MC morphology, protrusion dynamics, and migration is regulated by epidermal Par3. (A) Phase-contrast micrography and live-cell imaging of mouse KC–MC co-cultures (stills of Videos 1 and 2; 4 d co-culture; HC medium). Data are representative of two independent experiments. (B) TRP2 immunostaining of MC co-cultures with control or Par3^KO KCs. Data are representative of three independent experiments. (C) Quantification of B. MC dendritic length in direct co-cultures 7 d in HC medium (>20 images/condition) is shown. Data are pooled from independent experiments represented as mean ± SD. n = 3 biological replicates per group. *, P = 0.0196; Student’s t test. (D) TRP2 immunostaining of tail whole mounts of control and Par3^eKO;H2B-GFP⁺ mice (age: P58). (E) Quantification of in vivo MC morphology in tail whole mounts (>10 hair follicles per mouse). Data are pooled from independent experiments and are depicted as mean ± SD. n = 5 biological replicates per group. *, P = 0.0208; unequal variances Student’s t test with Welch’s correction. (F) Automated, unbiased quantification of MC compactness using CellProfiler (>20 images/condition). A low value for compactness reflects a MC shape that is closer to a perfect circle than higher values for compactness. Data are pooled from independent experiments and depicted as mean ± SD. n = 3 biological replicates per group. **, P = 0.0078; Student’s t test. (G) Live-cell imaging of membraneTomato (mT/mG)-expressing control MCs co-cultured with control or Par3^KO KCs (4-d co-culture; HC medium). From left to right: immunofluorescent images of tracked MCs over time representative of three independent experiments. The white outline shows the perimeter of tracked cells using Quimp11b. Far right: perimeter overlay of the same MCs at different time points during live-cell imaging to visualize protrusion dynamics over time. (H) Quantification of total distance traveled and mean speed of co-cultured mTomato⁺ MCs as represented in G. (>600 cells/condition). Data are pooled from independent experiments and are represented as mean ± SD. n = 3 biological replicates per group. Distance traveled: *, P = 0.0286. Mean speed: *, P = 0.0286. Mann Whitney U test was used. Bars: (A) 20 µm; (B and D) 10 µm; (G) 50 µm. Ctrl, control; rel., relative. See also Videos 1 and 2.