Journal: bioRxiv

Article Title: Clonal analysis reveals differential PGC contributions to the early germline and ovarian reserve

doi: 10.1101/2025.04.30.651497

Figure Lengend Snippet: (A) A schematic overview depicting zebrafish germline development, from PGCs to mature gametes. Arrows indicate developmental timing of the ziwi and cmlc2 promoters used in transgenic constructs. ( B) Schematic of the Germ traCre transgenic constructs and photoactivatable Cre (pACre) with nos3, 3’utr control element for germline-specific translation. (C-C”) Germ traCre transgenic lines were validated by injection of Cre mRNA. (C) Juveniles were examined at d21 for broad germline-specific mCherry expression (red) and pGH green heart marker expression. Scale bar: 1000 μm. Dashed box outlining enlarged image of heart and gonad expression. (C’-C”) Enlarged heart and germline view. Gut autofluorescence indicated by asterisk. Scale bar: 500 μm. (D) Schematic of the Germ traCre experimental workflow used to generate mosaic germline labeling. pACre- nos3,3’utr mRNA was injected into one-cell embryos and activated with blue light at 30h to induce recombination and marker expression. (E-G) Mosaic Germ traCre gonads. ( E-E’ ) d16 gonadal lobe with bright field imaging, mosaic mCherry expression (red), and Ddx4 germline labeling (blue). Scale bar: 50 μm. (F-G) Mosaic mCherry expression in adult ovary and testis (∼100d). Scale bars: 500 μm.

Article Snippet: The pCSDest2-pACre-nos3,3’utr plasmid (Addgene, #226790) used to synthesize pACre-nos3,3’utr mRNA was generated by recombining pCSDest2 (Addgene, #22424) ( ) and pME-pACre (Addgene, #226789).

Techniques: Transgenic Assay, Construct, Control, Injection, Expressing, Marker, Labeling, Imaging

Journal: bioRxiv

Article Title: Clonal analysis reveals differential PGC contributions to the early germline and ovarian reserve

doi: 10.1101/2025.04.30.651497

Figure Lengend Snippet: (A) Schematic of the Germbow transgenic construct. (B) Schematic of the Germbow experimental workflow used to generate multispectral mosaic labeling. One-cell stage embryos were injected with pACre-nos3,3’utr mRNA, pACre was activated with blue light exposure at 30h to induce random recombination and expression of multispectral markers. (C) Control, unactivated ovary expressing dTomato. Scale bar: 100μm. (D-D’) Activated adult ovary with mosaic color expression. Scale bars: 1000 μm, 100 μm. (E-E’) Activated adult testis with mosaic color expression. Scale bars: 500 μm, 100 μm. (F) Representative clutch of eggs/embryos produced from mosaic female matings. Scale bar: 1000 μm. (G) Bar graph showing egg color ratios for all mosaic examined Germbow females over the mating assay time course. Total number of eggs analyzed per female indicated. (H) Bar graph showing the total number of unique colors observed for each female. I . Bar graph comparison of colors initially observed in the first clutch that persisted to the final examined clutch across all females. ( J) Bar graph of colors that emerged after the first clutch in examined females.

Article Snippet: The pCSDest2-pACre-nos3,3’utr plasmid (Addgene, #226790) used to synthesize pACre-nos3,3’utr mRNA was generated by recombining pCSDest2 (Addgene, #22424) ( ) and pME-pACre (Addgene, #226789).

Techniques: Transgenic Assay, Construct, Labeling, Injection, Expressing, Control, Produced, Comparison