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

详细信息    查看全文

• 作者：Timothy Q DuBuc ; Ada A Dattoli ; Leslie S Babonis…
• 关键词：Nematostella vectensis ; Lifeact ; mTurquoise2 ; EB1 ; Cytoskeleton ; Microvilli ; Nuclear envelope ; Mitosis
• 刊名：BMC Cell Biology
• 出版年：2014
• 出版时间：December 2014
• 年：2014
• 卷：15
• 期：1
• 全文大小：4,879 KB
• 参考文献：1. Shimomura, O, Johnson, FH, Saiga, Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59: pp. 223-39 CrossRef
  2. Wikramanayake, AH, Hong, M, Lee, PN, Pang, K, Byrum, CA, Bince, JM, Martindale, MQ (2003) An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 426: pp. 446-450 CrossRef
  3. Saina, M, Genikhovich, G, Renfer, E, Technau, U (2009) BMPs and chordin regulate patterning of the directive axis in a sea anemone. PNAS 106: pp. 18592-18597 CrossRef
  4. Martindale, MQ, Pang, K, Finnerty, JR (2004) Investigating the origins of triploblasty: “mesodermal-gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa). Development 131: pp. 2463-2474 CrossRef
  5. Renfer, E, Amon-hassenzahl, A, Steinmetz, PRH, Technau, U (2009) A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis. PNAS 107: pp. 104-108 CrossRef
  6. Layden, MJ, R?ttinger, E, Wolenski, FS, Gilmore, TD, Martindale, MQ (2013) Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat Protoc 8: pp. 924-934 CrossRef
  7. Marlow, H, Roettinger, E, Boekhout, M, Martindale, MQ (2012) Functional roles of Notch signaling in the cnidarian Nematostella vectensis. Dev Biol 362: pp. 295-308 CrossRef
  8. Friedrich, CL, Moyles, D, Beveridge, TJ, Hancock, RE (2000) Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrob Agents Chemother 44: pp. 2086-2092 CrossRef
  9. Moelans, CB, Hoeve, N, Ginkel, J-W, Kate, FJ, van Diest, PJ (2011) Formaldehyde substitute fixatives. Analysis of macroscopy, morphologic analysis, and immunohistochemical analysis. Am J Clin Pathol 136: pp. 548-556 CrossRef
  10. Hejnol, A, Obst, M, Stamatakis, A, Ott, M, Rouse, GW, Edgecombe, GD, Martinez, P, Bagu?à, J, Bailly, X, Jondelius, U, Wiens, M, Müller, WEG, Seaver, E, Wheeler, WC, Martindale, MQ, Giribet, G, Dunn, CW (2009) Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc R Soc B 276: pp. 4261-4270 CrossRef
  11. Szymanski, DB, Cosgrove, DJ (2009) Dynamic coordination of cytoskeletal and cell wall systems during plant cell morphogenesis. Current Biology 19: pp. R800-R811 CrossRef
  12. Lancaster, OM, Berre, M, Dimitracopoulos, A, Bonazzi, D, Zlotek-Zlotkiewicz, E, Picone, R, Duke, T, Piel, M, Baum, B (2013) Mitotic rounding alters cell geometry to ensure efficient bipolar spindle formation. Dev Cell 25: pp. 270-283 CrossRef
  13. Güttinger, S, Laurell, E, Kutay, U (2009) Orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat Rev Mol Cell Biol 10: pp. 178-191 CrossRef
  14. Li, J, Shariff, A, Wiking, M, Lundberg, E, Rohde, GK, Murphy, RF (2012) Estimating microtubule distributions from 2D immunofluorescence microscopy images reveals differences among human cultured cell lines. PLoS ONE 7: pp. e50292 CrossRef
  15. Müller, M, Diensthuber, RP, Chizhov, I, Claus, P, Heissler, SM, Preller, M, Taft, MH, Manstein, DJ (2013) Distinct functional interactions between actin isoforms and nonsarcomeri
• 刊物主题：Cell Biology; Biological Microscopy; Life Sciences, general;
• 出版者：BioMed Central
• ISSN：1471-2121

文摘

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.