In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes

BMC Cell Biology

Methodology Article

Anna A Dattoli¹  Marten Postma¹  Miguel Salinas-Saavedra²  Mark Q Martindale²  Timothy Q DuBuc²  Leslie S Babonis²  Eric Röttinger³ 

[1] Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, NL-1098 XH, Amsterdam, The Netherlands;The Whitney Laboratory for Marine Bioscience, 9505 N. Ocean Shore Blvd, 32080-8610, St. Augustine, FL, USA;Kewalo Marine Laboratory, University of Hawaii, 41 Ahui St., 96813, Honolulu, HI, USA;Université Nice Sophia Antipolis, IRCAN, UMR 7284, 06107, Nice, France;CNRS, IRCAN, UMR 7284, 06107, Nice, France;INSERM, IRCAN, U1081, 06107, Nice, France;

关键词: Nematostella vectensis;    Lifeact;    mTurquoise2;    EB1;    Cytoskeleton;    Microvilli;    Nuclear envelope;    Mitosis;   

DOI  :  10.1186/s12860-014-0044-2

 received in 2014-06-12, accepted in 2014-11-19,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundCnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos.ResultsWe used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated ‘flash’ of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.ConclusionThe methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

【 授权许可】

Unknown   
© DuBuc et al.; licensee BioMed Central. 2016. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.