Fig. 5
From: Identification of dynamic undifferentiated cell states within the male germline
Stem cell potential and molecular characteristics of PDX1+ Aundiff. a Isolation of PDX1+ and PDX1− Aundiff from Plzf-mC/CreER; Pdx1GFP/+ adults by flow cytometry. Aundiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown (n = 7). b Pdx1-GFP+ and GFP− adult Aundiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− Aundiff fractions in transplantation assays from b. Data is presented as mean number of colonies per 105 donor cells ± s.e.m. (n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c. e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c (n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− Aundiff fractions isolated as in a (n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR) < 0.05 and absolute fold change ≥1.5. g Heatmap showing differentially expressed MAPK pathway genes from KEGG analysis of RNA-Seq data from f. h Quantitative RT-PCR analysis of indicated genes in mCherry+ CD9+ c-KIT− Aundiff isolated from Plzf-mC/CreER; Pdx1GFP/+ and Plzf-mC/CreER; Pdx1+/+ control adults. Mean values ± s.e.m. are shown (n = 6 mice per genotype). i Representative IF of testis sections from aged (8 months) mice of the indicated genotypes (n = 3 mice). VASA and PLZF staining identifies germ cell and spermatogonial populations respectively. Scale bar, 50 μm. Significance was calculated by two-tailed Student’s t-test (**P < 0.01, not significant (ns) P > 0.05)
