Supplementary Figure 4: Placode polarization and counter rotational movements require myosin II and Rho kinase activity.

(A-C) Representative immunofluorescence images of explants cultured from E15.5 in the presence of DMSO (A), blebbistatin (B), and Y-27632 (C) quantified in D. Follicles are labeled with Shh-Cre driving mGFP expression. 1st, 2nd, and 3rd wave follicles are present under all conditions. 1st wave follicles remain polarized in the presence of blebbistatin and Y-27632. Scale bar, 50 µm. (D) Quantification of hair follicle frequencies at each stage after 24 hours in culture. Control, n=649 follicles from 7 embryos and blebbistatin, n=611 follicles from 7 embryos (mean+SD. p= n.s. for all waves control vs blebbistatin, unpaired t-test); control, n=271 follicles from 4 embryos and Y-27632, n=244 follicles from 4 embryos (mean+SD. p= n.s. for all waves control vs. Y-27632). (E) Additional representative placode shown in Figure 4E from five treated explants. Y-27632 inhibits counter-rotational cell flows. Spinning disk confocal images from a 13.3 hour 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). Overall cell trajectories are shown in the schematic (right). Cells remain in their original rainbow pattern. Scale bar, 10μm. Anterior is to the left.