Fig. 6.

Afadin is required for spindle orientation in developing nephron tubules. (A) Schematic showing orientation of cell division. Oriented cell division occurs when the mitotic spindle is parallel to the longitudinal axis and perpendicular to the apical-basal axis of the tubule (a). Misorientation of cell division occurs with defects in the longitudinal (b) and/or apical-basal orientation (c) of cell division. (B,C) Diagrams representing how the orientation of cell division was determined within a nephron tubule. Angle θ is the angle between the longitudinal axis of the tubule and the spindle vector. Angle ϕ is the apical-basal component of the spindle angle measured as the angle between the spindle/chromatid vector and a radial vector, as seen in a transverse section of the tubule. For example, a measurement of ϕ=90° indicates that the cell is dividing perpendicular to the apical-basal axis and is correctly oriented. (D) Selected images of a z-stack of a control s-shaped body/tubule. F-actin (blue) and nuclei (green) are shown. z-axis depth is indicated and two mitotic cells in anaphase are shown (arrows in Db and Dc). The mitotic axes of the dividing cells in Db, and perhaps Dc, do not appear to be oriented along the longitudinal axis; however, transverse sections at b1 and c1 shows they are oriented nearly parallel to the apical and basal surfaces. Db1 also shows the dividing cell (and its chromatids) have moved into the lumen of the tubule (arrowhead) relative to other cells, whose nuclei are basal. The position of nuclei in non-dividing cells of an adjacent transverse section is shown for comparison. Scale bars: 10 µm. (E) Analysis of θ and ϕ mitotic angles in control (Afdn ^f/f) and mutant (Afdn ^f/f; Six2-cre) kidneys at E17.5 (n=3 for each genotype). In controls, θ is uniformly distributed, but ϕ is skewed toward higher angles (see text). The ϕ angles are different in controls and mutants (*P<0.001). NS, not significant. (F) Histogram analysis of ϕ in E. (G) Model of the role of afadin in lumen continuity and tubulogenesis. In control tubules, afadin regulates spindle orientation, thus allowing correct lumen placement and normal tubulogenesis. Without afadin, apical-basal spindle orientation is disrupted, leading to misplacement of lumens. We speculate that lumen misplacement leads to morphogenetic defects in the tubules.