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De Novo Production of Dermal Papilla Cells during the Anagen Phase of the Hair Cycle

2010; Elsevier BV; Volume: 130; Issue: 11 Linguagem: Inglês

10.1038/jid.2010.176

1523-1747

Woo Y., David Enshell‐Seijffers, Bruce Morgan,

melanin and skin pigmentation

connective tissue sheath or dermal sheath follicular dermal papilla yellow fluorescent protein Although keratinocytes are the primary constituents of the hair follicle and generate the hair shaft, mesenchymal cells also have important functions. These include the follicular dermal papilla (DP) and the connective tissue sheath or dermal sheath (CTS). The DP is embedded in the hair bulb during the anagen phase and forms a compact ball during the telogen phase, whereas CTS cells line the outside of the epithelial follicle from the bulge to its base. The central function of the DP in regulating the activity of keratinocytes during hair follicle regeneration and hair shaft morphogenesis has been established by extirpation and grafting studies (Ibrahim and Wright, 1977Ibrahim L. Wright E.A. Inductive capacity of irradiated dermal papillae.Nature. 1977; 265: 733-734Crossref PubMed Scopus (17) Google Scholar; Jahoda et al., 1984Jahoda C.A. Horne K.A. Oliver R.F. Induction of hair growth by implantation of cultured dermal papilla cells.Nature. 1984; 311: 560-562Crossref PubMed Scopus (466) Google Scholar, Jahoda et al., 1993Jahoda C.A. Reynolds A.J. Oliver R.F. Induction of hair growth in ear wounds by cultured dermal papilla cells.J Invest Dermatol. 1993; 101: 584-590Abstract Full Text PDF PubMed Google Scholar; McElwee et al., 2003McElwee K.J. Kissling S. Wenzel E. et al.Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla.J Invest Dermatol. 2003; 121: 1267-1275Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), and more recently by direct manipulation of gene expression in the DP in vivo (Enshell-Seijffers et al., 2010Enshell-Seijffers D. Kashiwagi M. Lindon C. et al.β-Catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair.Dev Cell. 2010; 18: 633-642Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). A strong correlation between DP size, hair bulb diameter and hair caliber has been noted (Van Scott and Ekel, 1958Van Scott E.J. Ekel T.M. Geometric relationships between the matrix of the hair bulb and its dermal papilla in normal and alopecic scalp.J Invest Dermatol. 1958; 31: 281-287Abstract Full Text PDF PubMed Scopus (139) Google Scholar; Ibrahim and Wright, 1982Ibrahim L. Wright E.A. A quantitative study of hair growth using mouse and rat vibrissal follicles. I. Dermal papilla volume determines hair volume.J Embryol Exp Morphol. 1982; 72: 209-224PubMed Google Scholar; Elliott et al., 1999Elliott K. Stephenson T.J. Messenger A.G. Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: implications for the control of hair follicle size and androgen responses.J Invest Dermatol. 1999; 113: 873-877Crossref PubMed Scopus (164) Google Scholar). The CTS is less accessible to experimental manipulation and its function in the intact follicle is more poorly defined. However, the proximal CTS shares properties with the DP that include the capacity to reform the DP in grafting studies (McElwee et al., 2003McElwee K.J. Kissling S. Wenzel E. et al.Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla.J Invest Dermatol. 2003; 121: 1267-1275Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). DP cells undergo comparatively few divisions and the constituents of a DP are a largely static population when compared with the dynamic changes in the keratinocyte populations that abut them. Modest expansion and contraction of cell numbers in the DP occurs over the course of the hair cycle. Tobin et al., 2003bTobin D.J. Gunin A. Magerl M. et al.Plasticity and cytokinetic dynamics of the hair follicle mesenchyme during the hair growth cycle: implications for growth control and hair follicle transformations.J Investig Dermatol Symp Proc. 2003; 8: 80-86Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) quantified these changes for the mouse hair cycle, reporting an increase in DP cell numbers during anagen I–V and a decrease in anagen VI–telogen. They noted that the increase in DP cell number observed in early anagen precedes detectable proliferation in the DP, and suggested that it results from the migration of CTS cells into the DP. We have generated a mouse line, Cor-cre, that expresses cre recombinase in the DP (Enshell-Seijffers et al., 2010Enshell-Seijffers D. Kashiwagi M. Lindon C. et al.β-Catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair.Dev Cell. 2010; 18: 633-642Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). When coupled with a cre-dependent reporter gene, this provides a method to trace the fate of DP cells. The reporter gene contains the sequences encoding yellow fluorescent protein (YFP) in the ubiquitously expressed Rosa26 locus that are separated from a promoter by a transcriptional termination cassette flanked by LoxP sites (Srinivas et al., 2001Srinivas S. Watanabe T. Lin C.S. et al.Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.BMC Dev Biol. 2001; 1: 4Crossref PubMed Scopus (2133) Google Scholar). When this stop cassette is excised in cells expressing cre-recombinase, YFP is expressed in the cell and its progeny regardless of their position in the tissue or changes in expression of the cell type-specific cre recombinase allele. In Cor-cre/+; rYFP/+ mice, YFP is not detected until p3 and effective deletion across the DP population is not complete until p7 (Enshell-Seijffers et al., 2010Enshell-Seijffers D. Kashiwagi M. Lindon C. et al.