GNDF augments mitosis in caudal nephric duct at the time of ureteric bud outgrowth. (A) Mitotic indices were determined as the percentage of pHH3-positive cells within b-catenin or E-cadherin positive epithelium. Graphs show indices of wild type (WT, black bars) and Gdnf hyper/hyper embryos (white bars) in caudal nephric duct (E10.5), ureteric bud epithelium (E11.5) and ureteric bud tips (E15.5). A statistically significant increase in mitotic indices was observed at E10.5 samples (7–9 vibratome sections per embryo, n = 3/genotype, p < 0.05, Student’s t-test). (B) Total cell counts in E11.5 WT and Gdnf hyper/hyper ureteric bud tips (p < 0.05). (C) Representative E10.5 WT and (D) Gdnf hyper/hyper kidney primordia shown after staining with pHH3 (red) and b-catenin (green). (E) E11.5 WT kidney shows typical T-shaped ureteric bud (E-cadherin, green), but (F) Gdnf hyper/hyper epithelium is abnormally enlarged without normal branching pattern. Arrows in (D) and (F) point to cell mass accumulated in the luminal side of the epithelium. (G) Regardless of the genotype, labeling for cleaved Caspase3 does not reveal apoptosis in the kidney primordia of E10.5 embryos (n = 7) while a statistically significant increase is seen at E11.5 in Gdnf hyper/hyper epithelium (white bar, n = 5 for WT and 4 for Gdnf hyper/hyper, Student’s t-test, p < 0.001). At E15.5 cleaved caspase 3 labeling reveals similar amount of apoptotic cells in the cortex of WT (n = 3) and Gdnf hyper/hyper (n = 2) kidneys. (H) An example of WT kidney stained with cleaved Caspase3 (red, n = 5) shows virtually no apoptosis, while (I) numerous apoptotic cells (arrowhead) are detected in the ureteric bud lumen of Gdnf hyper/hyper embryos at E11.5 (n = 4). Scale bars 50 µm. Error bars on all graphs represent standard deviation.