article  PubMed  CAS  Google Scholar 

  42. Cherry CM, Matunis EL (2010) Epigenetic regulation of stem cell maintenance in the Drosophila testis via the nucleosome-remodeling factor NURF. Cell Stem Cell 6:557–567

    Article  PubMed  CAS  Google Scholar 

  43. Xie T, Spradling AC (2000) A niche maintaining germ line stem cells in the Drosophila ovary. Science 290:328–330

    Article  PubMed  CAS  Google Scholar 

  44. Song X, Xie T (2002) DE-cadherin-mediated cell adhesion is essential for maintaining somatic stem cells in the Drosophila ovary. Proc Natl Acad Sci USA 99:14813–14818

    Article  PubMed  CAS  Google Scholar 

  45. Song X, Call GB, Kirilly D, Xie T (2007) Notch signaling controls germline stem cell niche formation in the Drosophila ovary. Development 134:1071–1080

    Article  PubMed  CAS  Google Scholar 

  46. Xie T, Spradling AC (1998) Decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94:251–260

    Article  PubMed  CAS  Google Scholar 

  47. Liu N, Han H, Lasko P (2009) Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3′ UTR. Genes Dev 23:2742–2752

    Article  PubMed  CAS  Google Scholar 

  48. Shen R, Weng C, Yu J, Xie T (2009) eIF4A controls germline stem cell self-renewal by directly inhibiting BAM function in the Drosophila ovary. Proc Natl Acad Sci USA 106:11623–11628

    Article  PubMed  CAS  Google Scholar 

  49. Wang L, Li Z, Cai Y (2008) The JAK/STAT pathway positively regulates DPP signaling in the Drosophila germline stem cell niche. J Cell Biol 180:721–728

    Article  PubMed  CAS  Google Scholar 

  50. Decotto E, Spradling AC (2005) The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals. Dev Cell 9:501–510

    Article  PubMed  CAS  Google Scholar 

  51. Nystul T, Spradling A (2007) An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement. Cell Stem Cell 1:277–285

    Article  PubMed  CAS  Google Scholar 

  52. Lu W, Casanueva MO, Mahowald AP, Kato M, Lauterbach D, Ferguson EL (2012) Niche-associated activation of rac promotes the asymmetric division of Drosophila female germline stem cells. PLoS Biol 10(7):e1001357

    Article  PubMed  CAS  Google Scholar 

  53. Rojas-Ríos P, Guerrero I, González-Reyes A (2012) Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain germline stem cells in Drosophila. PLoS Biol 10(4):e1001298

    Article  PubMed  Google Scholar 

  54. Li Y, Maines JZ, Tastan OY, McKearin DM, Buszczak M (2012) Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling. Development 139:1547–1556

    Article  PubMed  CAS  Google Scholar 

  55. Xing Y, Kurtz I, Thuparani M, Legard J, Ruohola-Baker H (2012) Loss-of-function screen reveals novel regulators required for Drosophila germline stem cell self-renewal. (G3) Bethesda 2:343–351

    Article  CAS  Google Scholar 

  56. Wang X, Pan L, Wang S, Zhou J, McDowell W, Park J, Haug J, Staehling K, Tang H, Xie T (2011) Histone H3K9 trimethylase eggless controls germline stem cell maintenance and differentiation. PLoS Genet 7(12):e1002426

    Article  PubMed  CAS  Google Scholar 

  57. König A, Yatsenko AS, Weiss M, Shcherbata HR (2011) Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation. EMBO J 30:1549–1562

    Article  PubMed  Google Scholar 

  58. Harris RE, Pargett M, Sutcliffe C, Umulis D, Ashe HL (2011) Brat promotes stem cell differentiation via control of a bistable switch that restricts BMP signaling. Dev Cell 20:72–83

    Article  PubMed  CAS  Google Scholar 

  59. Xia L, Jia S, Huang S, Wang H, Zhu Y, Mu Y, Kan L, Zheng W, Wu D, Li X, Sun Q, Meng A, Chen D (2010) The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response. Cell 143:978–990

    Article  PubMed  CAS  Google Scholar 

  60. Singh SR, Hou SX (2008) Immunohistological techniques for studying the Drosophila male germline stem cell. Methods Mol Biol 450:45–59

    Article  PubMed  CAS  Google Scholar 

  61. Lee T, Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24:251–254

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

M.K.S. is supported by the Knight’s Templar Eye Foundation and start-up support from the University of Dayton, OH. We thank Robin Permut for editing the manuscript.

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Authors and Affiliations

  1. Mouse Cancer Genetics Program, National Cancer Institute, NIH, Frederick, MD, USA

    Shree Ram Singh & Ying Liu

  2. Department of Biology, Center for Tissue Regeneration and Engineering at Dayton (TREND), University of Dayton, Dayton, OH, USA

    Madhuri Kango-Singh

  3. Institute of Evolution, University of Haifa, Haifa, Israel

    Eviatar Nevo

Authors
  1. Shree Ram Singh
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  2. Ying Liu
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  3. Madhuri Kango-Singh
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  4. Eviatar Nevo
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Editor information

Editors and Affiliations

  1. Regenerative Medicine Program, Ottawa Hospital Research Institute Sprott Centre for Stem Cell Research, Ottawa, Canada

    Kursad Turksen

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Singh, S.R., Liu, Y., Kango-Singh, M., Nevo, E. (2013). Genetic, Immunofluorescence Labeling, and In Situ Hybridization Techniques in Identification of Stem Cells in Male and Female Germline Niches. In: Turksen, K. (eds) Stem Cell Niche. Methods in Molecular Biology, vol 1035. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-508-8_2

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  • DOI: https://doi.org/10.1007/978-1-62703-508-8_2

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-507-1

  • Online ISBN: 978-1-62703-508-8

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