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 Wnt9b^neo/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). Wnt9b^neo/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 Wnt9b^neo/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 Wnt9b^neo/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;Wnt9b^flox/−, or Wnt9b ^neo/neo respectively. Wild type cells are from littermate controls.