Abstract
Primordial germ cells (PGCs) are the precursor cells that form during early embryogenesis and later differentiate into oocytes or spermatozoa. Abnormal development of PGCs is frequently a causative factor of infertility and germ cell tumors. However, our understanding of PGC development remains insufficient, and we have few pharmacological tools for manipulating PGC development for biological study or therapy. The zebrafish (Danio rerio) embryos provide an excellent in vivo animal model to study PGCs, because zebrafish embryos are transparent and develop outside the mother. Importantly, the model is also amenable to facile chemical manipulations, including scalable screening to discover novel compounds that alter PGC development. This chapter describes methodologies for manipulating the germline (i.e., PGCs) with small molecules and for monitoring PGC development. Utilizing the 3′UTR of PGC marker genes such as nanos3 and ddx4/vasa is a key component of these methodologies, which consist of expressing fluorescent or luminescent proteins in PGCs, treatment with small molecules, and quantitative observation of PGC development.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Strome S, Updike D (2015) Specifying and protecting germ cell fate. Nat Rev Mol Cell Biol 16:406–416. https://doi.org/10.1038/nrm4009
Siddiqui NU, Li X, Luo H et al (2012) Genome-wide analysis of the maternal-to-zygotic transition in Drosophila primordial germ cells. Genome Biol 13:R11. https://doi.org/10.1186/gb-2012-13-2-r11
Lebedeva LA, Yakovlev KV, Kozlov EN et al (2018) Transcriptional quiescence in primordial germ cells. Crit Rev Biochem Mol Biol 53:579–595. https://doi.org/10.1080/10409238.2018.1506733
Seydoux G, Dunn MA (1997) Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development 124:2191–2201
Yamaguchi S, Kimura H, Tada M et al (2005) Nanog expression in mouse germ cell development. Gene Expr Patterns 5:639–646. https://doi.org/10.1016/j.modgep.2005.03.001
Fang F, Angulo B, Xia N et al (2018) A PAX5–OCT4–PRDM1 developmental switch specifies human primordial germ cells. Nat Cell Biol 20:655. https://doi.org/10.1038/s41556-018-0094-3
Subtelny AO, Eichhorn SW, Chen GR et al (2014) Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508:66–71. https://doi.org/10.1038/nature13007
Winata CL, Łapiński M, Pryszcz L et al (2018) Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition. Development 145:dev159566. https://doi.org/10.1242/dev.159566
Kithcart AP, MacRae CA (2018) Zebrafish assay development for cardiovascular disease mechanism and drug discovery. Prog Biophys Mol Biol 138:126–131. https://doi.org/10.1016/j.pbiomolbio.2018.07.002
McCarroll MN, Gendelev L, Keiser MJ, Kokel D (2016) Leveraging large-scale behavioral profiling in zebrafish to explore neuroactive polypharmacology. ACS Chem Biol 11:842–849. https://doi.org/10.1021/acschembio.5b00800
Oikonomou G, Prober DA (2017) Attacking sleep from a new angle: contributions from zebrafish. Curr Opin Neurobiol 44:80–88. https://doi.org/10.1016/j.conb.2017.03.009
Rennekamp AJ, Peterson RT (2015) 15 years of zebrafish chemical screening. Curr Opin Chem Biol 24:58–70. https://doi.org/10.1016/j.cbpa.2014.10.025
Raz E (2003) Primordial germ-cell development: the zebrafish perspective. Nat Rev Genet 4:690. https://doi.org/10.1038/nrg1154
Paksa A, Raz E (2015) Zebrafish germ cells: motility and guided migration. Curr Opin Cell Biol 36:80–85. https://doi.org/10.1016/j.ceb.2015.07.007
Krøvel AV, Olsen LC (2002) Expression of a vas::EGFP transgene in primordial germ cells of the zebrafish. Mech Dev 116:141–150. https://doi.org/10.1016/S0925-4773(02)00154-5
Jin YN, Schlueter PJ, Jurisch-Yaksi N et al (2018) Noncanonical translation via deadenylated 3′ UTRs maintains primordial germ cells. Nat Chem Biol 14:844. https://doi.org/10.1038/s41589-018-0098-0
Mishima Y, Giraldez AJ, Takeda Y et al (2006) Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr Biol 16:2135–2142. https://doi.org/10.1016/j.cub.2006.08.086
Corish P, Tyler-Smith C (1999) Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng Des Sel 12:1035–1040. https://doi.org/10.1093/protein/12.12.1035
Leclerc GM, Boockfor FR, Faught WJ, Frawley LS (2000) Development of a destabilized firefly luciferase enzyme for measurement of gene expression. BioTechniques 29:590–601. https://doi.org/10.2144/00293rr02
Thorne N, Inglese J, Auld DS (2010) Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem Biol 17:646–657. https://doi.org/10.1016/j.chembiol.2010.05.012
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jin, Y.N., Peterson, R.T. (2021). Chemical Genetics: Manipulating the Germline with Small Molecules. In: Dosch, R. (eds) Germline Development in the Zebrafish. Methods in Molecular Biology, vol 2218. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0970-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0970-5_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0969-9
Online ISBN: 978-1-0716-0970-5
eBook Packages: Springer Protocols