Although many vertebrate organs, such as kidneys, lungs and liver, are composed of epithelial tubules, little is known of the mechanisms that establish the length or diameter of these tubules. In the kidney, defects in the establishment or maintenance of tubule diameter are associated with one of the most common inherited human disorders, polycystic kidney disease. Here we show that attenuation of Wnt9b signaling during kidney morphogenesis affects the planar cell polarity of the epithelium and leads to tubules with significantly increased diameter. Although previous studies showed that polarized cell divisions maintain the diameter of postnatal kidney tubules, we find that cell divisions are randomly oriented during embryonic development. Our data suggest that diameter is established during early morphogenetic stages by convergent extension processes and maintained by polarized cell divisions. Wnt9b, signaling through the non-canonical Rho/Jnk branch of the Wnt pathway, is necessary for both of these processes.
PubMed ID: 19543268
PMC ID: PMC2761080
Article link: Nat Genet.
Genes referenced: axin2 cdh1 cdkn2b ctnnb1 dvl2 epha8 mapk9 prkci rho rho.2 wnt4 wnt7b wnt9b
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|Figure 2. Characterization of cyst origin in Wnt9bneo/neo kidneysSections of P1 (a, b, e, f, i, j) and P15 (c, d, g, h, k, l) kidneys stained with the collecting duct specific marker Dolichos biflorus agglutinin (DBA) in a–d, the loop of Henle marker Tamm Horsfall protein (THP) in e–h, and the proximal tubule marker Lotus tetragolonobus lectin (LTL) in i–l. In all panels arrows denote normal tubules and asterisks denote cystic tubules. At birth, cysts are found primarily in the proximal tubules (compare i to j). Cysts are also found in the collecting ducts although the majority of DBA positive epithelia appear normal (see arrows in b). Cysts were not observed in the loop of Henle at birth (compare e to f). By P15 cysts are present in all segments of the nephron (compare c to b, g to h, and k to l). Nuclei were counterstained with DAPI (blue).|
|Figure 4. Wnt9b is required for the elongation and narrowing of kidney tubulesRepresentative sections through wild type DBA positive tubules from E13.5 (a), E15.5 (b), E17.5 (c), and P1 mice (d) showing the number of nuclei composing the wall of the tubule. Outlined tubules represent transverse sections. Quantitation reveals that the number of cells within the wall of wild-type collecting duct (black bars in e, n=563, 606, 844, and 692 for E13.5, 15.5, 17.5 and P1 respectively) and proximal tubules (black bars in f, n=425, 1030, 791, and 778 for E13.5, 15.5, 17.5 and P1 respectively) significantly decreases during the embryonic period. The number of cells within the tubule wall is significantly increased in Wnt9b mutant kidneys (white bars in e and f, n=384 or 412, 521 or 424, 915 or 902, and 665 or 635 for DBA or LTL at E13.5, 15.5 17.5 or P1 respectively). N=3 kidneys for each stage, tubular segment and genotype. Error bars represent standard error of the mean.|
|Figure 6. Wnt9b signals through the noncanonical pathway to regulate tubule diameterWestern blots of total protein extracted from wild-type and Wnt9bneo/neo kidneys probed with an antibody specific to the dephosphorylated (active) form of β-catenin show no significant differences in canonical Wnt activity compared to wild type (a). Section in situ hybridization with a probe for the β−catenin target axin-2 also shows no significant decrease in canonical activity in P1 Wnt9bneo/neo kidneys (c) compared to wild type (b). Note that there is no ectopic axin2 expression in cystic proximal tubules (asterisks in c). (d) Western blots indicate that activated Rho is significantly decreased in Wnt9bneo/neo kidneys at P1 relative to total (+GTP control) Rho levels. Addition of GDP (+GDP) to inactivate Rho was used as a negative control. Phosphorylated Jnk2 is also significantly decreased in the Wnt9bneo/neo kidneys at P1 relative to total levels of Jnk2 (d). Blots shown are representative examples of data gathered from at least 3 different blots from 3 independent protein extractions.|