Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation
(A, B) Transcripts were measured by qRT-PCR in wild-type (Wt1+/+) and Wt1-deficient (Wt1-/-) XX and XY gonads. Genes are classified as being expressed at similar levels in both sexes or as exhibiting a clear predominance in either XX or XY gonads. Loss of Wt1 showed a significant change in expression of (A) Dax1, Nr5a1(Sf1), Amhr2, Star, and Gata4 in both sexes. Expression of (B) Fst, Foxl2, Amh2, Sox9, and Ctnnb1 was changed sex-specifically. (C) Representative morphology of Wt1+/+ and Wt1-/- XX and XY gonads (marked by dashed lines) with attached mesonephroi. Tissues were obtained from embryos at 13.5 dpc. Note the disrupted morphology (dashed lines) in the Wt1-/- XY and XX gonads. Scale bars indicate 500 μm. For qRT-PCR data (A, B) relative transcript levels were normalized to Gapdh (2-ΔCt) and shown in percent. Error bars indicate S.E.M. calculated from independent biological replicates (n ≥ 5). Statistical significances are marked by brackets (ANOVA with Tukey’s post hoc test) and asterisk (*p<0.05, t-test). n.d. = not detectable.