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Enrichment of Undifferentiated Germline and Somatic Cells from Drosophila Testes

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Germline Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2677))

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Abstract

The Drosophila male germline provides a strong model system to understand numerous developmental and cell-biological processes, owing to a well-defined anatomy and cell type markers in combination with various genetic tools available for the Drosophila system. A major weakness of this system has been the difficulty of approaches for obtaining material for biochemical assays, proteomics, and genomic or transcriptomic profiling due to small-size and complex tissues. However, the recent development of techniques has started allowing us the usage of a low amount of material for these analyses and now we can strategize many new experiments. The method for enrichment or isolation of rare populations of cells is still challenging and should meaningfully influence the reliability of the results. Here, we provide our semi-optimized protocol of enrichment of undifferentiated germ cells and somatic cells from non-tumorous Drosophila testis, which we have successfully improved after multiple trials.

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References

  1. Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG et al (2019) CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10(1):1930. https://doi.org/10.1038/s41467-019-09982-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hicks SC, Townes FW, Teng M, Irizarry RA (2018) Missing data and technical variability in single-cell RNA-sequencing experiments. Biostatistics 19(4):562–578. https://doi.org/10.1093/biostatistics/kxx053

    Article  PubMed  Google Scholar 

  3. de Cuevas M, Matunis EL (2011) The stem cell niche: lessons from the drosophila testis. Development 138(14):2861–2869. https://doi.org/10.1242/dev.056242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li L, Xie T (2005) Stem cell niche: structure and function. Annu Rev Cell Dev Biol 21:605–631

    Article  CAS  PubMed  Google Scholar 

  5. Yamashita YM, Jones DL, Fuller MT (2003) Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301(5639):1547–1550. https://doi.org/10.1126/science.1087795

    Article  CAS  PubMed  Google Scholar 

  6. Tulina N, Matunis E (2001) Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science (New York, NY) 294(5551):2546–2549. https://doi.org/10.1126/science.1066700

    Article  CAS  Google Scholar 

  7. Bate M (1993) Arias AM. Cold Spring Harbor Laboratory Press, The development of Drosophila melanogaster

    Google Scholar 

  8. Demarco RS, Eikenes ÅH, Haglund K, Jones DL (2014) Investigating spermatogenesis in Drosophila melanogaster. Methods 68(1):218–227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Spradling A, Fuller MT, Braun RE, Yoshida S (2011) Germline stem cells. Cold Spring Harb Perspect Biol 3(11):a002642

    Article  PubMed  PubMed Central  Google Scholar 

  10. Fischer JA, Giniger E, Maniatis T, Ptashne M (1988) GAL4 activates transcription in drosophila. Nature 332(6167):853–856. https://doi.org/10.1038/332853a0

    Article  CAS  PubMed  Google Scholar 

  11. Buszczak M, Paterno S, Lighthouse D, Bachman J, Planck J, Owen S et al (2007) The Carnegie protein trap library: a versatile tool for drosophila developmental studies. Genetics 175(3):1505–1531. https://doi.org/10.1534/genetics.106.065961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Venken KJ, Schulze KL, Haelterman NA, Pan H, He Y, Evans-Holm M et al (2011) MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nat Methods 8(9):737–743. https://doi.org/10.1038/nmeth.1662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. McGuire SE, Mao Z, Davis RL (2004) Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in drosophila. Sci STKE 2004(220):l6. https://doi.org/10.1126/stke.2202004pl6

    Article  Google Scholar 

  14. Jensen L, Venkei ZG, Watase GJ, Bisai B, Pletcher S, Lee CY et al (2021) me31B regulates stem cell homeostasis by preventing excess dedifferentiation in the Drosophila male germline. J Cell Sci 134(14). https://doi.org/10.1242/jcs.258757

  15. Chau J, Kulnane LS, Salz HK (2012) Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during drosophila oogenesis. Proc Natl Acad Sci U S A 109(24):9465–9470. https://doi.org/10.1073/pnas.1120473109

    Article  PubMed  PubMed Central  Google Scholar 

  16. Eun SH, Feng L, Cedeno-Rosario L, Gan Q, Wei G, Cui K et al (2017) Polycomb Group Gene E(z) Is Required for Spermatogonial Dedifferentiation in Drosophila Adult Testis. J Mol Biol 429(13):2030–2041. https://doi.org/10.1016/j.jmb.2017.04.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Padrón A, Iwasaki S, Ingolia NT (2019) Proximity RNA Labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules. Mol Cell 75(4):875–887.e5. https://doi.org/10.1016/j.molcel.2019.07.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA et al (2013) Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339(6125):1328–1331. https://doi.org/10.1126/science.1230593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mannix KM, Starble RM, Kaufman RS, Cooley L (2019) Proximity labeling reveals novel interactomes in live Drosophila tissue. Development 146(14). https://doi.org/10.1242/dev.176644

  20. Chen C, Yamashita YM (2020) Alstrom syndrome gene is a stem-cell-specific regulator of centriole duplication in the drosophila testis. elife 9. https://doi.org/10.7554/eLife.59368

  21. Blatt P, Wong-Deyrup SW, McCarthy A, Breznak S, Hurton MD, Upadhyay M et al (2021) RNA degradation is required for the germ-cell to maternal transition in Drosophila. Curr Biol 31(14):2984–2994. https://doi.org/10.1016/j.cub.2021.04.052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chen C, Yamashita YM (2020) Alstrom syndrome gene is a stem-cell-specific regulator of centriole duplication in the drosophila testis. elife 9:e59368. https://doi.org/10.7554/eLife.59368

