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Fig. 3. Gastrulation defects from inhibition of Xclaudin 1. (A) Verification of Xclaudin 1 MO in translation blocking efficiency by using Western blotting co-injection of Xclaudin 1 MO (20 ng) completely blocked the translation of co-injected Xclaudin 1 mRNA (500 pg/embryo) that bears MO-targeted 5′-UTR sequence, but not the one that misses MO-targeted sequence (500 pg/embryo) but bears only the whole coding region. Animal cap explant lysates from early gastrula stages following 2-cell stage injection at animal pole region were used for immunoblot (IB) analysis with anti-myc antibodies. β-Actin served as a loading control. (B) Four blastomeres at 8-cell stage embryos were injected at the dorso-animal blastomeres with Xclaudin 1 MO (20 ng). Unlike control embryos injected with control MO embryos (n = 70), embryos injected with Xclaudin 1 MO (n = 65) displayed slightly opened neural folds, dorsally bending trunk and shortened axis. These phenotypes were rescued by co-injection of Xclaudin 1 mRNA (ORF Xclaudin 1-myc) and MO (n = 67). (C) Gastrulation defects caused by Xclaudin 1 MO were rescued distinctively by the co-injection of Xclaudin 1 mRNA. The number of rescued embryos was increased when 20 pg of Xclaudin mRNA (n = 58) was injected from that with 10 pg (n = 54). Graph of numbers of embryos, which showed normal embryo (open bar), a short (slanting line bar), shorter (gray solid bar) anteroposterior and dorsally bending trunk axis (black solid bar) in injection of Xclaudin 1 mRNAs. (D) Xclaudin 1 knockdown retarded blastopore closure (n = 72). Frames from a representative time-lapse sequence showed that blastopore closure was delayed in either Xclaudin 1 MO (20 ng)- or mRNA-injected embryos until sibling control embryos reach equivalent midgastrula stage 13. Image published in: Chang DJ et al. (2010) Copyright © 2010. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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