Figure 5. Planar cell fate asymmetry arises from directional cell rearrangements.

(A) Representative immunofluorescence images of early placodes showing radial symmetry with inner tip cells expressing P-Cadherin and ShhGFP (green) and outer SC progenitors expressing Sox9 (magenta) from one of two embryos (top) and one of four embryos (bottom). (B) Representative image of Sox9 (magenta) expression in control and Shh KO placodes from one of two embryos (het) and one of three embryos (KO). All cells express mTomato (blue). Additional examples of Shh KO placode morphology are shown in Supplemental Figure 5A–C. (C) Shh mutant placode cells undergo atypical cell rearrangements. Representative placode from one of two embryos. Spinning disk confocal images from a time series of placode cells expressing mGFP (top). Overall cell trajectories are shown in the schematic (right). Cells were segmented and false colored in a rainbow pattern of vertical lines perpendicular to the AP axis at t=0. Cell tracks show the movement of cells during the designated time window (bottom). See Supplemental Video 8. Additional example shown in Supplemental Figure 5D. (D) Representative immunofluorescence images of early, mid, and late/germ wild-type placodes (left) stained for Hoechst (blue) and asymmetry markers Sox9 (magenta), P-Cadherin (green) quantified in E. Placodes are initially radially symmetric before becoming planar polarized. Asymmetry markers in Vangl1;Vangl2 dcKO and Fz6 KO placodes fail to polarize or polarize in random orientations (right) quantified in E. (E) Quantification of Sox9 asymmetry in early (n=7), mid (n=11), and late/germ (n=9) wild-type placodes (p=0.048 early vs. mid; p=0.0002 early vs. late; unpaired t-test, from four embryos), and Vangl2 KO (p=7.22×10⁻⁵ vs. late, n=7 placodes from three embryos) and Fz6 KO (p=n.s. vs. late, n=9 placodes from three embryos) germ placodes represented as the posterior/anterior ratio of Sox9 immunofluorescence intensity (mean+SD). Randomly oriented, polarized Fz6 KO placodes were chosen for analysis. Fz6 KO hair germ orientation was determined based on morphology. Scale bar, 10µm. Anterior is to the left.