Co-injection of dnmt1 MO partially rescues the h2afv morphant phenotype (A) Bright field images comparing embryos injected with control morpholino-2 (0.67 ng) or with h2afv (0.67 ng), dnmt1 (0.43 ng) or dnmt1-h2afv co injected embryos exhibiting the severe phenotype at 24 and 48 hpf. The dnmt1 morphants do not exhibit any significant phenotype either at 24 hpf or 48 hpf (n = 5) (B) Phenotype scored in a minimum of 5 clutches as Severe, Mild or Normal and displayed as percentages show that the percentage of h2afv morphants exhibiting the severe phenotype is significantly reduced in a dnmt MO background. (C) Percent survival of h2afv morphants with and without addition of dnmt1 MO at 24 hpf and 48 hpf. The survival of the h2afv morphants is not significantly affected with knockdown of Dnmt1 protein (p-value between the dead vs. alive numbers of h2afv and dnmt1- h2afv double morphants at 24 hpf = 0.4855 and at 48 hpf = 0.05. (D) Western blot analysis of protein lysates of 16-18 hpf embryos demonstrates complete depletion of the H2afv protein in both h2afv morphants and dnmt1- h2afv morphants while the expression of H2afv protein is unaffected in control and dnmt1 morphant embryos (n = 2). (E) Slot blot analysis of 5-MeC levels using genomic DNA from control, h2afv morphants and dnmt1- h2afv morphants at 14-18 somite stage (16-18 hpf). The h2afv morphants exhibit significant hypermethylation and the inhibition of dnmt1 in this background does not have a significant effect on the global DNA hypermethylation. One way ANOVA was performed comparing each of the morphants with the uninjected controls. Number of clutches (n) = 4. Scale bar: 1000 μm.
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