Abundance of Early Embryonic Primordial Germ Cells Promotes Zebrafish Female Differentiation as Revealed by Lifetime Labeling of Germline

Abstract

Teleost sex differentiation largely depends on the number of undifferentiated germ cells. Here, we describe the generation and characterization of a novel transgenic zebrafish line, Tg ( piwil1 : egfp-UTRnanos3 ) ihb327Tg , which specifically labels the whole lifetime of germ cells, i.e., from primordial germ cells (PGCs) at shield stage to the oogonia and early stage of oocytes in the ovary and to the early stage of spermatogonia, spermatocyte, and spermatid in the testis. By using this transgenic line, we carefully observed the numbers of PGCs from early embryonic stage to juvenile stage and the differentiation process of ovary and testis. The numbers of PGCs became variable at as early as 1 day post-fertilization (dpf). Interestingly, the embryos with a high amount of PGCs mainly developed into females and the ones with a low amount of PGCs mainly developed into males. By using transient overexpression and transgenic induction of PGC-specific bucky ball ( buc ), we further proved that induction of abundant PGCs at embryonic stage promoted later ovary differentiation and female development. Taken together, we generate an ideal transgenic line Tg ( piwil1 : egfp-UTRnanos3 ) ihb327Tg which can visualize zebrafish germline for a lifetime, and we have utilized this line to study germ cell development and gonad differentiation of teleost and to demonstrate that the increase of PGC number at embryonic stage promotes female differentiation.

Retention of paternal DNA methylome in the developing zebrafish germline

“…While these results argue against the existence of active TET-mediated DNA demethylation in the developing zebrafish germline, the possibility of earlier TET-independent removal of DNA methylation as reported in mouse zygotes 17 , cannot be completely excluded. Transgenic tools allowing for lifetime labelling of embryonic, juvenile and male and female germlines will be crucial to answering questions related to 5mC remodelling during the teleost life cycle 108 . Assuming that there is no DNA methylome reprogramming in the zebrafish germline, one might wonder whether more variability in 5mC patterning could be observed among individual fish.…”

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

“…5a)—we find consistent methylation levels in both the male and female zebrafish germline from the gonad transformation period until the point of sexual maturity for cells in the early stages of gametogenesis. Isolation of male germline cells from late gametogenesis, using either single cell analysis or a marker more versatile than vasa:EGFP 51 , will refine our understanding of when gross sexually-dimorphic methylation patterns become apparent in zebrafish. In addition, full-coverage sequencing during the gonad transformation period may uncover sex-specific methylation at single-copy loci, however, the fact that global methylation remains similar between sexes supports previous claims that germline stem cells remain plastic in adults and can rapidly switch to producing the alternative gamete 52 .…”

Knockout of myoc Provides Evidence for the Role of Myocilin in Zebrafish Sex Determination Associated with Wnt Signalling Downregulation

“…Fluorescence immunohistochemistry was performed as previously described [ 43 , 44 ]. Briefly, gelatin embedded histological sections of juvenile (28 dpf) or adult zebrafish (7 months) were treated with immunoblocking solution [10% fetal bovine serum (FBS), 1% DMSO and 1% Triton X-100 in DPBS] at room temperature for 1 h. Gonadal tissue and myocilin were identified using an antibody against the germ cell marker vasa (1:200 [ 45 ], GTX128306, GeneTex, Hsinchu City, Taiwan) or the anti-myocilin (TNT, 1:150) antibody, followed, respectively, by incubation with a Cy2-conjugated anti-chicken IgY (1:1000) or anti-rabbit IgG secondary antibody (1:1000). After that, sections were incubated with a secondary antibody, counterstained, mounted and visualized as described earlier.…”