Figure 2.
Spatially distinct basement membrane expansion rates synchronize with directional cell movement. (A) Schematic of region definitions in the developing hair follicle. Tip: Interface region between the prematrix and dermal condensate, defined by two bending points at the follicle tip. Lower stalk: The lower half of the follicle extends to the tip. Upper stalk: The upper half extends to the junction region. Junction: Bending neck region where follicular epidermis meets interfollicular epidermis. BM dynamics were measured in these regions. (B) Confocal images of hair follicles in dorsal skin explants from eGFP-Col4a2 mice show that BM length changes over 7 h. ROIs (dashed rectangles) indicate photobleached areas. Re-photobleaching of the targeted BM was performed every 2–3 h to maintain clear photobleached edges. See Fig. S2 for details. BM length changes were measured at the tip (blue lines), lower stalk (orange lines), and upper stalk (gray lines). Scale bar: 20 μm. (C) Percent BM length changes over a 7-h (n = 9 follicles from seven explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (D) Measurement of BM thickness in hair follicles of dorsal embryonic skin explants from eGFP-Col4a2 mice. BMs at the tip (blue lines), lower stalk (orange lines), and upper stalk (gray lines) were measured. Scale bars: 20 μm (left two images), 2 μm (right three images). (E) Quantification of BM thickness (n = 13 follicles from three explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (F) EdU incorporation assays. Dividing cells were labelled with EdU (magenta) with Hoechst counterstaining (blue). EdU-positive nuclei in the basal layer of the tip (blue line), lower stalk (orange line), and upper stalk (gray line) were measured across z-planes (−10, 0, +10, and +15 μm). Scale bar: 20 μm. (G) Percentage of EdU-positive nuclei in the tip, lower stalk, and upper stalk regions (n = 18 follicles from seven explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (H) Snapshot images of 3D time-lapse videos of developing hair follicles in eGFP-Col4a2; mem-tdTomato explants. The relative locations of the BM (lower bleached edge) and epithelial basal progenitors (white arrowheads) from reference points on the BM (upper bleached edge, yellow arrowheads) in lower (orange lines) and upper stalk regions (gray lines) were measured at 0 and 9 h 30 min. Re-photobleaching of the targeted BM was performed. See Fig. S4 for detailed tracking procedures. Scale bar: 20 μm. (I) Normalized length changes in cell displacement and BM expansion (n = 7 cells and BM regions from five follicles, each from a separate explant in an independent experiment). Values: means ± SD. Two-tailed unpaired t tests were used. (J) Normalized length changes (relative to cell displacement) of BM expansion and the value subtracted BM expansion from the cell displacement (data from Fig. 2 I). Values: means ± SD. Two-tailed unpaired t tests were used. (K) A schematic of the composite cell and BM movement. All statistical tests in this figure assumed normal data distribution, but this was not formally tested.

Spatially distinct basement membrane expansion rates synchronize with directional cell movement. (A) Schematic of region definitions in the developing hair follicle. Tip: Interface region between the prematrix and dermal condensate, defined by two bending points at the follicle tip. Lower stalk: The lower half of the follicle extends to the tip. Upper stalk: The upper half extends to the junction region. Junction: Bending neck region where follicular epidermis meets interfollicular epidermis. BM dynamics were measured in these regions. (B) Confocal images of hair follicles in dorsal skin explants from eGFP-Col4a2 mice show that BM length changes over 7 h. ROIs (dashed rectangles) indicate photobleached areas. Re-photobleaching of the targeted BM was performed every 2–3 h to maintain clear photobleached edges. See Fig. S2 for details. BM length changes were measured at the tip (blue lines), lower stalk (orange lines), and upper stalk (gray lines). Scale bar: 20 μm. (C) Percent BM length changes over a 7-h (n = 9 follicles from seven explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (D) Measurement of BM thickness in hair follicles of dorsal embryonic skin explants from eGFP-Col4a2 mice. BMs at the tip (blue lines), lower stalk (orange lines), and upper stalk (gray lines) were measured. Scale bars: 20 μm (left two images), 2 μm (right three images). (E) Quantification of BM thickness (n = 13 follicles from three explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (F) EdU incorporation assays. Dividing cells were labelled with EdU (magenta) with Hoechst counterstaining (blue). EdU-positive nuclei in the basal layer of the tip (blue line), lower stalk (orange line), and upper stalk (gray line) were measured across z-planes (−10, 0, +10, and +15 μm). Scale bar: 20 μm. (G) Percentage of EdU-positive nuclei in the tip, lower stalk, and upper stalk regions (n = 18 follicles from seven explants, each from an independent experiment). Values: means ± SD. One-way ANOVA with Tukey’s post-hoc tests was used. (H) Snapshot images of 3D time-lapse videos of developing hair follicles in eGFP-Col4a2; mem-tdTomato explants. The relative locations of the BM (lower bleached edge) and epithelial basal progenitors (white arrowheads) from reference points on the BM (upper bleached edge, yellow arrowheads) in lower (orange lines) and upper stalk regions (gray lines) were measured at 0 and 9 h 30 min. Re-photobleaching of the targeted BM was performed. See Fig. S4 for detailed tracking procedures. Scale bar: 20 μm. (I) Normalized length changes in cell displacement and BM expansion (n = 7 cells and BM regions from five follicles, each from a separate explant in an independent experiment). Values: means ± SD. Two-tailed unpaired t tests were used. (J) Normalized length changes (relative to cell displacement) of BM expansion and the value subtracted BM expansion from the cell displacement (data from Fig. 2 I). Values: means ± SD. Two-tailed unpaired t tests were used. (K) A schematic of the composite cell and BM movement. All statistical tests in this figure assumed normal data distribution, but this was not formally tested.

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