Extended Data Fig. 3: Generation of Stag3 knockout in GSCs and characterization of their phenotype.
From: The mitotic STAG3–cohesin complex shapes male germline nucleome
a, (top) Scheme of PAM and guide RNA in the Stag3 exon 2. (bottom) Sequences of the targeted loci in two Stag3 KO lines. b, Western blot for STAG3 and α-tubulin in WT and Stag3 KO GSCs. c, STAG3 enrichment at STAG3-peaks defined in WT GSCs in WT and Stag3 KO GSCs (one experiment). d, DEGs between WT and Stag3 KO GSCs by MA plot. e, Two-sided Fisher’s exact test of the overlap of DEGs between E12.5 male germ cells38 and P7 KIT− spermatogonia24 with DEGs between WT and Stag3 KO GSCs. p-values and the proportion of overlapped genes are indicated, and colors represent the odds ratio. f, A model of spermatogonia subpopulations44,45,46. Undifferentiated spermatogonia contain spermatogonial stem cells (SSCs) (“stem-cell” spermatogonia: Gfrα1+/Plvap+), which either self-renew or form “intermediate” (Gfrα1+/Plvap−) and “differentiation-primed” (Gfrα1−/Ngn3+/Rarg+) spermatogonia. The “differentiation-primed” spermatogonia are immediate precursors for “differentiating” spermatogonia (Kit+) for spermatogenesis. Both “intermediate” and “differentiation-primed” spermatogonia retain a capacity to revert to “stem-cell” spermatogonia. The expression of key markers is described at the bottom of the panel. g, Uniform manifold approximation and projection (UMAP) for dimensionality reduction and Louvain clustering of the mouse “spermatogonia” scRNA-seq dataset43. “Spermatogonia” are classified into: “stem-cell” spermatogonia (2), “differentiation-primed” spermatogonia (3), “differentiating” spermatogonia 1 (1), “differentiating” spermatogonia 2 (0), and “early meiotic” spermatocytes (4). h, UMAP embedding as in (g) for marker expression (normalized expression levels). i, Marker gene expression in the five cell populations in (g). j, Mean expression of the Stag1/2/3 in the “stem-cell” spermatogonia (StemCellSg), “differentiation-primed” spermatogonia (Diff. Primed), and “differentiating” spermatogonia 1 (DiffSg1). k, UMAP embedding and Louvain clustering of a human “spermatogonia” scRNA-seq dataset131. Cells were classified into 12 populations. DiffSg: differentiating spermatogonia; StemCellSg: “stem-cell” spermatogonia; Diff.Primed: “differentiation-primed” spermatogonia; NonSg: non-spermatogonia. l, Marker gene expression in the 12 cell populations in (k).
