Zebrafish preserve preserve germline epigenetic memory globally but demethylate and amplify sex-linked rDNA during feminisation

The germline is unique because it is the only cellular lineage capable of transferring genetic information from one generation to the next. Intergenerational transmission of epigenetic memory is also possible; however, in mammals this is largely prevented by extensive epigenetic erasure during germline definition from somatic precursors. We report that the ‘preformed’ germline of zebrafish, defined by inheritance of cellular components from the egg and not cellular reprogramming, shows consistently high levels of DNA methylation throughout development. This demonstrates epigenetic erasure is not a barrier to transgenerational epigenetic inheritance in zebrafish and likely other vertebrates with preformed germlines. Our analysis also uncovered female-specific germline amplification and demethylation of an 11.5-kb repeat region encoding 45S ribosomal RNA (fem-rDNA). The peak of fem-rDNA expansion coincided with the initial expansion of stage IB oocytes, the poly-nucleolar cell type responsible for zebrafish feminisation. Given that fem-rDNA overlaps with the only zebrafish locus identified thus far as sex-linked, we hypothesize fem-rDNA expansion is intrinsic to sex determination in this species. vasa:eGFP positive cells were isolated from zebrafish since day 1 post fertilisation (1 dpf) until sexual maturity (70 dpf) using fluorescence assisted cells sorting (FACS). Low coverage whole genome bisulfite sequencing from eGFP positive and negative using a modified PBAT protocol was used to assess DNA methylation in the zebrafish germline.