In situ hybridization
We examined expression of three Wnt genes in Pygo mutants. Wnt7b is expressed in the stalks [20], Wnt11 in the tips [4], and Wnt9b in the stalks and weakly in the tips of the branching ureteric bud [2]. All three genes showed similar expression patterns in Pygo2+/+ and Pygo1/Pygo2 double-mutant kidneys (Figure 4). In addition, these in situ hybridization patterns confirmed the confocal microscopy results, showing a reduced number of tips (Wnt11-positive) per surface area in the Pygo1/Pygo2 double-homozygous mutants.
Figure 4  Expression of Wnt7b, Wnt9b, and Wnt11 in E18.5 Pygo1/Pygo2 compound null kidneys. Whole-mount in situ of hybridizations of (A-C) E18.5 wild-type kidneys and (D-F) Pygo1-/-/Pygo2-/- mutant kidneys with the ureteric bud derivative markers: (A, D) Wnt7b, (B, E) Wnt9b, and (C, F) Wnt11. The mutant kidneys showed normal expression patterns for the ureteric stalk markers Wnt7b and Wnt9b, and reduced density of ureteric tips as measured by Wnt11. Original magnification × 32.