Journal: Nature Communications
Article Title: Identification of dynamic undifferentiated cell states within the male germline
Figure Lengend Snippet: Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P
Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).
Techniques: Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Injection, Expressing, Quantitative RT-PCR, In Vitro, Activity Assay, Isolation, Transplantation Assay, Cell Culture, Two Tailed Test