Fig. 5

Modulating physiological Ca²⁺ oscillations alters feather elongation process. a Virtual sections of 4D ratiometric Ca²⁺ imaging of elongating feather buds on H&H 34 skins (n = 3). ROI analysis of GCaMP6s and mCherry fluorescence ratio exhibited synchronized Ca²⁺ oscillations in anterior (cyan rectangle) and posterior (magenta rectangle) mesenchyme. The high Ca²⁺ area expanded anteriorly over time (the intersection of cyan and magenta lines).′ in x-axis denotes minutes. Pseudocolor ratiometric images were shown at selected times (arrowheads). 50 μM Nifedipine or 150 μM Carbenoxolone treatment dramatically reduced the number of cells showing elevated Ca²⁺ (n = 2). Scale bar, 50 μm. b Feather buds on H&H 31 skins became elongated filaments oriented posteriorly after 4-day culture with DMSO (red arrows, n = 10/10). Nifedipine-treated feather buds were shorter and had no apparent anterior–posterior polarity (n = 8/8). 150 μM Carbenoxolone treatment inhibited feather elongation and disrupted feather polarities (red arrows, n = 6/6). H&E: Hematoxylin and Eosin staining. Scale bars, 500 μm (whole-mount skin), 50 μm (section). c In H&H 35 Skins, the regions (n = 4/4) infected with lentivirus encoding Connexin-43 shRNA (pLL3.7-Cx43-SH) exhibited dramatically inhibited elongation of young feather buds (cyan ellipses) compared to the control side (magenta ellipses, n = 23 for each group, **P < 0.01, Wilcoxon rank test). Rectangles highlight magnified area. Scale bar, 500 µm. Customized boxplot: Mean (red) ± s.d. (pink), 95% confidence interval (violet). Dots denote individual data points