Abstract
Tight junctions (TJs) function as a physiological barrier between epithelial and endothelial sheets by restricting the diffusion of fluid through the intercellular space. Recent morphological studies associated with TJs have revealed that the TJ is a dynamic rather than a static structure; indeed, several in vitro studies indicate that proper TJ function requires dynamic TJ behavior. Direct observation of the dynamic behavior of TJs is necessary to understand the essential roles of TJs in physiological contexts, such as during embryogenesis and metastasis. Here we describe a protocol for the generation of transgenic medaka (Oryzias latipes) that express claudin–EGFP. This fluorescent fusion protein enables real-time imaging of TJs in the living embryo. Claudin–EGFP transgenic medaka will be a useful tool to screen for mutations and for small molecules affecting cell–cell adhesion.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2: 285–93.
Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occluding. J Cell Biol 141: 1539–50.
Furuse M, Sasaki H, Fujimoto K, Tsukita S (1998) A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 143: 391–401.
Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP (1999) Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 283: 103–6.
Konrad M, Schaller A, Seelow D, Pandey AV, Waldegger S, Lesslauer A, Vitzthum H, Suzuki Y, Luk JM, Becker C, Schlingmann KP, Schmid M, Rodriguez-Soriano J, Ariceta G, Cano F, Enriquez R, Juppner H, Bakkaloglu SA, Hediger MA, Gallati S, Neuhauss SC, Nurnberg P, Weber S (2006) Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement. Am J Hum Genet 79(5): 949–57.
Sasaki H, Matsui C, Furuse K, Mimori-Kiyosue Y, Furuse M, Tsukita S (2003) Dynamic behavior of paired claudin strands within apposing plasma membranes. Proc Natl Acad Sci USA 100: 3971–6.
Matsuda M, Kubo A, Furuse M, Tsukita S (2004) A peculiar internalization of claudins, tight junction-specific adhesion molecules, during the intercellular movement of epithelial cells. J Cell Sci 117: 1247–57.
Miyamoto T, Momoi A, Kato K, Tsukita S, Furuse M, Furutani-Seiki M (2009) Generation of transgenic medaka expressing claudin7-EGFP for imaging tight junction in living medaka embryos. Cell Tissue Res 335: 465–471.
Kato K, Takagi M, Tamaru Y, Akiyama S, Konishi T, Murata O, Kumai H (2007) Construction of an expression vector containing a beta-actin promoter region for gene transfer by microinjection in red sea bream Pagrus major. Fish Sci 73: 440–445.
Furutani-Seiki M, Wittbodt J (2004) Medaka and Zebrafish, an evolutionary twin study. Mech Dev 121: 629–637.
Ozato K, Kondoh H, Inohara H, Iwamatsu T, Wakamatsu Y, Okada TS (1986) Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos. Cell Differ 19: 237–244.
Furutani-Seiki M, Sasado T, Morinaga C, Suwa H, Niwa K, Yoda H, Deguchi T, Hirose Y, Yasuoka A, Henrich T, Watanabe T, Iwanami N, Kitagawa D, Saito K, Asaka S, Osakada M, Kunimatsu S, Momoi A, Elmasri H, Winkler C, Ramialison M, Loosli F, Quiring R, Carl M, Grabher C, Winkler S, Del Bene F, Shinomiya A, Kota Y, Yamanaka T, Okamoto Y, Takahashi K, Todo T, Abe K, Takahama Y, Tanaka M, Mitani H, Katada T, Nishina H, Nakajima N, Wittbrodt J, Kondoh H (2004) A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes. Mech Dev 121: 647–658.
Kasahara M, Naruse K, Sasaki S, Nakatani Y, Qu W, Ahsan B, Yamada T, Nagayasu Y, Doi K, Kasai Y, Jindo T, Kobayashi D, Shimada A, Toyoda A, Kuroki Y, Fujiyama A, Sasaki T, Shimizu A, Asakawa S, Shimizu N, Hashimoto S, Yang J, Lee Y, Matsushima, Sugano S, Sakaizumi M, Narita T, Ohishi K, Haga S, Ohta F, Nomoto H, Nogata K, Morishita T, Endo T, Shin IT, Takeda H, Morishita S, Kohara Y (2007) The medaka draft genome and insights into vertebrate genome evolution. Nature 7145: 714–719.
Porazinski S, Wang H, Furutani-Seiki M (In press) Embryology and genetic methods-medaka. Methods in Molecular Biology.
Acknowledgment
We wish to thank all members of the Shoichiro Tsukita laboratory for helpful discussions.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Miyamoto, T., Furuse, M., Furutani-Seiki, M. (2011). In Vivo Imaging of Tight Junctions Using Claudin–EGFP Transgenic Medaka. In: Turksen, K. (eds) Claudins. Methods in Molecular Biology, vol 762. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-185-7_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-185-7_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-184-0
Online ISBN: 978-1-61779-185-7
eBook Packages: Springer Protocols