(
A)
TCF/Lef:H2B-GFP time-lapse data of a nephron developing through post-MET Pretubular Aggregate (PTA), Renal Vesicle (RV), Comma-shaped (CB), and S-shaped (SB) stages. These data are shown as time-lapse in
Video 2. (
B) S-shaped body nephron from
Figure 1B shown at four different brightness settings that represent a doubling in brightness for each field going left to right. This clearly shows that all segments of the nephron are positive for the
TCF/Lef:H2B-GFP reporter but follow a visual gradient. Green arrows point to positive cells in the dimmest portion of the nephron and an area of the kidney that even in at the brightest settings show up as negative. (
C) Quantification of antibody stain for β-catenin phosphorylated at Ser33/Ser33/Thr41 in the distal, medial, and proximal segments (nine measurements per nephron in five nephrons). Error bars represent SEM, p-values derived from t-tests. (
D–
F) Lef1 and Ccnd1 (CycD1) antibody stains in S-shaped body nephrons; Pax2 is used as a structural marker against all nuclei within the nephron, Jag1 is used to detect the medial segment, Wt1 is used to detect the developing podocytes in the proximal segment.