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) Dissected XX and XY gonad/ mesonephroi regions (12.5 dpc) were incubated for 72 h in a droplet of medium supplemented with either Wt1 or Gata4 vivo-morpholino. Transfection with the corresponding mismatch vivo-morpholinos was performed as control. Efficient gene silencing was assessed by immunostaining of WT1 (B) and GATA4 (C) in XX and XY gonad/ mesonephroi explants. Cell proliferation was determined by means of BrdU incorporation, and nuclei were visualized with Dapi. Proliferating cells were reduced in both XX and XY gonad/ mesonephroi explants upon WT1 knockdown (B), and knockdown of GATA4 only showed an effect on XY explants (C). BrdU-positive cells were counted in at least 5 tissue sections obtained from 3 different embryos and normalized to Dapi-stained nuclei. Scale bars indicate 100 μm. Error bars represent S.E.M., *p<0.05, **p<0.01, ***p<0.001, t-test. mo = vivo-morpholino. mism = vivo-morpholino-mismatch. G = gonad. M = mesonephros.