Fig. 7

DNA methylation is essential for normal embryogenesis in O. fusiformis. a Schematic representation of the experimental design. Independent batches of embryos were treated after fertilization with either 1% DMSO (control) or 50 µM zebularine and 10 µM 5-azacytidine for 24 h until the larval stage, when phenotypes were scored. b Z-projections of confocal stacks of zebularine-treated and DMSO-control embryos fixed at the early larval stage and stained for acetylated tubulin (magenta), actin (yellow) and nuclei (cyan). Zebularine treatment impairs annelid embryogenesis as treated larvae fail to undergo normal organogenesis in O. fusiformis. c Zebularine but not 5-azacytidine treatment significantly decreases global methylation levels in O. fusiformis as measured with shallow coverage EM-seq (two-tailed unpaired t-tests). d Bar plots depicting the number of differentially expressed genes and transposable elements (TEs) after zebularine treatment. Reduction of normal methylation levels reactivates TE expression, mainly in intergenic regions. e The evolution of DNA methylation in bilaterally symmetrical animals and Spiralia. Gene body methylation is likely the ancestral stage in animals, and the presence of adult de-methylation in deuterostomes and annelids suggests that this might be an ancestral bilaterian feature. TE methylation might have evolved multiple times independently, similarly to the levels of DNA methylation, with numerous independent transitions to a hypermethylation and unmethylation state in different animal lineages