|
Fig. 3 Involvement of sall4, rec8a and rec8b in mediating RA effects in the zebrafish testis. (A) Modulation of sall4, rec8a and rec8b mRNA levels in response to retinoids. Testicular explants were cultured for 4 days with RA, RE or DEAB (each at 10 µM). Data are mean fold change±s.e.m. (n=6-15; *P<0.05, **P<0.01) and are shown relative to the control condition, which is set at 1 (dashed line). DEAB effects were also tested using testicular cell suspensions cultured for 3 days in the absence or presence of 10 µM DEAB (n=3, technical replicates; *P<0.05). (B-D) Effects of GapmeR (1 µM)-mediated knockdown of sall4, rec8 or rec8b after 4 days of tissue culture, on the targeted and selected germ cell-marker genes. Data are mean fold change±s.e.m. (n=3, technical replicates; *P<0.05, **P<0.01) and are normalized to the GapmeR control (Ctrl), which is set at 1. (E) A pooled testicular cell suspension was obtained from 20 Tg(vasa:EGFP) testes and subjected to FACS. Black and white arrowheads indicate EGFP− and EGFP+ cells, respectively. Localization of the vasa:EGFP signal by CLSM in a whole-mount preparation of adult testis shows preferential expression of EGFP in single germ cells and in small germ cell groups, demonstrating expression in type Aund and Adiff spermatogonia. (F) Relative mRNA expression of sall4, rec8a and rec8b in FACS-sorted fractions. Data are shown as mean fold change±s.e.m. (n=3, technical replicates; *P<0.05) and are expressed relative to the EGFP− condition, which is set at 1. Scale bars: 25 μm.