Fig 1: GDNF-RET signaling tunes branching in mouse and human ureteric bud tissues.A. GDNF-RET signaling interactions involved in kidney branching.B. Immunofluorescence of cleared E14 mouse kidney. Top left, ECAD and SIX2. Bottom left, RET and GFRA1. Top right, isolated RET. Bottom right, isolated GFRA1 signal.C. Air-liquid interface (ALI) culture of mouse embryonic kidney explants.D. Design of GDNF-RET signaling perturbation experiments in ALI culture.E. Immunofluorescence of E13 kidneys grown in ALI culture for 4 days in 100 nM Selpercatinib (+RETi), 100 ng ml−1 GDNF (+GDNF), or control media. Inset, close-up of tip domains. Explants are immunostained for EpCAM (epithelium), RET (tip cells), and JAG1 (early nephrons).F. Tip number on days 1-4 across all conditions, N = 4 kidneys per condition. P-values by one-way Kruskal-Wallis test with Dunn’s post hoc test.G. Differentiation of iUB organoids from hiPSCsH. Immunofluorescence of epithelial (ECAD) and tip (RET) markers in a day 12 iUB organoid.I. Design of GDNF-RET signaling perturbations for iUB organoids.J. Merged brightfield and GATA3:mCherry images of iUB organoids at days 9 and 12. Organoids were cultured in control (−GDNF) or complete branching medium (+GDNF, 50 ng ml−1), excess GDNF (++GDNF, 250 ng ml−1), or complete branching medium with 100 nM Selpercatinib (+RETi). See: Fig. S4F.K. Projected area (x104 μm2) on days 9 and 12 for all conditions, n = 47, 56, 40, 46 iUB organoids (−GDNF, +RETi, +GDNF, ++GDNF) from 2 independent biological replicates.L. Circularity (a.u.) on days 9 and 12.M. Bud number on days 9 and 12. P-values in panels K, L, M by one-way ANOVA with Dunnett’s post hoc test using +GDNF as reference group.