Fig. 5. H2BGFP-dilution analysis in tail epidermis.

a Structure of the mouse tail epidermis. 3D reconstruction of confocal z stacks (top panel) and orthogonal xyz view (mid panel). Apical blister-shaped regions (scale) alternate with deeper regions (interscale) (boundaries delineated by dotted lines), being arranged in lines separated by hair follicles (asterisks). Green: H2BGFP expression; white: KRT14 immunostaining (as a marker of basal layer); red: CD45 immunostaining; blue: DAPI. Scale bars, 200 μm. Bottom panel: Cartoon illustrating the tail skin structure. b Representative confocal z stacks of scale and interscale regions of tail epidermis during H2BGFP chase experiments in R26^M2rtTA/TetO-H2BGFP mice, showing H2BGFP (green), immunostaining for KRT14 (white) and immunostaining for pan-leukocyte marker CD45 (red). Images from the same time point correspond to different mice to illustrate inter-animal variation. Label-retaining cells (LRCs) are highlighted with arrowheads (CD45+ cells) or full arrows (CD45− cells) (details in inserts; blue: DAPI). Scale bars, 20 μm. c Time course of basal-layer keratinocyte H2BGFP intensity distributions from scale and interscale regions of tail. Experimental data shown as boxplots per individual biologically independent mouse (intensities normalized to average keratinocyte intensity at time 0, raw H2B-GFP intensity values are given in Supplementary Data 2). n = 3 animals at each time point except 18 days where n = 2 mice. Centre line of box is median value, box indicates 25th and 75th centiles and whiskers indicate minimum and maximum values. Solid black lines: average H2BGFP-dilution rates (within 95% CI limits—shaded gray areas—where the value of λ = 1.2/week reported by Mascre et al.⁴ falls). Insert: Detail of H2BGFP intensity distributions separated per field of view for the single tissue found to be bimodal in the overall, per-animal modality test (see Fig. 3d). Analyses per field of view all resulted non-significant (unimodality).