Fig. 3
From: Identification of dynamic undifferentiated cell states within the male germline
Identification and characterization of distinct Aundiff populations. a Oct4-GFP− and Oct4-GFP+ Aundiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. Aundiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ Aundiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a. Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). c viSNE maps derived from single cell analysis of 150 single Aundiff cells isolated from pooled adult Plzf-mC/CreER testis. Based on expression of a total of 71 genes identified from analysis of Aundiff fractions in b plus genes previously linked with Aundiff function. Relative expression of selected genes associated with stem and progenitor fractions by each plotted cell is indicated (red = high, blue = low). d viSNE maps from single cell analysis of c. Spermatogonial and germ cell markers are in left panels. Middle panels show stem cell markers not identified as differentially expressed in Oct4-GFP− and GFP+ Aundiff fractions by microarray. Differentiation markers are in right panels. e Aundiff isolated from 2 Plzf-mC/CreER; Oct4-GFP adults were analysed by single cell RNA-Seq and developmental trajectory calculated using Monocle. Cells were classified as follows: Stem cells (Gfra1 or Etv5 positive), progenitors (Sox3 or Upp1 positive), transitional cells (positive for combinations of stem and progenitor genes) and undefined undiff (negative for these stem and progenitor genes). f−h Expression of indicated genes across cell trajectory (pseudotime) from single cell analysis of Aundiff in e. Expression of housekeeping gene Ubc is shown
