Extended Data Fig. 8. Characterization of K10rtTA; pTRE-Cdkn1b mice.
(A) Threshold used to define K10 reporter positivity for fate imbalance analysis (Fig. 4A, B). Red line indicates normalized K10 reporter levels in cells one timepoint (24 h) prior to division; blue line indicates K10 reporter levels one timepoint prior to delamination. (B) Strategy to block proliferation in differentiating basal cells. K10 promoter-controlled rtTA was used to drive expression of both the G1 cell cycle inhibitor Cdkn1b (pTRE-Cdkn1b) and a fluorescent reporter (pTRE-H2BGFP) in a Doxycycline-inducible manner. All Cdkn1b experiments were performed in ear epidermis. (C) K10 reporter positive mitotic figures within 200 µm × 200 µm basal regions after 24 hours of Doxycycline. Student’s two-sided t-test, p < 0.020. (D) Average epidermal thickness after 4 days of Doxycycline administration. Student’s two-sided t-test, p > 0.05. (E) Involucrin (top row) and Loricrin (bottom row) staining after 10 days of Doxycycline. (F) Proportion of basal cells undergoing delamination between Day 3 and Day 4 of Doxycyline. Student’s two-sided t-test, p > 0.05. Scale bars=15 µm. (G) Percent tracked cells scored as K10 reporter positive at the timepoint prior to delamination between Day 3 and Day 4 of Doxycyline administration. Student’s two-sided t-test, p > 0.05. (H) New K10 induction events, indicated by white arrows, between 9 and 10 days of Doxycycline administration. Scale bar = 10 µm. (I) Average number of new K10 induction events within 500 µm x 500 µm basal regions between 9 and 10 days of Doxycycline administration. Student’s two-sided t-test, p < 0.031. (J) smFISH staining for Krt10 and Krt14 mRNA together with IHC for H2BGFP and a membrane stain in both dorsal and ear skin. Note that due to extremely high levels of Krt10 mRNA expression in suprabasal and delaminating cells, they look overexposed when increasing channel brightness in order to show Krt10-dim cells. Arrows indicate Krt10-dim H2BGFP-negative cells. Dashed line marks the basement membrane. Scale bars = 15 µm. (K) H2BGFP-negative Krt10-dim basal cells in dorsal and ear tissue. Graph represents average from one independent immunostaining experiment using n = 3 mice. Student’s two-sided t-test, p = 0.011 (dorsal), p > 0.05 (ear). (L) Average basal density within 200 µm x 200 µm regions after 4 days of Doxycycline administration. Graph represents average of n = 3 imaged mice. Student’s two-sided t-test, p > 0.05. For bar graphs in (C), (D), (F), (G), (I), (K) and (L), data is from n = 3 mice and error bars are mean ±S.D.