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Figure 11. tfap2a knockdown phenocopies ERK3 knockdown in Xenopus laevis embryos. A, B, Control MO (40 (A) or 20 (A, B) ng) or TFAP2A MO1/2 (20 (A) or 10 (A, B) ng each of MO1 and MO2) were co-injected with dextran-fluorescein into both (A) or the left (B) V2 blastomere(s) at the 8-cell stage. Embryos with pronephric fluorescence were further analyzed. A, Upper panels, stage 42 embryos injected with control MO (40 ng) or TFAP2A MO1/2 (20 ng each of MO1 and MO2). The black arrowhead indicates edema. The graph shows the percent of embryos with edema from two independent experiments. **P<0.01 by z-test. B, atp1b1 expression at stage 33/34 was inhibited by TFAP2A MO1/2 (N=21/21) but not by control MO (N=0/18) in three independent experiments. pn, pronephros. C, Control MO (80 ng) or TFAP2A MO1/2 (40 ng each of MO1 and MO2) was injected into animal regions of both ventral blastomeres at the 4-cell stage. At stage 39, epidermal disintegration was induced by TFAP2A MO1/2 (N=31/31) but not by control MO (N=0/39) in three independent experiments. A-C, Lateral views with anterior to the left and dorsal up. Scale bars, 1 (A), 0.2 (B) or 0.5 (C) mm. D-F, Control MO (60 ng in (D, F) or 40 ng in (E)) 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. D, 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 um. CAAX-GFP mRNA (200 pg) was co-injected to visualize the plasma membrane. E, 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. 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 um. Dorsal is up. D, F, Essentially the same results were obtained for 8-10 embryos from two independent experiments. The data are representative of 13-15 images. |
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Figure 13. Phenotypes induced by ERK3 knockdown are partially rescued by tfap2a overexpression in Xenopus laevis. A, B, Control MO (20 ng) or ERK3 MO1/2 (10 ng each of MO1 and MO2) was injected with or without tfap2a.L mRNA (400 pg) into the left V2 blastomere at the 8- cell stage. All injections were carried out using dextran-fluorescein as a lineage tracer. Embryos with pronephric fluorescence were fixed at stage 33/34 for whole mount in situ hybridization. A, Lateral views with anterior to the left and dorsal up. pn, pronephros. Scale bars, 200 m. B, The percent of embryos with normal or aberrant atp1b1 expression in pronephros was scored in five independent experiments. Severe, no expression; Moderate, weak and discontinuous expression; Mild, weak but continuous expression. *P<0.05 by Mann-Whitney U test. C, D, Control MO (40 ng) or ERK3 MO1/2 (20 ng each of MO1 and MO2) was injected with or without tfap2a.L mRNA (800 pg) into the animal regions of both ventral blastomeres at the 4-cell stage. CAAX-GFP mRNA (200 pg) was also co-injected in all conditions to visualize the plasma membrane. Injected embryos were fixed at stage 13 for immunofluorescence using anti-E-cadherin. Apical surfaces of the epidermal epithelia were observed by confocal microscopy. Scale bars, 10 m. D, Each point represents the average fluorescence intensity of junctional E-cadherin in one cell (control MO, 49 cells from 16 microscopic fields; ERK3 MO1/2, 51 cells from 20 microscopic fields; ERK3 MO1/2 plus tfap2a.L mRNA, 58 cells from 19 microscopic fields; from two independent experiments). The bars represent the average±S.D. *P<0.05 by Mann- Whitney U test. |
