Xenbase Image ID: 172386

Figure 4. ERK3 knockdown leads to defective adherens and tight junctions in the epithelia of the Xenopus laevis embryonic epidermis. Control MO (60 ng in (A, C-F) or 40 ng in (B)), ERK3 MO1/2 (20 ng each of MO1 and MO2), ERK3 MO3 (60 ng) or TFAP2A MO1/2 (20 ng each of MO1 and MO2) was injected into the animal regions of both ventral blastomeres at the 4-cell stage. A, Injected embryos were fixed at stage 13 for immunofluorescence using the anti-E-cadherin antibody. Apical surfaces of the epidermal epithelia were observed by confocal microscopy. Scale bars, 10 m. CAAX-GFP mRNA (200 pg) was co-injected to visualize the plasma membrane. B, Real-time quantitative RT-PCR analysis of E-cadherin and ZO-1 expression. Injected embryos were harvested at stage 14. The expression levels of E-cadherin or ZO-1 were normalized to those of odc. The bars represent the average±S.D. The normalized E-cadherin or ZO-1 expression level in control embryos was defined as 1.0 in each experiment. Shown are all data points from four independent experiments. **P<0.01 by t test. n.s., not significant by t test. C-E, Injected embryos were fixed at stage 13 for immunofluorescence using the anti-ZO-1 antibody. Apical surfaces of the epidermal epithelia were observed by confocal microscopy. C, Scale bars, 10 m. The yellow arrowheads indicate gaps in ZO-1 signals. D, Each point indicates the number of gaps in ZO-1 signals per microscopic field (control MO, N=23; ERK3 MO1/2, N=25; ERK3 MO3, N=27; TFAP2A MO1/2, N=27) from two independent experiments. The bars represent the average±S.D. **P<0.01 by Tukey test. E, Each point represents the average fluorescence intensity of junctional ZO-1 in one cell (control MO, 80 cells from 27 microscopic fields; ERK3 MO1/2, 80 cells from 27 microscopic fields; ERK3 MO3, 81 cells from 27 microscopic fields; TFAP2A MO1/2, 81 cells from 27 microscopic fields; from two independent experiments). The bars represent the average±S.D. **P<0.01 by Tukey test. F, Injected embryos after vitelline membrane removal at stage 23 were exposed to 1 mg/ml EZ-link Sulfo-NHS-LC-Biotin for 10 min at 15°C and then fixed for transverse sectioning. EZ-link Sulfo-NHS-LC-Biotin was detected with streptavidin FITC. Scale bars, 100 m. Dorsal is up. A, F, Essentially the same results were obtained for 8-11 embryos from three independent experiments. The data are representative of 19-23 images. Image published in: Takahashi C et al. (2018) Copyright © 2018. Image 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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