Fig. 2

Functional VGCCs, gap junctions and CRAC channels in elongating feathers. a Schematic drawing exhibiting the skin explant and strip configuration used for time-lapse Ca²⁺ imaging. Bright field images of feather buds at different developmental stages in skin explants and strips are shown. Dotted lines highlight epithelial-mesenchymal boundaries. Scale bars, 50 μm. b–d Time-lapse Ca²⁺ imaging of feathers from skin strips at different stages (n = 3 for each stage). Dotted lines indicate regions of interest (ROI) for quantification (′′ denotes seconds). Pseudocolor images of the ratios are presented at selected time points (arrowheads). Scale bar, 50 μm. e Quantifying change of mesenchymal Ca²⁺ increases in feathers from skin strips. R_o: baseline F GCaMP6s/F mCherry ratio before KCl application. ΔR: the peak ratio subtracts R_o. Mean ± s.e.m. *P < 0.05 (Wilcoxon rank test). Dots represent individual data points. f Time-lapse Ca²⁺ imaging of feathers in skin explants at H&H 35 (n = 16/16). g Pretreatment with 50 μM Nifedipine for 3 min greatly dampened the KCl-induced Ca²⁺ elevation (n = 4/4). h Pretreatment with 150 µM Carbenoxolone for 30 min significantly reduced the KCl response (n = 4/4). i Feather buds pretreated for 30 min with 0 mM Ca²⁺ solution including 10 µM Thapsigargin exhibited notable Ca²⁺ influx after perfusion of 2 mM Ca²⁺ solution (n = 3/3). j 5 µM BTP2 significantly reduced the Ca²⁺ influx (n = 3/3). k Quantification of Ca²⁺ response in feathers from skin explants under different treatments. Mean ± s.e.m. *P < 0.05 (Wilcoxon rank test)