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Figure 4


Fig. 4. Depletion of Shroom3 abolishes the apical localization of Rock1 in the neural tube. (A) Co-localization of Shroom3 and Rock1 at the apical junctions of chicken neural tubes. A transverse section of stage 9 chicken embryo at the hindbrain level was double-immunostained for these proteins. (B) Test for RNAi-mediated depletion of chicken Shroom3. MDCK cells were co-transfected with RNAi vectors for the chicken Shroom3 (Shrm RNAi), scrambled sequences (Control RNAi) or empty vector (Vec), and with either an EGFP-tagged middle region of chicken Shroom3 (EGFP-ch-Shrm/M) or EGFP alone. The cell lysates were immunoblotted with anti-GFP or anti-{alpha}-tubulin antibodies. There is effective and specific depletion of Shroom3 by the RNAi vector. (C) Effects of Shroom3 knockdown on the localization of Rock1 in the neural tube. Chicken embryos were co-electroporated with EGFP and RNAi vectors, as shown in Fig. S3 in the supplementary material, and fixed at stage 9. A section of an embryo at the forebrain-midbrain level was double-immunostained for EGFP and Shroom3, and adjacent sections were immunostained for Rock1, pMLC and ZO-1 in each experiment. Rock1 and pMLC levels are reduced at the apical junctions in the Shroom3-depleted tube (Shrm RNAi), the closure of which is also perturbed. Scale bars: 20 µm.





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