Fig 1: Identification of novel cellular markers from single-cell data.(A) Across major cell-type clusters, we identified 22 gene expression markers specific to one cell type or aspect of spermatogenesis and not previously reported. Here we show the expression levels of these genes. Expected protein expression patterns for Nol8, Lrrc34, Abhd5, and Kif5b were confirmed, but the antibodies for Acyp1, Ccdc62, and Unc80 did not show positive staining in any testicular cell types,which could be an antibody-related problem or an indication that these RNAs were not translated. (B–D) Thin scale bar, 50 µm; thick scale bar, 20 µm. (B) Nol8, a nucleolar protein, marks primary spermatocytes while Lrrc34 marks nucleoli in round spermatids (white arrowheads) (C) Within the tubules, Abhd5 marks specific cytoplasmic regions of elongating spermatids destined to form the residual body (white arrow head) and staining intensity peaks during seminiferous tubule stages IV-V. (D) Kif5b marks Sertoli cells within seminiferous tubules (white arrow head). We co-stained Kif5b with a well-known Sertoli cell marker, Vimentin (red arrow head), and indeed both proteins colocalize to Sertoli cells (orange arrow head). Co-staining also reveals that Kif5b staining extends further out in the cell body (blue-green arrow head) than Vimentin.