Figure 1.
Figure 1. Hras mutant cells outcompete wild-type neighbors yet are integrated into normal follicular tissue. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Left: Cartoon schematic depicting targeted activation to the HFSC compartment using the K19CreER system. Right: Two-photon images of Hras+/+ and HrasG12V/+ follicles during the growth phase. (B) Western blot analysis reveals that Hras signaling is active in the HrasG12V/+ K19CreER skin during the growth phase of follicle cycling as assessed by phospho-MAPK (n = 7 Hras+/+, 8 HrasG12V/+) and phospho-PI3K expression (n = 3 Hras+/+, 4 HrasG12V/+ mice). (C) Two-photon representative images of the same Hras+/+ and HrasG12V/+ follicles from first to second rest phase (∼1 mo) show a significant expansion of tdTomato in the HrasG12V/+ follicles. (D) Top: Imaris tdTomato volumetric analysis schematic showing (from left to right) a two-photon image of the HFSC during rest phase, a volumetric mask of the entire follicle, a volumetric mask of the tdTomato population within the entire follicle, and an isolated volumetric mask of only the tdTomato population. Bottom: The tdTomato expansion in HrasG12V/+ follicles is statistically significantly higher than in Hras+/+ follicles (n = 20 follicles per genotype across four mice per genotype). (E) Analysis of the average number of mitotic (left) and apoptotic (right) events per hair follicle from two-photon images reveals enhanced behaviors in HrasG12V/+ follicles yet proper temporal maintenance of these behaviors within the appropriate cycle phases (mitotic events: n = 64 follicles across 4 Hras+/+ mice, average follicle length 149.6 µm and n = 45 follicles across 5 HrasG12V/+ mice, average follicle length 166.85 µm; apoptotic events: n = 25 follicles across 5 Hras+/+ mice, average follicle length 243.27 µm and n = 40 follicles across 6 HrasG12V/+ mice, average follicle length 251.09 µm). (F) Targeted dermal papilla (DP) ablation during the rest phase locks follicles in permanent quiescence. Follicle 1 is ablated, while follicle 2 is not, and the asterisk denotes the autofluorescent halo produced after ablation (n = 50 follicles across 4 HrasG12V/+ mice, n = 30 follicles across 3 Hras+/+ mice; no ablated follicles ever regrew in either group). Insets are at higher exposure of the red channel to highlight the dermal papilla before and after ablation. (G) Cartoon summary depicting follicular HrasG12V/+ integration and obedience of normal tissue programs. For significance values, *, P < 0.05 and ****, P < 0.0001, as determined by an unpaired, two-tailed t test. Error bars indicate standard deviation and n.d. indicates not detected. All scale bars represent 50 µm.

Hras mutant cells outcompete wild-type neighbors yet are integrated into normal follicular tissue. All epithelial nuclei are in green (K14H2BGFP), and recombined cells are in red (tdTomato). (A) Left: Cartoon schematic depicting targeted activation to the HFSC compartment using the K19CreER system. Right: Two-photon images of Hras+/+ and HrasG12V/+ follicles during the growth phase. (B) Western blot analysis reveals that Hras signaling is active in the HrasG12V/+ K19CreER skin during the growth phase of follicle cycling as assessed by phospho-MAPK (n = 7 Hras+/+, 8 HrasG12V/+) and phospho-PI3K expression (n = 3 Hras+/+, 4 HrasG12V/+ mice). (C) Two-photon representative images of the same Hras+/+ and HrasG12V/+ follicles from first to second rest phase (∼1 mo) show a significant expansion of tdTomato in the HrasG12V/+ follicles. (D) Top: Imaris tdTomato volumetric analysis schematic showing (from left to right) a two-photon image of the HFSC during rest phase, a volumetric mask of the entire follicle, a volumetric mask of the tdTomato population within the entire follicle, and an isolated volumetric mask of only the tdTomato population. Bottom: The tdTomato expansion in HrasG12V/+ follicles is statistically significantly higher than in Hras+/+ follicles (n = 20 follicles per genotype across four mice per genotype). (E) Analysis of the average number of mitotic (left) and apoptotic (right) events per hair follicle from two-photon images reveals enhanced behaviors in HrasG12V/+ follicles yet proper temporal maintenance of these behaviors within the appropriate cycle phases (mitotic events: n = 64 follicles across 4 Hras+/+ mice, average follicle length 149.6 µm and n = 45 follicles across 5 HrasG12V/+ mice, average follicle length 166.85 µm; apoptotic events: n = 25 follicles across 5 Hras+/+ mice, average follicle length 243.27 µm and n = 40 follicles across 6 HrasG12V/+ mice, average follicle length 251.09 µm). (F) Targeted dermal papilla (DP) ablation during the rest phase locks follicles in permanent quiescence. Follicle 1 is ablated, while follicle 2 is not, and the asterisk denotes the autofluorescent halo produced after ablation (n = 50 follicles across 4 HrasG12V/+ mice, n = 30 follicles across 3 Hras+/+ mice; no ablated follicles ever regrew in either group). Insets are at higher exposure of the red channel to highlight the dermal papilla before and after ablation. (G) Cartoon summary depicting follicular HrasG12V/+ integration and obedience of normal tissue programs. For significance values, *, P < 0.05 and ****, P < 0.0001, as determined by an unpaired, two-tailed t test. Error bars indicate standard deviation and n.d. indicates not detected. All scale bars represent 50 µm.

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