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Figure 3


Fig. 3. Effect of Runx1 disruption on HF cycle and keratinocyte growth. (A) X-Gal stained skin (blue) from Rosa26R mice shows efficiency of K14-Cre. (B) PCR of genomic DNA shows detection of K14-Cre transgene (top) and Runx1 alleles (bottom): loxP unexcised (Fl), loxP excised ({Delta}4) and loxP untargeted (+). Lane 1, homozygous floxed mouse with no excision; lane 2, heterozygous floxed with no excision (both designated wild type); lane 3, homozygous floxed and excised (mutant {Delta}4). (C) Western blot of total skin protein extract probed with N-terminal Runx1 antibody. (D) Skin sections from PD21 mice show nuclear Runx1 protein (red) in hair germ cells in wild-type but not {Delta}4 animals. Asterisk indicates hair shaft autofluorescence. (E) Quantification of HFs with nuclear Runx1 expression (over 50 follicles from nonadjacent sections counted/point). (F) Hematoxylin and Eosin stained skin sections at indicated ages demonstrate prolonged telogen in {Delta}4 mice. (G) Summary of hair cycle stage determined by microscopy of Hematoxylin and Eosin stained skin sections. In brackets are numbers of mice analyzed. At PD21, telogen or catagen VIII were designated Tel. (H) Wild-type but not {Delta}4 mouse skin at PD25 produces new hair during first hair cycle following morphogenesis. After gently shaving far from the skin the hair was carefully clipped with scissors to avoid injury produced by close shaving. (I) One hundred and one wild-type and {Delta}4 mice analyzed by skin color at PD29 show {Delta}4 mice in telogen (pink skin) when virtually all wild-type mice are in anagen (black skin color) (P<0.001). (J) Bright-field images of Hematoxylin and Eosin stained keratinocytes on feeder cells, 2 weeks post-plating. Wild-type keratinocyte colony is outlined. (K) Growth curve from 100,000 live keratinocytes plated on feeders. Runx1{Delta}4/{Delta}4 keratinocyte proliferation is impaired (P<0.0001) after ~3 weeks in culture. (L) Arrow indicates an example of colony imaged by phase contrast (L). (M) Quantification of primary keratinocyte colonies obtained from equal numbers of wild type and {Delta}4 plated cells. {Delta}4 mutant show impaired colony formation Pexp1=0.012; Pexp2=0.019. Ep, epidermis; Hf, hair follicle; DP, dermal papillae; hg, hair germ; Bu, bulge. Scale bars: 50 µm.





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