Abstract
Time-lapse 4D imaging of fluorescently tagged proteins to follow the dynamics of cellular structures (chromosomes, microtubules, actin, centrosomes, cortical structures like the CAB in ascidians, etc.) combined with targeted gene knockdown during embryonic development is a powerful technique to understand the mechanisms of embryonic development. The eggs and embryos of the primitive marine chordate Phallusia mammillata are an excellent model system for combining live cell imaging with gene knockdown experiments. Here we describe simple methods for microinjecting Phallusia eggs with mRNA encoding fluorescent fusion proteins combined with 4D time-lapse imaging techniques we use to follow all of embryonic development from the egg to late tailbud stage.
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References
Olivier N, Luengo-Oroz MA, Duloquin L et al (2010) Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy. Science 329:967–971
Segalen M, Bellaïche Y (2009) Cell division orientation and planar cell polarity pathways. Semin Cell Dev Biol 20:972–977
Satoh N (2003) The ascidian tadpole larva: comparative molecular development and genomics. Nat Rev Genet 4:285–295
Negishi T, Takada T, Kawai N et al (2007) Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians. Curr Biol 17:1014–1025
Dumollard R, Hebras C, Besnardeau L, McDougall A (2013) Beta-catenin patterns the cell cycle during maternal-to-zygotic transition in urochordate embryos. Dev Biol 384: 331–342
Dehal P, Satou Y, Campbell RK et al (2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298:2157–2167
Tassy O, Daian F, Hudson C et al (2006) A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis. Curr Biol 16:345–358
Satou Y, Kawashima T, Shoguchi E et al (2005) An integrated database of the ascidian, Ciona intestinalis: towards functional genomics. Zool Sci 22:837–843
Endo T, Ueno K, Yonezawa K et al (2010) CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality. http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/21071393
Hotta K, Mitsuhara K, Takahashi H et al (2007) A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva. Dev Dyn 236:1790–1805
Zalokar M, Sardet C (1984) Tracing of cell lineage in embryonic development of Phallusia mammillata (Ascidia) by vital staining of mitochondria. Dev Biol 102:195–205
Prodon F, Chenevert J, Hébras C et al (2010) Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors. Development 137:2011–2021
Kiehart DP (1982) Microinjection of echinoderm eggs: apparatus and procedures. Methods Cell Biol 25(Pt B):13–31
Acknowledgements
We would like to thank Celine Hebras for all molecular biology reagents, Francois Prodon for providing images in Fig. 2a, and the ANR (08-BLAN-0136-02) for financial support.
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McDougall, A., Lee, K.Wm., Dumollard, R. (2014). Microinjection and 4D Fluorescence Imaging in the Eggs and Embryos of the Ascidian Phallusia mammillata . In: Carroll, D., Stricker, S. (eds) Developmental Biology of the Sea Urchin and Other Marine Invertebrates. Methods in Molecular Biology, vol 1128. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-974-1_11
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DOI: https://doi.org/10.1007/978-1-62703-974-1_11
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