In vitro analysis of ureteric bud and trunk growth. Time-lapse imaging of 24 h cultured E11.5 (A) wild type (WT) and (B) Gdnf hyper/hyper kidneys where nephric ducts and ureteric bud epithelia are visualized by transgenic Hoxb7CreGFP expression. Yellow arrow marks cleft-like furrow between the distinct ends of ureteric bud tips, arrowhead points to ureteric trunk. (C) Measurement of trunk lengths, presented as the average primary trunk length ± SEM, in WT and Gdnf hyper/hyper kidneys at E12 reveals that UB trunks are significantly shorter in Gdnf hyper/hyper kidneys (728.6 ± 91.3 µm in WT, 286.5 ± 28.0 µm in Gdnf hyper/hyper, n = 4/genotype, p = 0.013, unpaired two-tailed Student’s t-test). (D) Trunk measurements in WT and Gdnf wt/ko kidneys show that trunk length in Gdnf wt/ko kidneys is increased (408.9 ± 27.9 µm in WT (n = 13), 439.8 ± 16.0 µm in Gdnf wt/ko (n = 16), p = 0.019, unpaired two-tailed Student’s t-test). Analysis of (E) EdU-positive cell counts and (F) total epithelial cell counts (average cell numbers depicted inside the bars on (E–H) in ureteric bud tips (marked by black circles in A and B at 0 and 10 h images) are shown as percentage of growth rates (WT, n = 5; Gdnf hyper/hyper, n = 4). Growth rate at 0 h was set to 100% in both genotypes. The increase ratio in wild type cell numbers was approximately the same in EdU+ (38.9 ± 20.5%, n = 5) and total cell counts (46.6 ± 25.2%, n = 4), while both EdU+ cells (87.5 ± 38.3%, n = 3) and total cells (63.9 ± 30%, n = 3) increased remarkably more in Gdnf hyper/hyper tips. (G) Corresponding analysis of growth rates in the ureter trunks of E11.5 WT and (H) Gdnf hyper/hyper kidneys. Dramatic increase in both EdU+ cells (77.9 ± 28.1%, n = 4) and total cell numbers (99.2 ± 6.6%, n = 3) were observed in WT kidney trunks. Gdnf hyper/hyper trunk cells failed to increase either EdU+ (91.6 ± 14.7%, n = 3) or total cell numbers (92.6 ± 2.6%, n = 3). The area of measurements is depicted in A and B images of 0 and10h as black lines. Scale bar: 200 µm.