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Additional file 6: Figure 2. Targeted RNAi knockdown of FN generates defects in notochord morphogenesis. (A-D) Representative Bra:FNHP1998 phalloidin stained embryos fixed at approximately 12 HPF. (A) Bra:GFP negative control condition. (B-D) FN knockdown embryos representative of each hairpin phenotype: (B) “Normal” (C) “Mildly Defective” and (D) “Severely Defective.” Scale bar: 20 μm. (E) Bar graphs comparing the percentage of each phenotype in Bra:GFP vs. Bra:ScFNHP1998 vs Bra:FNHP1998 samples. N>300 per condition spanning at least four trials. Arrow bars: standard error, (PGFP, Brac>scFNHP1998, and Brac>FNHP1998 data (X2 = 861.65, df = 6, p < 2.2e-16). Then, to determine significance for comparisons between control and experimental samples, we conducted two-tailed, two-sample proportion test. Severely defective Brac>GFP vs Brac>FNHP1998 (Z=18.0549, df=1, p < 2.2e-16). Mildly defective Brac>scFNHP1998 vs Brac>FNHP1998 (Z=-10.1005, df=1, p< 2.2e-16). Movies 1–6. Time lapse confocal projections of representative U6>FngRNA6, Brac>nls::Cas9::nls, Brac>GFP transgenic embryos. Membranes stained by incubation with FM4-64 (50μl of 100μg/1 ml H20 stock solution added to 1 ml FSW in a cover-slip bottom imaging dish (MatTek). Samples imaged at 10-minute intervals (Movies 1-4) or 1-minute intervals (Movies 5-6). |
