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First published online 6 February 2008
doi: 10.1242/dev.012799

Development 135, 1059-1068 (2008)
Published by The Company of Biologists 2008
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Runx1 modulates developmental, but not injury-driven, hair follicle stem cell activation

Karen M. Osorio, Song Eun Lee, David J. McDermitt, Sanjeev K. Waghmare, Ying V. Zhang, Hyun Nyun Woo and Tudorita Tumbar*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA.


Figure 1
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  		Fig. 1. HF organization and the hair cycle. (A,B) The follicle cell layers are depicted in color with appropriate protein marker (boxed). Stem cells (SCs) are in the bulge and progenitor cells are in the matrix. Differentiated hair lineages: Cp, companion cell layer; IRS, inner root sheath; He, Henle's layer; Hu, Huxley's layer; Ci, cuticle of IRS; Ch, cuticle of hair shaft; Co, cortex of hair shaft; Me, medulla. Exogen is hair shaft loss.

 

Figure 2
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  		Fig. 2. Runx1 expression in HF during SC activation. (A) FACS of skin cells after 4 weeks of H2B-GFP repression shows GFP epifluorescence, and surface CD34 and {alpha}6-integrin expression. (B) RT-PCR for Runx1b in the HFSC pool (CD34+/{alpha}6+/GFPhigh) relative to other epithelial skin cells (CD34-/{alpha}6+/GFP+). (C) Skin at second telogen-anagen transition (a) from mice in A. Skin at first telogen-anagen transition (PD21) from Runx1lacZ/+ (b) and wild-type (c-e) mice. Staining for Runx1 and Ki67 (d,e) show HFs from serial sections. Arrows (c,d) indicate bulge CD34+/nuclear Runx1+ cell, enlarged in inset. Arrow in e points to a Ki67+ bulge cell, which is indicative of early stage of stem/progenitor cell proliferation (activation). Ep, epidermis; Bu, bulge; hg, hair germ, DP, dermal papillae. Scale bars: 20 µm. Blue is DNA DAPI staining.

 

Figure 3
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  		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.

 

Figure 4
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  		Fig. 4. Analyses of Runx1{Delta}4/{Delta}4 bulge SC numbers and gene expression. (A) Skin sections from wild-type and {Delta}4 mice at PD21 show expression of markers indicated at the top (yellow). Bu, bulge; hg, hair germ; DP, dermal papillae. Asterisk indicates background signal of hair shaft. (B) Surface expression of CD34 and {alpha}6-integrin by FACS of skin cells at PD20. (C) Summary of FACS experiments in B shows frequency of {Delta}4 and wild-type CD34+/{alpha}6-integrin+ bulge cells in the skin (P=0.2 demonstrates no significant differences). (D) Bulge (Bu) and outside the bulge (O/Bl) sorted cells from (B) were used to prepare total RNA and cDNA. RT-PCR analyses show expression levels for genes indicated on the left. +/+ and -/+ designate CD34 and {alpha}6-integrin expression in each population. The last four lanes are negative controls without reverse transcriptase. (E) Summary of phenotypes for mutant mice indicated (left column) and gene expression level obtained consistently in wild-type and {Delta}4 mice tested (right column). Tm, targeted mutation (knockout); Tg, transgenic (overexpression). Level of expression in Runx1{Delta}4/{Delta}4 bulge is indicated in the right-hand column by + (increase), - (decrease) or N/C (no change). N/A, not applicable.

 

Figure 5
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  		Fig. 5. Effect of Runx1{Delta}4/{Delta}4 on bulge SC proliferation. (A) Sections from 3- or 4-day-old BrdU-labeled skin (PD20-PD23 or 24) show cells that proliferated during anagen onset. Early anagen wild-type follicle (a,b) shows multiple BrdU+ (red) cells in hair germ and several BrdU+(red) and CD34+ (green) bulge cells (arrows). Telogen Runx1{Delta}4/{Delta}4 follicle shows complete lack of BrdU+ cells in CD34+ bulge cells or germ cells (c,d). Ep, epidermis; Bu, bulge; hg, hair germ; DP, dermal papillae; De, dermis. Asterisk shows hair shaft autofluorescence. (B) Fraction of follicles scored on skin section shown in A that displayed BrdU+ cells in bulge or germ. Sixty-seven percent of follicles have BrdU+ bulge cells for wild-type mice and there is a complete lack of BrdU+ bulge cells for {Delta}4 mice. Follicles with BrdU+ germ cells are further subdivided into those with more than two BrdU+ cells/germ and one or two BrdU+ cells/germ. Total number of HFs analyzed from five wild-type (black) and five {Delta}4 (gray) littermates is shown (802 wild type & 737 {Delta}4). Error bars underscore variability of BrdU+ follicle fractions in each category. (C) CD34+/{alpha}6-integrin+ cells from mice in A,B were sorted on slides, fixed and stained as described (Tumbar, 2006Go). There is a high frequency of cells that are double positive for keratin 5 (K5, red) and β4-integrin (β4, green, bottom panel). BrdU+ (red) and DAPI (blue) staining (top panel) shows lack of proliferation in {Delta}4 but not wild-type bulge cells. (D) Sorted bulge cells from C counted for double expression of epithelial K5 and β4 markers. Un, unsorted live cell control. Number of cells is at the top, ID of mice is at the bottom. (E) Quantification of proliferating (BrdU+) sorted bulge cells from (C). Number of cells is at the top, mouse ID is at the bottom. Negative controls were from BrdU-negative mice.

 

Figure 6
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  		Fig. 6. Injury reverses Runx1{Delta}4/{Delta}4 HFSCs block in quiescence. (A) Schematic of HFSC activation. (B) Back region of {Delta}4 mice post-hair plucking shows hair growth in injured area (arrow). (C) Hematoxylin and Eosin staining of {Delta}4-skin sections collected from wounded and unwounded (opposite) back regions at time points indicated show progression through the hair cycle. (D) Runx1{Delta}4/{Delta}4 injured skin shows proliferating Ki67+ (red, arrows) of CD34+ bulge cells (green). (E) Staining of skin sections 18 days post-wounding shows normal expression of differentiated hair lineage markers. There is a lack of Runx1 staining (performed in serial sections) in {Delta}4 but not in wild-type follicles. (F) Schematic of long-term functional HFSC assay.

 

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© The Company of Biologists Ltd 2008