Figure 3.

DAZL targets are enriched for SSC-associated genes and loss of DAZL leads to a defect in spermatogonial progenitor maintenance. (A) Schematic illustration of SSC-associated genes bound by DAZL. (B) Genome browser tracks showing binding peak distributions (DAZL-CLIP) and transcript levels (RNA-seq) of SSC-associated target genes. (C) Immunohistochemical staining of the SSC marker PLZF in Dazl VKO testes. Scale bar: 20 μm. (D) Gradual germ cell loss in testes of Dazl VKO mice. Testes sections from 3, 5, 10 and 15-week-old mice were stained for VASA expression to detect the number of VASA-positive tubules. A seminiferous tubule containing at least one VASA-positive cell was classified as a VASA-positive tubule. The number of mice at each time point is shown in parentheses. More than 100 cross-sections were scored for samples taken from random slides. (E) Comparison of the number of LIN28A⁺ and PLZF⁺ SSCs in cross-sections of testes from Dazl heterozygotes (Het) and Dazl VKO mice at different ages. The number of mice is the same as in (D). A seminiferous tubule containing at least one SSC was classified as tubule with SSCs. Data are mean ± SEM for 20 round seminiferous tubules of both genotypes at each age. *P < 0.05, **P < 0.01, ***P < 0.005. (F) Dazl knockdown led to a significant reduction in the size of SSC clones at day 4 following shRNA lentiviral transduction. Scale bar: 200 μm. (G) Cell count of SSCs at day 4 following lentiviral transduction. (H) SSC markers (LIN28A, PLZF) were downregulated at day 4 following lentiviral transduction. (I) Established SSC clones immunostained for DAZL and PLZF after 4 days of culture of Thy1⁺ cells from 7dpp Dazl heterozygotes and Dazl VKO testes. Scale bar: 50 μm. (J-L) Fluorescence intensity of DAZL targets was compared between Dazl heterozygotes and Dazl VKO testes at 7dpp by immunofluorescent staining in TRA98-positive cells. Scale bar: 20 μm. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.005.