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gärtner SMK, Hundertmark T, Nolte H, Theofel I, Eren-Ghiani Z, Tetzner C et al (2019) Stage-specific testes proteomics of Drosophila melanogaster identifies essential proteins for male fertility. Eu J Cell Biol 98(2):103–115. https://doi.org/10.1016/j.ejcb.2019.01.001

    Article  CAS  Google Scholar 

  24. Chen D, McKearin D (2003) Dpp Signaling Silences bam Transcription Directly to Establish Asymmetric Divisions of Germline Stem Cells. Curr Biol 13(20):1786–1791. https://doi.org/10.1016/j.cub.2003.09.033

    Article  CAS  PubMed  Google Scholar 

  25. 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(5551):2542–2545. https://doi.org/10.1126/science.1066707

    Article  CAS  PubMed  Google Scholar 

  26. Schulz C, Kiger AA, Tazuke SI, Yamashita YM, Pantalena-Filho LC, Jones DL et al (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(2):707–723. https://doi.org/10.1534/genetics.103.023184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shivdasani AA, Ingham PW (2003) Regulation of stem cell maintenance and transit amplifying cell proliferation by TGF-β signaling in drosophila spermatogenesis. Curr Biol 13(23):2065–2072. https://doi.org/10.1016/j.cub.2003.10.063

    Article  CAS  PubMed  Google Scholar 

  28. Leatherman JL, DiNardo S (2010) Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in drosophila testes. Nat Cell Biol 12(8):806–811. https://doi.org/10.1038/ncb2086

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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 (Cambridge, England) 131(6):1365–1375. https://doi.org/10.1242/dev.01025

    Article  CAS  PubMed  Google Scholar 

  30. Li Y, Zhang Q, Carreira-Rosario A, Maines JZ, McKearin DM, Buszczak M (2013) Mei-p26 cooperates with Bam, Bgcn and Sxl to promote early germline development in the Drosophila ovary. PLoS One 8(3):e58301-e. https://doi.org/10.1371/journal.pone.0058301

    Article  CAS  Google Scholar 

  31. Shi Z, Lim C, Tran V, Cui K, Zhao K, Chen X (2020) Single-cyst transcriptome analysis of Drosophila male germline stem cell lineage. Development 147(8). https://doi.org/10.1242/dev.184259

  32. DeLuca SZ, Ghildiyal M, Pang L-Y, Spradling AC (2020) Differentiating drosophila female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins. elife 9:e56922

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Witt E, Benjamin S, Svetec N, Zhao L (2019) Testis single-cell RNA-seq reveals the dynamics of de novo gene transcription and germline mutational bias in drosophila. elife 8:e47138

    Article  PubMed  PubMed Central  Google Scholar 

  34. Grmai L, Harsh S, Lu S, Korman A, Deb IB, Bach EA (2021) Transcriptomic analysis of feminizing somatic stem cells in the Drosophila testis reveals putative downstream effectors of the transcription factor Chinmo. G3 11(4):jkab067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lim RSM, Osato M, Kai T (2012) Isolation of undifferentiated female germline cells from adult Drosophila ovaries. Curr Protoc Stem Cell Biol 22(1):2E. 3.1–2E. 3.14

    Google Scholar 

  36. Kai T, Williams D, Spradling AC (2005) The expression profile of purified drosophila germline stem cells. Dev Biol 283(2):486–502

    Article  CAS  PubMed  Google Scholar 

  37. Xie J, Wooten M, Tran V, Chen BC, Pozmanter C, Simbolon C et al (2015) Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the drosophila male germline. Cell 163(4):920–933. https://doi.org/10.1016/j.cell.2015.10.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Van Doren M, Williamson AL, Lehmann R (1998) Regulation of zygotic gene expression in drosophila primordial germ cells. Curr Biol 8(4):243–246. https://doi.org/10.1016/s0960-9822(98)70091-0

    Article  PubMed  Google Scholar 

  39. Inaba M, Yuan H, Yamashita YM (2011) String (Cdc25) regulates stem cell maintenance, proliferation and aging in drosophila testis. Development 138(23):5079–5086. https://doi.org/10.1242/dev.072579

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kelso RJ, Buszczak M, Quiñones AT, Castiblanco C, Mazzalupo S, Cooley L (2004) Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster. Nucleic Acids Res 32(Database issue):D418–D420. https://doi.org/10.1093/nar/gkh014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Evan Jellison and the Flow Cytometry Core at the UConn Health Center and Hideyuki Oguro for their assistance with cell sorting and helpful discussion and optimization of the sorting protocol, Active motif technical support team for suggestions for tissue dissociation method, Bo Reese and UConn Center for Genome Innovation for library quality check.

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Authors and Affiliations

  1. Department of Cell Biology, University of Connecticut Health, Farmington, CT, USA

    Sharif M. Ridwan, Matthew Antel & Mayu Inaba

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  1. Sharif M. Ridwan
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  2. Matthew Antel
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  3. Mayu Inaba
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Correspondence to Mayu Inaba .

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Editors and Affiliations

  1. Department of Molecular Biology, The University of Texas Southwestern Medical Center, Dallas, TX, USA

    Michael Buszczak

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Ridwan, S.M., Antel, M., Inaba, M. (2023). Enrichment of Undifferentiated Germline and Somatic Cells from Drosophila Testes. In: Buszczak, M. (eds) Germline Stem Cells. Methods in Molecular Biology, vol 2677. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3259-8_7

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  • DOI: https://doi.org/10.1007/978-1-0716-3259-8_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3258-1

  • Online ISBN: 978-1-0716-3259-8

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