5-hmC and 5mC Staining in Zebrafish Early Embryos and Mice Zygote, Related to Figure3 (A-E) 5-hmC and 5-mC were stained for zebrafish 16-cell, 32-cell, 1,000-cell, tail of the 24hr embryos (A-D) and mice zygote at PN4 (pre-nuclear 4) (E). Zebrafish embryos are shown with their animal poles at the top of the image. All zebrafish embryos were stained in parallel under identical conditions and imaged with identical exposure time. Mouse zygote was stained as whole-mounts using the same antibody dilutions and incubation time. Male and female symbols indicate male and female pronuclear, respectively. Scale bars are 30 μm in(A-D) and 20 μm in (E).

Functional Analysis of DNA Methylation in Zebrafish, Related to Figure 5 (A) Anti-correlations between gene expression and DNA methylation level in promoter region (2 k upstream TSS) and 5′UTR for sperm, oocyte, 1,000-cell embryos, and germ-ring embryos (P value for Pearson correlation < 0.0001). Relative DNA methylation density in exon, intron and 3′UTR and corresponding gene expression are displayed. Each row represents one gene. DNA methylation level is colored white to red to indicate low to high. Gene expression level is also colored white to red to indicate low to high. (B) Gene expression varies inversely with methylation level of TSS-proximal region. Gene was aligned at TSS or TSE (transcription end site). The average methylation level for each 100bp interval was calculated. Genes were grouped into deciles by gene expression. The average methylation levels for four representative deciles are showed. (C) Genes with lower methylation level in promoters are highly expressed compared to genes with higher methylation level in each stage of sperm, oocyte, 1,000-cell and germ-ring embryos. ML represents methylation level. Genes with methylation level < 0.2 or > 0.8 were assigned into two groups. Expression levels between the two groups of genes were compared using two tailed student t test with P value cutoff < 0.05.(D) Images for wild-type and androgenetic zebrafish 18 hr after fertilization. Androgenetic zebrafish shows haploid syndromes seen a shortened body phenotype.