Abstract
In situ hybridization (ISH) methods remain the most popular approach for profiling the expression of a gene at high spatial resolution and have been broadly used to address many biological questions. One compelling application is in the field of evo-devo, where comparing gene expression patterns has offered insight into how vertebrate development has evolved. Gene expression profiling in the invertebrate chordate amphioxus (cephalochordate) has been particularly instrumental in this context: its key phylogenetic position as sister group to all other chordates makes it an ideal model system to compare with vertebrates and for reconstructing the ancestral condition of our phylum. However, while ISH methods have been developed extensively in vertebrate model systems to fluorescently detect the expression of multiple genes simultaneously at a cellular and subcellular resolution, amphioxus gene expression profiling is still based on single-gene nonfluorescent chromogenic methods, whose spatial resolution is often compromised by diffusion of the chromogenic product. This represents a serious limitation for reconciling gene expression dynamics between amphioxus and vertebrates and for molecularly identifying cell types, defined by their combinatorial code of gene expression, that may have played pivotal roles in evolutionary innovation. Herein we overcome these problems by describing a new protocol for application of the third-generation hybridization chain reaction (HCR) to the amphioxus, which permits fluorescent, multiplex, and quantitative detection of gene expression in situ, within the changing morphology of the developing embryo, and in adult tissues. A detailed protocol is herein provided for whole-mount preparations of embryos and vibratome sections of adult tissues.
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References
Graham A, Papalopulu N, Krumlauf R (1989) The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell 57(3):367–378
Dubrulle J, Pourquié O (2004) fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature 427:419–422
Riddle RD, Johnson RL, Laufer E et al (1993) Sonic Hedgehog mediates the polarizing activity of the ZPA. Cell 75(7):1401–1416
Nelson C, Morgan B, Burke AC et al (1996) Analysis of Hox gene expression in the chick limb bud. Development 122:1449–1466
Akam M, Martinez-Arias A (1985) The distribution of Ultrabithorax transcripts in Drosophila embryos. EMBO 4:1689–1700
Baker NE (1987) Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos. EMBO 6:1765–1773
McGinnis W, Krumlauf R (1992) Homeobox genes and axial patterning. Cell 68:283–302
Benito-Gutiérrez È (2011) Amphioxus as a model for mechanisms in vertebrate development. In: eLS (ed). https://doi.org/10.1002/9780470015902.a0021773
Meuleman D, Bronner-Fraser M (2004) Gene-Regulatory Interactions in neural crest evolution and development. Dev Cell 7:291–299
Holland ND, Holland LZ (1993) Embryos and larvae of invertebrate deuterostomes. In: Stern CD, Holland PWH (eds) Essential developmental biology, a practical approach. IRL Press, Oxford, pp 21–32
Holland PW, Holland LZ, Williams NA et al (1992) An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution. Development 116:653–661
Irimia M, Piñeiro C, Maeso I et al (2010) Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer. EvoDevo 1:7
Choi HMT, Schwarzkopf M, Fornace ME et al (2018) Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145:dev165753
Choi HMT, Chang JY, Trinh LA et al (2010) Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat Biotechnol 28:1208–1212
Choi HMT, Beck VA, Pierce NA (2014) Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano 8:4284–4294
Choi HMT, Calvert CR, Husain N et al (2016) Mapping a multiplexed zoo of mRNA expression. Development 143:3632–3637
Yu JK, Holland LZ (2009) Amphioxus whole-mount in situ hybridization. Cold Spring Harb Protoc 2009:pdb.prot5286
Yu JK, Holland LZ (2009) Amphioxus (Branchiostoma floridae) spawning and embryo collection. Cold Spring Harb Protoc 2009:pdb.prot5285
Benito-Gutiérrez E, Nake C, Llovera M et al (2005) The single AmphiTrk receptor highlights increased complexity of neurotrophin signalling in vertebrates and suggests an early role in developing sensory neuroepidermal cells. Development 132:2191–2202
Benito-Gutiérrez È, Stemmer M, Rohr SD et al (2018) Patterning of a telencephalon-like region in the adult brain of amphioxus. bioRxiv. 307629
Rybak-Wolf A, Solana J (2014) In: Nielsen BS (ed) Whole-Mount in situ hybridization using DIG-labelled probes in planarian in in situ hybridization protocols, methods in molecular biology, vol 1211. Springer, New York, pp 41–50
Fuentes R, Fernández J (2014) Fixation/permeabilization procedure for mRNA in situ hybridisation of zebrafish whole-mount oocytes, embryos and larvae. In: Nielsen BS (ed) In situ hybridization protocols, methods in molecular biology, vol 1211. Springer, New York, pp 1–12
Saint-Jeannet JP (2017) Whole-mount in situ hybridization of xenopus embryos. From the xenopus collection, edited by Hazel L. Sive. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot097287
Benito-Gutiérrez E, Weber H, Bryant DV et al (2013) Methods for generating year-round access to amphioxus in the laboratory. PLoS One 8:e71599
Acknowledgments
The authors would like to thank Ben Steventon for encouraging us to develop the HCR protocol in amphioxus; to Christo Christov for technical support to our lab and amphioxus facility, the latter supported by a Sir Isaac Newton Trust Research Grant (Ref. 15.07(r)); to everybody in the histopathology and imaging facilities at the CRUK-CI; and to Matt Wayland in the imaging facilities at the Department of Zoology, which are supported by a Sir Isaac Newton Trust Research Grant (Ref. 18.07ii(c)). We also acknowledge support from CRUK (C9545/A29580) to EBG, Wellcome Trust Grant (203806/Z/16/A) to TGA, and the Claire Barnes Trust to GG.
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Andrews, T.G.R., Gattoni, G., Busby, L., Schwimmer, M.A., Benito-Gutiérrez, È. (2020). Hybridization Chain Reaction for Quantitative and Multiplex Imaging of Gene Expression in Amphioxus Embryos and Adult Tissues. In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_11
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DOI: https://doi.org/10.1007/978-1-0716-0623-0_11
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