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Fig. 4. The failure to initiate renal medulla development in Wnt7b
mutants reflects alterations in the plane of epithelial cell division.
(A) Cell proliferation was analyzed in wild-type and Wnt7b
mutant kidneys at E15.5 by pulse-labeling with BrdU. BrdU incorporation was
visualized by anti-BrdU antibodies in conjunction with specific markers, as
indicated, to identify specific epithelial compartments. E-Cadherin (E-cad)
and cadherin 6 (Cdh6) immunostainings together with DBA lectin demarcate the
cell boundaries between the nephron and collecting duct epithelia. No
significant difference in the proliferation rate is observed between wild-type
(n=3) and Wnt7b mutants (n=4). Scale bar: 40 µm.
(B) Diagrams illustrating the effects of oriented cell division on
tubule morphogenesis and three-dimensional reconstructions of optical sections
showing mitotic configurations in the collecting duct epithelium. Anaphase
chromosomes were visualized with phospho-histone H3 staining (PH3, green,
arrow) and the collecting epithelium with pan-cytokeratin staining (red;
A'-D'). Stacks of single optical sections (A',B') were
rendered to generate 3D images (C',D') for the measurement of
mitotic angles. (C) Histogram of mitotic angles in the prospective
medullary collecting ducts of wild-type and Wnt7b mutant kidneys at
E15.5. Mitotic angles were determined relative to the longitudinal axis of the
perspective collecting duct epithelium delineated with pan-cytokeratin
immunostaining as detailed in the Materials and methods. (D) 3D
reconstruction of stacks of optical sections of acetylated 
-tubulin
staining demonstrate that normally sized primary cilia (arrow) were present in
the collecting duct epithelia of Wnt7b mutants. Moreover, the
intraflagellar transport protein Polaris localized to the primary cilium as
expected. (E) Apoptosis was compared by TUNEL analysis (red) in the
collecting duct epithelium (DBA lectin, green) of kidney sections from
wild-type and Wnt7b mutant embryos at E17.5. The apoptosis rate
increased markedly and significantly in Wnt7b mutants (n=4)
relative to in wild-type controls (n=4).
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