Zebrafish preserve global germline DNA methylation while sex-linked rDNA is amplified and demethylated during feminisation

Abstract

The germline is the only cellular lineage capable of transferring genetic information from one generation to the next. Intergenerational transmission of epigenetic memory through the germline, in the form of DNA methylation, has been proposed; however, in mammals this is largely prevented by extensive epigenetic erasure during germline definition. Here we report that, unlike mammals, the continuously-defined 'preformed' germline of zebrafish does not undergo genome-wide erasure of DNA methylation during development. Our analysis also uncovers oocyte-specific germline amplification and demethylation of an 11.5-kb repeat region encoding 45S ribosomal RNA (fem-rDNA). The peak of fem-rDNA amplification coincides 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 hypothesise fem-rDNA expansion could be intrinsic to sex determination in this species.

Fig. 1

Isolation and quantitation of DNA methylation in the zebrafish germline. a–d Fluorescence microscopy of tg(vasa:EGFP) zebrafish embryos and larvae. 1.5 h post-fertilisation (hpf) (a), 24 hpf (b), 48 hpf (c–d). 1.8× view of EGFP +ve labelled cells is shown inset (dashed lines). Scale bars are 500 µm. Forward scatter height (FSC-H) e Flow cytometry plot of 10 zebrafish larvae at 48 hpf. The red dashed square indicates the EGFP +ve population gated for isolation. Blue dots indicate discrete data points (i.e., cellular events), whereas green, yellow and red colouring indicate increasing data density. f Percentage of methylation in CG context from 1 to 28 days post-fertilisation (dpf) in both vasa:EGFP +ve germline cells (green line) and control cells (black line). For each sample type and timepoint, n = 3 independent biological replicates were used, except for 28 dpf vasa:EGFP +ve, which has n = 5 independent biological replicates

Fig. 2

Fluorescence microscopy of germline cells and their methylation during gonad transformation. a–f Phenotypic sex in zebrafish can be identified using vasa:EGFP expression: during the ‘juvenile ovary’ stage, expression of EGFP is low but consistent between individuals (a, b). Later, expression of EGFP vastly increases in presumptive females (c, d) relative to presumptive males (e, f). This enables sex phenotyping in early stages of sexual differentiation. Scale bars are 500 µm. g, h Methylation in the vasa:EGFP +ve germline cells of female (g) and male (h) fish from the gonad transformation stage until sexual maturity (35–70 dpf). Non-germline control cells were also tested (black lines and dots). For each sample type and timepoint, n = 3 independent biological replicates were used

Fig. 3

Amplification of oocyte-specific fem-rDNA in a previously identified sex-linked region. a Number of CG calls mapping to windows of 1 Mb in the whole zebrafish genome. A peak is observed at the right tip of chromosome 4 following 25-dpf and 28-dpf. b Reads map to both the complementary original top (CTOT, red) complementary original bottom (CTOB, blue) strand of the 45S fem-rDNA unit on chromosome 4. Components of the rDNA repeat are indicated (External transcribed spacer (ETS), Internal transcribed spacer (ITS), Intergenic spacer (IGS)). c The amplified region is located within the most significant sex-linked SNPs from non-domesticated zebrafish strains, Cooch Behar, Ekkwill, Nadia and WIK

Fig. 4

Amplification and methylation of oocyte-specific fem-rDNA during gonad transformation. a Percentage of reads mapping to fem-rDNA in the germline prior to sexual differentiation (green), and in presumptive males (blue) and females (red) during gonad transformation and after sexual maturation. b Relationship between the amplification and methylation of fem-rDNA for vasa:EGFP −ve control samples (grey dots), and vasa:EGFP +ve germline cells from sexually undifferentiated fish at 1–21 (green dots) and 25–28 dpf (magenta dots); presumptive female fish at 35–45 (pink) and 50–70 (red) dpf; presumptive male fish at 35–45 (light blue) and 50–70 (blue) dpf. Samples have been divided into 3 clusters based on rDNA level and methylation; (1) ‘background’ non-amplified and methylated (2) moderately amplified and lowly methylated, (3) highly amplified and unmethylated. These consist of n = 75, n = 15, and n = 14 samples, respectively

Fig. 5

Global DNA methylation and fem-rDNA dynamics in the zebrafish germline. a CG methylation in the mouse and zebrafish germline. In stark contrast to mice, the zebrafish germline does not undergo extensive erasure of DNA methylation and germline DNA from females (red), males (blue) and fish of undifferentiated sex (green) are similar. Note, data for mouse were taken from the meta-analysis provided by Lee, Hore and Reik. b Striking amplification of fem-rDNA occurs in germline cells during the critical period of gonad transformation in females. Beginning around 28 dpf perinucleolar oocytes amplify in at least 2 orders of magnitude oocyte-specific 45S rDNA. ICM, inner cell mass; non-growing (NG) oocyte