Supplementary Figure 5: Characterization of placode morphology and cell movements in Shh mutants.

(A-C) Shh KO embryos display expanded and irregular shaped placodes. (A) In controls, Shh-GFP (green) and P-Cadherin (magenta) mark the inner placode. In Shh KO embryos, the zone of Shh-GFP and P-Cadherin is expanded, irregularly shaped, and lacks a smooth boundary between GFP+ and GFP- cells. Representative mild and severe Shh KO examples along with a control, quantified in B. (B) Quantification of placode morphologies. The area and perimeter of Shh KO placodes, as measured by the zone of Shh-GFP expression, are expanded compared to controls. Shh KO placodes are less circular and more anisotropic than controls. n=30 control placodes from 3 embryos; n= 40 ShhKO placodes from 4 embryos. Lines show mean+SD. p=1.25x10⁻¹⁶ for area, p=1.06x10⁻¹⁶ for perimeter, p= 8.82x10⁻⁷ for circularity, p= 8.78x10⁻⁷ for aspect ratio, two-tailed t-tests with Welch’s correction for unequal variances (except for aspect ratio, where variances are not significantly different). (C) Shh-Cre>mGFP highlights the irregular morphology and poor compartmentalization of Shh KO placodes. E15.5 explants were cultured overnight before fixation. Representative images from two control and two mutant embryos. (D) Shh KO placode cells undergo atypical cell rearrangements. Additional example to Figure 5C. Spinning disk confocal images from a time series of placode cells expressing mTomato (top). Cells were segmented and false coloured in a rainbow pattern of vertical lines perpendicular to the AP axis at the beginning of the video. Cell tracks show the movement of cells during the designated time window (bottom). Additional placode representative of two embryos. Overall cell trajectories are shown in the schematic (right). Scale bar, 10μm. Anterior is to the left.