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Whole-Mount In Situ Hybridization for Detection of Migrating Zebrafish Endodermal Cells

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Cell Migration in Three Dimensions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2608))

Abstract

One of the most important events in early vertebrate development is the formation and positioning of the endoderm, the embryonic progenitor cell population that gives rise to the internal organs. Recent years have seen renewed interest in the mechanisms underlying the specification and migration of endodermal progenitor cells. The zebrafish is a well-established, accessible, and powerful model to study this cell population. Zebrafish endodermal cells are specified around 4 h after fertilization and subsequently migrate as evenly spaced single cells in a stereotypical manner in the next 6 h. Given the large numbers of fertilized eggs that can be obtained from a single breeding pair and the ease of chemical and genetic perturbations, the zebrafish is an excellent model to study mechanisms underlying endoderm specification and migration. An easy approach to visualizing and quantitating endodermal cells and their migratory routes is by whole-mount in situ hybridization (WISH) on fixed embryos, collected in time series. This chapter provides basic information on the organization and staging of the embryos, with an emphasis on the migrating endodermal cell population. In addition, optimized protocols for the isolation and fixation of staged embryos are provided as well as detailed probe synthesis and WISH protocols, specific for migrating endoderm. Finally, details are provided on how to approach these experiments quantitatively, and some common pitfalls are discussed.

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References

  1. Schier AF, Talbot WS (2005) Molecular genetics of Axis formation in zebrafish. Annu Rev Genet 39:561–613

    Article  CAS  Google Scholar 

  2. Solnica-Krezel L, Sepich DS (2012) Gastrulation: making and shaping germ layers. Annu Rev Cell Dev Biol 28:687–717

    Article  CAS  Google Scholar 

  3. Pinheiro D, Heisenberg CP (2020) Zebrafish gastrulation: putting fate in motion. Curr Top Dev Biol 136:343–375

    Article  CAS  Google Scholar 

  4. Nowotschin S, Hadjantonakis A-K, Campbell K (2019) The endoderm: a divergent cell lineage with many commonalities. Development 146(11):150920

    Article  Google Scholar 

  5. Zorn AM, Wells JM (2009) Vertebrate endoderm development and organ formation. Annu Rev Cell Dev Biol 25:221–251

    Article  CAS  Google Scholar 

  6. Probst S, Sagar, Tosic J, Schwan C, Grün D, Arnold SJ (2021) Spatiotemporal sequence of mesoderm and endoderm lineage segregation during mouse gastrulation. Development 148(1):193789

    Article  Google Scholar 

  7. van Boxtel AL, Economou AD, Heliot C, Hill CS (2018) Long-range signaling activation and local inhibition separate the mesoderm and endoderm lineages. Dev Cell 44:179–191

    Article  Google Scholar 

  8. Kondow A, Ohnuma K, Kamei Y, Taniguchi A, Bise R, Sato Y, Yamaguchi H, Nonaka S, Hashimoto K, Akiko Kondow C (2020) Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development. Dev Growth Diff 62:495–502

    Article  CAS  Google Scholar 

  9. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assunção JA, Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Eliott D, Threadgold G, Harden G, Ware D, Mortimer B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden P, Barker N, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, Ellwood M, Woodmansey R, Clark G, Cooper J, Tromans A, Grafham D, Skuce C, Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Ürün Z, Eser C, Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberländer M, Rudolph-Geiger S, Teucke M, Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C, Yang F, Carter NP, Harrow J, Ning Z, Herrero J, Searle SMJ, Enright A, Geisler R, Plasterk RHA, Lee C, Westerfield M, de Jong PJ, Zon LI, Postlethwait JH, Nüsslein-Volhard C, Hubbard TJP, Crollius HR, Rogers J, Stemple DL (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496:498–503

    Article  CAS  Google Scholar 

  10. Nair S, Schilling TF (2008) Chemokine signaling controls endodermal migration during zebrafish gastrulation. Science 322:89–92

