Abstract
In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and resolution by fluorescent in situ hybridization coupled with labeling of membranes and macromolecular complexes. Oocytes and embryos of ascidians – marine chordates closely related to vertebrates – are ideal models to understand how maternal mRNAs pattern the simple ascidian tadpole. More than three dozen cortically localized maternal mRNAs have been identified in ascidian eggs. They include germ cell markers such as vasa or pem-3 and determinants of axis (pem-1), unequal cleavage (pem-1), and muscle cells (macho-1). High resolution localization of mRNAs, proteins, and lipids in whole eggs and embryos and their cortical fragments shows that maternal mRNA determinants pem-1 and macho-1 are anchored to cortical endoplasmic reticulum and segregate with it into small posterior somatic cells. In contrast, mRNAs such as vasa are associated with granular structures which are inherited by the same somatic cells plus adjacent germ cell precursors. In this chapter, we provide detailed protocols for simultaneous localization of mRNAs and proteins to determine their association with cellular structures in eggs and embryos. Using preparations of isolated cortical fragments with intact membranous structures allows unprecedented high resolution analysis and identification of cellular anchoring sites for key mRNAs. This information is necessary for understanding the mechanisms for localizing mRNAs and partitioning them into daughter cells after cleavage.
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Acknowledgments
We thank H. Yasuo, C. Hudson, T. Lepage, P. Dru, A. McDougall, E. Houliston (Developmental Biology unit, Villefranche-sur-Mer Marine Station, CNRS/UPMC UMR7009, France), and L. Yamada (Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Japan) for helpful discussions and advice about ISH. This work was supported by grants to Christian Sardet from ANR (Agence Nationale de la Recherche), CNRS-ACI (Centre National de la Recherche Scientifique – Action Concertée Incitative), ARC (Association pour la Recherche sur le Cancer), and AFM (Association Française contre les Myopathies). Alexandre Paix was supported by CNRS, ARC, and ANR (Post-doctoral fellowship, A. McDougall laboratory).
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Paix, A., Chenevert, J., Sardet, C. (2011). Localization and Anchorage of Maternal mRNAs to Cortical Structures of Ascidian Eggs and Embryos Using High Resolution In Situ Hybridization. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_4
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DOI: https://doi.org/10.1007/978-1-61779-005-8_4
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