Abstract
Whole-mount in situ hybridization is able to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain–hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain–hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Change history
12 January 2024
A correction has been published.
References
Dworkin S, Darido C, Georgy SR, Wilanowski T, Srivastava S, Ellett F et al (2012) Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways. Development 139(3):525–536. https://doi.org/10.1242/dev.066522
Picker A, Scholpp S, Böhli H, Takeda H, Brand M (2002) A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines. Development 129(13):3227–3239. https://doi.org/10.1242/dev.129.13.3227. PMID: 12070097.
Giudicelli F, Taillebourg E, Charnay P, Gilardi-Hebenstreit P (2001) Krox-20 patterns the hindbrain through both cell-autonomous and non cell-autonomous mechanisms. Genes Dev 15(5):567–580. https://doi.org/10.1101/gad.189801. PMID: 11238377; PMCID: PMC312642
Dworkin S, Heath JK, deJong-Curtain TA, Hogan BM, Lieschke GJ, Malaterre J et al (2007) CREB activity modulates neural cell proliferation, midbrain-hindbrain organization and patterning in zebrafish. Dev Biol 307(1):127–141. https://doi.org/10.1016/j.ydbio.2007.04.026
Dworkin S, Simkin J, Darido C, Partridge DD, Georgy SR, Caddy J et al (2014) Grainyhead-like 3 regulation of endothelin-1 in the pharyngeal endoderm is critical for growth and development of the craniofacial skeleton. Mech Dev 133:77–90. https://doi.org/10.1016/j.mod.2014.05.005
Gasperoni J, Fuller J, Darido C, Wilanowski T, Dworkin S (2022) Grainyhead-like (Grhl) target genes in development and cancer. Int J Mol Sci 23:2735. https://doi.org/10.3390/ijms23052735
Mathiyalagan N, Dworkin S (2022) Wholemount in-situ hybridization (WISH) in zebrafish embryos to analyze craniofacial development. Methods Mol Biol 2403:19–32. https://doi.org/10.1007/978-1-0716-1847-9_2. PMID: 34913113
Miles LB, Darido C, Kaslin J, Heath JK, Jane SM, Dworkin S (2017) Mis-expression of grainyhead-like transcription factors in zebrafish leads to defects in enveloping layer (EVL) integrity, cellular morphogenesis and axial extension. Sci Rep 7(1):17607. https://doi.org/10.1038/s41598-017-17898-7
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Fuller, J., Dworkin, S. (2024). In Situ Hybridization to Characterize Neurulation and Midbrain–Hindbrain Boundary Formation in Zebrafish. In: Dworkin, S. (eds) Neurobiology. Methods in Molecular Biology, vol 2746. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3585-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3585-8_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3584-1
Online ISBN: 978-1-0716-3585-8
eBook Packages: Springer Protocols