Re-introduction of RNaseH2a is unable to rescue the developmental phenotype. (A) Embryos resulting from various crosses with homozygous rnaseh2a−/− parents. (B) Survival curve of embryos with various homozygous parents (n = 110). (C) Schematic of predicted mechanism of detrimental phenotypes resulting from each parental cross. (D) RTqPCR analysis of a DNA damage sensitive p53 reporter (Δ113p53) shows significant upregulation of expression in all embryos resulting from a homozygote parent. ±SD, one-way ANOVA, **P <0.001 (n = 3, 20 pooled embryos). (E) Microinjection of RNaseH2a mRNA caused a more severe phenotype in embryos from a rnaseh2a−/− incross compared with the control or uninjected embryos. Embryos from a wild-type incross were uninjected. (F) Quantification of embryo size, ±SD, two-way ANOVA, ****P <0.0001 (n = 16). (G) Gel analysis of alkaline treated DNA from testes of adult males show an increase in ribonucleotides in rnaseh2a-/- adult males. (H) Density plot of alkaline treatment of testes from adult zebrafish. (I) Ratio of long to short DNA fragments is significantly reduced in rnaseh2a−/− testes DNA after alkaline treatment. Student's t-test, n = 3 ± SEM, *P< 0.05. (J) Microinjection of a p53 morpholino visibly improves the development of all embryos with one or two homozygous parents (injected embryos are from same batch as the embryos shown panel A).
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