Fig 1.
Generation of the transgenic zebrafish with the NTR-EGFP expression under the control of zebrafish dnd promoter.
A) genomic structure of zebrafish dnd gene, including 5’UTR region, 4 exons and 3’ UTR region identified for its PGC-specific presence in the fourth exon. The positions for the region in zebrafish DNA sequence BX890575.23 are labeled in blue. The position sites for the region used related to the transcriptional starting site of dnd are labeled in black. B) schematic representation of the transgenic construct containing the 5’-dnd:ntr-egfp-3’UTR of dnd-3’ fragment flanked by I-Sce1 sites. C) Schematic representation of the transgenic construct containing the 5’-dnd:ntr-egfp without dnd-3’ fragment flanked by I-Sce1 sites. D) Fluorescence microscopic image showing EGFP-expressing PGCs of a F0 progeny at 72 hpf. E) in situ hybridization signals of vasa probe showing the position of PGC at 72 dpf.
Fig 2.
Germ line expression of the dnd+3’UTR:NTR-EGFP and dnd-3’UTR:NTR-EGFP transgene in zebrafish.
The fluorescent protein expression was monitored using fluorescence microscopy. A-D) Expression pattern of the transgene in dnd+3’UTR:NTR-EGFP transgenic fish. A and C, the expression pattern following female transmission, with PGC-specific expression at 24 hpf stage (the inset in C shows the EGFP-positive PGCs in transgenic embryos); B and D, no fluorescence expression following male transmission. E-H) Expression pattern of the transgene in dnd-3’UTR: NTR-EGFP transgenic fish. E and G, the expression pattern following female transmission, without PGC-specific expression at 24 hpf stage (G); F and H, no fluorescence expression following male transmission either. I) early germ cell-specific expression patterns of transgene at 18 dpf in both dnd+3’UTR:NTR-EGFP or dnd-3’UTR: NTR-EGFP transgenic fish, following either female or male transmission.
Fig 3.
Gonadal expression of the dnd+3’UTR:NTR-EGFP and dnd-3’UTR:NTR-EGFP transgene in adult zebrafish.
Anatomical views of the gonadal expression of the NTR-EGFP transgene are presented in A-D; Views of NTR-EGFP trangene expression and histological features with HE staining in the cyosectioned gonadal tissue are presented in E-H and I-L; A and B) Expression of the transgene in zebrafish ovary (A) and testis (B) in dnd+3’UTR:NTR-EGFP transgenic fish at 100 dpf; C and D) Expression of the transgene in zebrafish ovary (C) and testis (D) in dnd-3’UTR:NTR-EGFP transgenic fish at 100 dpf; E and F) NTR-EGFP transgene expression in the cryosections of the ovary (E) and testis (F) of the F1 progenies of dnd+3’UTR:NTR-EGFP transgenic fish at 100 dpf; G and H) NTR-EGFP transgene expression in the cryosections of the ovary (G) and testis (H) of the F1 progenies of dnd-3’UTR:NTR-EGFP transgenic fish at 100 dpf; I-J) histological features with HE staining for the I-J) shows the HE staining for the cryosections of the ovary (I) and testis (J) of the F1 progenies of dnd+3’UTR:NTR-EGFP transgenic fish at 100 dpf; K-L) shows the HE staining for the cryosections of the ovary (K) and testis (L) of the F1 progenies of dnd-3’UTR:NTR-EGFP transgenic fish at 100 dpf; Fluorescence was observed in the oocytes at all stages (E, G, I, K) and in spermatocytes(sc) and sperm(sp) (F, H, J, L).
Fig 4.
Detection of the transcripts of dnd and ntr-egfp transgene in transgenic fish at different developmental stages.
Endogenous dnd or exogenous ntr-egfp transcript was detected using RT-PCR. A) endogenous dnd (a) or exogenous ntr-egfp (b) transcript detected in the fish body at different developmental stages. B) Endogenous dnd (middle) or exogenous ntr-egfp (bottom) transcript detected in various tissues from adult fish at 120 dpf. Transcripts of β-actin were amplified from the same samples as an internal control to test the quality of the cDNA template.
Fig 5.
Induced cell death in transition gonads of the dnd:NTR-EGFP transgenic fish via MTZ treatments.
The MTZ-induced cell death were evaluated by an in situ cell death assay in the transition gonads of the dnd:NTR-EGFP transgenic fish at 26 dpf stage after 1 week MTZ treatment. Transgenic fish at 18 dpf were treated with 5 mM MTZ/0.2% DMSO or 0.2% DMSO solution for 1 week. The fish were cryosectioned at 6μm for in situ TUNEL assay. A-D) transition gonad after MTZ treatments with apoptosis signals under fluorescent microscope (red fluorescence, A), merge images under fluorescent microscope and white light microscope (B), DAPI staining (C), HE staining (D); E-H) transition gonad in control groups with apoptosis signals under fluorescent microscope (red fluorescence, E), merge images under fluorescent microscope and white light microscope (F), DAPI staining (G), HE staining (H); The white frame indicates the gonad position.
Fig 6.
The absence of females developed from MTZ-treated transgenic juveniles.
The dnd-3’UTR:NTR-EGFP transgenic or wild-type sibling control juveniles at 18 dpf were treated with 5 mM MTZ/2% DMSO or 2% DMSO solution for 20 days. The sex ratios of adult fish at 120 dpf were recorded for each of the groups after treatment. A) No female recorded in the groups from 3 independent transgenic lines, line 1 (MTZ1), line 2 (MTZ2) and line 3 (MTZ3) after MTZ-treatment. Various sex ratios of the transgenic fish after DMSO-treatment as the controls were recorded. B) Various sex ratios recorded in the groups of wild-type zebrafish after MTZ-treatments for different periods, 10-day (MTZ1), 20-day (MTZ2), 25-day (MTZ3), or DMSO-treatment for 25 days. The total numbers of fish were labeled under each group.
Fig 7.
Fertile with impaired fecundity in MTZ-treated transgenic zebrafish males.
The fertilization rates of the adult males from 3 independent transgenic lines after MTZ-treatment or DMSO-treatment were recorded. Each group consisted of 10 fishes. The average fertilization rates of the ten fishes of each experiment were measured. The data shown here represents the mean ± the standard error of the mean from three separate experiments.
Fig 8.
The sufficient number of early germ cells required for the female fate sustain in zebrafish.
The dnd-3’UTR:NTR-EGFP transgenic juvenile siblings were treated with 0.2% DMSO solution from 18 dpf to 39 dpf (con), or 5 mM MTZ/0.2% DMSO solution from 18 dpf to 39 dpf (3-week treatment group), from 25 dpf to 39 dpf (2-week treatment group), and from 32 dpf to 39 dpf stages (1-week treatment group). Total RNA of 5 fish were collected at 39 dpf from each groups. A) relative dnd expression levels via quantitative real-time PCR in fish from each groups; B) numbers of the male and female of the rest fish in each groups at their 70 dpf stage. The letters a, b, c, represent the statistically significant difference among groups (p<0.001, student T-test). The results represent 3 independent lines.
Table 1.
Several transgenic zebrafish for germ cell visualization in vivo.