Whole mount in situ hybridization techniques for analysis of the spatial distribution of mRNAs in sea urchin embryos and early larvae.

Autor:	Erkenbrack EM; Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, United States; Yale Systems Biology Institute, Yale University, West Haven, CT, United States., Croce JC; Laboratoire de Biologie du Développement de Villefranche-sur-Mer (LBDV), Sorbonne Université, CNRS, Evolution of Intercellular Signaling in Development (EvoInSiDe) team, Villefranche-sur-Mer, France., Miranda E; Department of Biology, Duke University, Durham, NC, United States., Gautam S; Department of Biological Sciences, Auburn University, Auburn, AL, United States., Martinez-Bartolome M; Department of Biological Sciences, Auburn University, Auburn, AL, United States., Yaguchi S; Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan., Range RC; Department of Biological Sciences, Auburn University, Auburn, AL, United States. Electronic address: range@auburn.edu.
Jazyk:	angličtina
Zdroj:	Methods in cell biology [Methods Cell Biol] 2019; Vol. 151, pp. 177-196. Date of Electronic Publication: 2019 Mar 20.
DOI:	10.1016/bs.mcb.2019.01.003
Abstrakt:	A critical process in embryonic development is the activation and spatial localization of mRNAs to specific cells and territories of the embryo. Revealing the spatial distribution of mRNAs and how it changes during development is a vital piece of information that aids in understanding the signaling and regulatory genes driving specific gene regulatory networks. In the laboratory, a cost-efficient, reliable method to determine the spatial distribution of mRNAs in embryos is in situ hybridization. This sensitive and straightforward method employs exogenous antisense RNA probes to find specific and complementary sequences in fixed embryos. Antigenic moieties conjugated to the ribonucleotides incorporated in the probe cross-react with antibodies, and numerous staining methods can be subsequently employed to reveal the spatial distribution of the targeted mRNA. The quality of the data produced by this method is equivalent to the experience of the researcher, and thus a thorough understanding of the numerous steps comprising this method is important for obtaining high quality data. Here we compile and summarize several protocols that have been employed chiefly on five sea urchin species in numerous laboratories around the world. Whereas the protocols can vary for the different species, the overarching steps are similar and can be readily mastered. When properly and carefully undertaken, in situ hybridization is a powerful tool providing unambiguous data for which there currently is no comparable substitute and will continue to be an important method in the era of big data and beyond. (© 2019 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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