    Article  CAS  Google Scholar 

  11. Stark BC, Gao Y, Sepich DS, Belk L, Culver MA, Hu B, Mekel M, Ferris W, Shin J, Solnica-Krezel L, Lin F, Cooper JA (2022) CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish. Dev Biol 481:148–159

    Article  CAS  Google Scholar 

  12. Kondow A, Ohnuma K, Kamei Y, Taniguchi A, Bise R, Sato Y, Yamaguchi H, Nonaka S, Hashimoto K (2020) Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development. Dev Growth Diff 62:495–502

    Article  CAS  Google Scholar 

  13. Shah G, Thierbach K, Schmid B, Waschke J, Reade A, Hlawitschka M, Roeder I, Scherf N, Huisken J (2019) Multi-scale imaging and analysis identify pan-embryo cell dynamics of germlayer formation in zebrafish. Nature Comm 10:1–12

    Article  CAS  Google Scholar 

  14. Thisse C, Thisse B (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Prot 3:59–69

    Article  CAS  Google Scholar 

  15. Thisse B, Pflumio S, Fürthauer M, Loppin B, Heyer V, Degrave A, Woehl R, Lux A, Steffan T, Charbonnier XQ, Thisse C (2001) Expression of the zebrafish genome during embryogenesis. ZFIN Direct Data Submission. http://zfin.org

    Google Scholar 

  16. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310

    Article  CAS  Google Scholar 

  17. Carvalho L, Heisenberg CP (2010) The yolk syncytial layer in early zebrafish development. Trends Cell Biol 20:586–592

    Article  CAS  Google Scholar 

  18. Warga RM, Nusslein-Volhard (1999) Origin and development of the zebrafish endoderm. Development 126(4):827–838

    Article  CAS  Google Scholar 

  19. Dumortier JG, Martin S, Meyer D, Rosa FM, David NB (2012) Collective mesendoderm migration relies on an intrinsic directionality signal transmitted through cell contacts. PNAS 109:16945–16950

    Article  CAS  Google Scholar 

  20. Montero JA, Carvalho L, Wilsch-Bräuninger M, Kilian B, Mustafa C, Heisenberg CP (2005) Shield formation at the onset of zebrafish gastrulation. Development 132:1187–1198

    Article  CAS  Google Scholar 

  21. Giger FA, David NB (2017) Endodermal germ-layer formation through active actin-driven migration triggered by N-cadherin. PNAS 114:10143–10148

    Article  CAS  Google Scholar 

  22. Alexander J, Rothenberg M, Henry GL, Stainier DYR (1999) Casanova plays an early and essential role in endoderm formation in zebrafish. Dev Biol 215:343–357

    Article  CAS  Google Scholar 

  23. Alexander J, Stainier DYR (1999) A molecular pathway leading to endoderm formation in zebrafish. Curr Biol 9:1147–1157

    Article  CAS  Google Scholar 

  24. Aoki TO, David NB, Minchiotti G, Saint-Etienne L, Dickmeis T, Persico GM, Strähle U, Mourrain P, Rosa FM (2002) Molecular integration of casanova in the nodal signalling pathway controlling endoderm formation. Development 129:275–286

    Article  Google Scholar 

  25. Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene

    Google Scholar 

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Authors and Affiliations

  1. Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands

    Antonius L. van Boxtel

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  1. Antonius L. van Boxtel
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Correspondence to Antonius L. van Boxtel .

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Editors and Affiliations

  1. Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam University Medical Center, Amsterdam, The Netherlands

    Coert Margadant

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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van Boxtel, A.L. (2023). Whole-Mount In Situ Hybridization for Detection of Migrating Zebrafish Endodermal Cells. In: Margadant, C. (eds) Cell Migration in Three Dimensions. Methods in Molecular Biology, vol 2608. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2887-4_9

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  • DOI: https://doi.org/10.1007/978-1-0716-2887-4_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2886-7

  • Online ISBN: 978-1-0716-2887-4

  • eBook Packages: Springer Protocols

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