Figure 4: Lineage tracing supports the basal to luminal differentiation.

(a) Scheme summarizing the genetic strategy used to target RFP expression in K14-expressing basal cells and the protocol used to tracing the fate of basal cells during early prostate development. (b) Immunostaining of CK5 and CK8 with RFP 3 days after tamoxifen injection in newborn K14CreER/RosaRFP mice (1 week). See Supplementary Table S7 for further data. (c) Immunostaining of p63 and CK8 with RFP 3 weeks after tamoxifen injection in newborn K14CreER/RosaRFP mice. (d) Confocal analysis of cell fates during tolephase of RFP-positive basal cells by staining RFP, Survivin with p63 or CK8. (e) Confocal detection of dividing luminal cell 3 weeks after tamoxifen injection in K14CreER/RosaRFP mice at birth by staining P-H3. Yellow arrowheads point to the dividing RFP⁺ cell that is a CK8-positive luminal cell. (f) Grafts of p63^+/+ and p63^−/− UGS in renal capsules 10 days post implantation. Dotted lines denote the boundary of prostate grafts. (g) Histological analysis of basal and luminal compartments from p63^+/+ and p63^−/− grafts stained for AR and p63. (h) Analysis of dividing luminal cells in p63^+/+ and p63^−/− UGS grafts by staining for CK5 and P-H3. (i) Immunodetection of horizontal divisions of luminal cells in p63^−/− grafts by staining AR and P-H3, E-cadherin and Survivin. A short line indicates a horizontal spindle orientation. (j) Wet weight of p63^+/+ (n=4) and p63^−/− (n=3) grafts. (k) Quantification of dividing luminal cells in p63^−/− (n=3) and p63^+/+ (n=3) grafts from histological analysis of P-H3 staining. P10, postnatal day 10; R (2d), androgen replacement was administrated for 2 days from day 14 post castration. Dashed lines in d, e, i denote the positions of the basement membrane (bold) and the boundary of luminal cells (light). Data in j, k are presented as mean±s.e.m., and P values were determined by Student’s t-test using Prism5. Scale bars, 10 μm (c,e,i), 25 μm (b,d), 50 μm (g) and 100 μm (h), 1 mm (f).