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

BACKGROUND: Cnidarians 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. RESULTS: We 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. CONCLUSION: The 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.