Karner CM , Chirumamilla R , Aoki S , Igarashi P , Wallingford JB , Carroll TJ .

	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 3. Cell division becomes oriented after birth in a Wnt9b–dependent process(a) Graphical representation of the angle between the mitotic spindles and the longitudinal axis of DBA-positive tubules at E15.5 indicates that cell division in both wild type (black bars) and Wnt9bneo/neo tubules (white bars) is randomly oriented at E15.5 when compared to the expected random distribution by the Kolmogorov-Smirnov (KS) test. P > 0.55 for both wild type (N=109) and mutant (n=96). (b) At P5, the orientation of dividing cells in KspCre;Wnt9b−/flox DBA positive cells (white bars, n=50) is significantly different (p < 0.01, Mann-Whitney U test) from wild type (black bars, n=45) indicating that Wnt9b is necessary for orientation of cell division that occurs post-natally.
	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 5. Wnt9b is necessary for the orientation of polarized cells perpendicular to the axis of extension. Confocal images (a and d), cell outlines (b and e) and 3D reconstructions (c and f) of frontal sections through E15.5 wild type (a–c) and Wnt9bneo/neo (d–f) kidneys stained with anti-E-cadherin (green), anti-aPKC (red) and DBA (blue). In all cases, proximal is up and distal is down. The images in a and d represent sections just basal to the apical membrane as marked by anti-aPKC (red, not seen). Mediolaterally elongated cells are marked in white, proximal-distally elongated cells in black and unelongated cells in gray in b and e. The majority of wild-type cells are seen to be mediolaterally elongated perpendicular to the axis of extension (white in b). Wnt9bneo/neo cells are still elongated but the direction of elongation appears to be random (note increased number of black, proximal-distally elongated cells in e relative to b). (c and f) 3D reconstructions of Wild type (c) or Wnt9bneo/neo (f) e15.5 tubules to allow for visualization of cell orientation. Arrows indicate angle of orientation for marked cells. Quantitation of the angle of cellular elongation relative to the proximal distal axis of the tubule for wild-type (left in g and h) and Wnt9bneo/neo mutant (right in g) or KspCre;Wnt9b−/flox (right in h) cells. White bars indicate cells that are perpendicular to the axis of elongation (45–90°) while black bars represent cells that are parallel (0–45°). The percentage of cells within each 10 degree increment is indicated. There is a significant change in the orientation of the elongated cells between wild type and mutants. (P<0.001, KS test). The data was gathered from at least 3 different animals. The total number of oriented cells analyzed is 92, 86, or 93 for wild type, KspCre;Wnt9bflox/−, or Wnt9b neo/neo respectively. Wild type cells are from littermate controls.
	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.

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