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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Nephron Exp Nephrol. 2014 Nov 19;128(0):80–88. doi: 10.1159/000368411

Fig. 2.

Fig. 2

wnt5a knockdown by antisense morpholinos results in abnormal body curvature. a Diagram showing the zebrafish wnt5a gene, the coding region, and the regions targeted by the AUG- and splice-MO. The sequences of MOs complementary to the gene target were: AUG-MO: 5 ′-ACTTCAGCTTCAGCAGCATCATAAC-3 ′ (GI_BC150218) and splice-MO: 5 ′-gtgtttttgcggcgtacatacCGAT-3 ′ (GI_BC150218; capitalized letters: targeted exon; non-capitalized letters: intron). b Phenotype of injection control (phenol red) embryos and AUG- and splice-site wnt5a morphants at 3 dpf with dorsal to the top and anterior to the left. The AUG- and splice-site morphants phenocopied each other with a downward curved tail, small eyes, and pericardial edema. ‘Class 1’ morphants exhibits a ‘curly tail down’ body shape and pericardial edema (90% of total abnormal morphants, first row middle and right column). ‘Class 2’ morphants exhibits more severe defects including a reduced and abnormally shaped body axis and significant pericardial edema (10% of total abnormal morphants, second row middle and right column). Bar = 2 mm. c A dose-response occurs, with increasing amounts of morpholino resulting in a worsening phenotypes for both the AUG- and splice-MOs (p = 0.064 in AUG-MO 7.5 ng vs. control, p < 0.001 in AUG-MO 15 ng vs. control, p < 0.001 in AUG-MO 22 ng vs. control, p = 0.062 in splice-MO 7.5 ng vs. control, p < 0.001 in splice-MO 15 ng vs. control, p < 0.001 in splice-MO 22 ng vs. control). The use of two MOs with unrelated sequences demonstrates the specificity of the observed phenotype. d The AUG-MO phenotype can be partially rescued with mouse Wnt5a mRNA, which is resistant to the AUG-MO due to a difference in primary base pair structure, thereby demonstrating that the phenotypes are not due to off-target effects. Abnormal phenotypes as described above are counted at 72 hpf in different injection groups (we tried mouse mRNA dose from 20 to 120 pg and identified 40 pg RNA to be the dose at which nonspecific or toxic effects of the RNA do not occur). The difference is statistically significant at p < 0.05 (p < 0.001 in AUG-MO 15 ng vs. control group, p = 0.005 in AUG-Mo 15 ng + 40 pg mWnt5a mRNA group vs. AUG-MO 15 ng group).