β-Catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair.Dev Cell. 2010; 18: 633-642Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). By the end of the anagen phase, virtually all DP cells express YFP, while the proximal CTS is variably labeled. Corin is not expressed in catagen or telogen, and although Corin expression returns during early anagen, cre recombinase is not reliably detected until anagen V (data not shown) (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease Corin is a novel modifier of the Agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (82) Google Scholar). If all DP cells in a follicle are labeled with YFP at the end of one growth phase, the appearance of unlabeled cells in the subsequent anagen phase would provide evidence of recruitment of new cells to the DP. Mice of the genotype Cor-cre/+; rYFP/+ were killed at p13 and the extent of labeling in the DP was determined in tissue sections (Figure 1a). In a survey of follicles from five mice, no unlabeled DP cells were observed in 94±5% of the follicles scored (n=129) (Figure 2). Although most DP cells were labeled in the remaining follicles, one or two cells were unlabeled. During the catagen phase, a compact ball of YFP+ cells is associated with the regressing epithelial strand. In telogen phase, the descendents of the anagen DP form a compact ball of contiguous labeled cells, surrounded by more elongated cells that are variably labeled (Figure 1b). The variable labeling of these peripheral cells is consistent with the assumption that they derive from the CTS and confirms that most are not descendents of the DP. However, definitive distinction between DP and CTS derivatives at this stage is not possible.Figure 2The frequency of follicular dermal papilla (DP) with unlabeled cells at p13 and during the first anagen (p25–29). Error bars represent SD; n=5 mice per sample.View Large Image Figure ViewerDownload (PPT) In contrast to p13, many unlabeled DP cells are observed during the early anagen phase of the first post-natal hair cycle (Figure 1c and d). Skins in the early anagen phase were harvested (n=5), and follicles in anagen stage III–IV were analyzed (Muller-Rover et al., 2001Muller-Rover S. Handjiski B. van der Veen C. et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Crossref PubMed Google Scholar). Before stage III, distinction between DP and adjacent CTS is open to debate. At stage III, the DP is engulfed by the hair bulb, thereby allowing unambiguous definition of the boundary between DP and CTS with a line between the keratinocytes at the base of the follicle. Overall, 75%±8 of the follicles showed one or more DP cells that did not express YFP (n=256) (Figure 2). To confirm that this was not a failure of the rYFP reporter transgene, mice in which rYFP had been activated in the germ line were examined. No unlabeled DP cells were observed (n=68 follicles). These data demonstrate either that new cells are recruited to the DP during early anagen, or that a rare unlabeled population of DP cells in each follicle escaped detection in the random section analysis at P13. To rule out this latter possibility, 10 follicles were optically sectioned at 1-μ intervals through the entire hair bulb. All of the DP cells were labeled in each of these follicles. Given the preponderance of follicles containing unlabeled DP cells in the first true anagen, this result clearly demonstrates that these unlabeled cells do not arise from unlabeled DP cells persisting from the previous hair cycle (P<0.0001). We therefore conclude that most follicles incorporate new cells into the DP during the early anagen phase. Previous work had demonstrated that engrafted DP/CTS cells could be incorporated into the DP of existing follicles (McElwee et al., 2003McElwee K.J. Kissling S. Wenzel E. et al.Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla.J Invest Dermatol. 2003; 121: 1267-1275Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar; Biernaskie et al., 2009Biernaskie J. Paris M. Morozova O. et al.SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.Cell Stem Cell. 2009; 5: 610-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Induction of anagen enhanced the efficiency with which engrafted SKPs, a multipotent population that can be derived from DP in culture, are incorporated into existing hair follicles (Biernaskie et al., 2009Biernaskie J. Paris M. Morozova O. et al.SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.Cell Stem Cell. 2009; 5: 610-623Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). This suggests that the early anagen environment includes conditions conducive to recruitment of new cells to the DP. The results reported here demonstrate that new cells are incorporated into the DP during the early anagen phase of a natural hair cycle. It has been suggested that emigration from the DP to the CTS and/or dermis also occurs to achieve the reduction of DP cell numbers observed during anagen VI–telogen (Tobin et al., 2003bTobin D.J. Gunin A. Magerl M. et al.Plasticity and cytokinetic dynamics of the hair follicle mesenchyme during the hair growth cycle: implications for growth control and hair follicle transformations.J Investig Dermatol Symp Proc. 2003; 8: 80-86Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Both Corin and cre recombinase are detected in the proximal CTS, and sporadic labeling of the proximal CTS is observed during morphogenesis of the follicle as early as p4 (Enshell-Seijffers et al., 2010Enshell-Seijffers D. Kashiwagi M. Lindon C. et al.β-Catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair.Dev Cell. 2010; 18: 633-642Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), well before the mature anagen stages when export of cells from the DP is postulated. As a result, we cannot address the possibility of export to the CTS. If YFP-expressing CTS cells are incorporated into the DP during early anagen, this would lead to an underestimate of the magnitude of cellular recruitment to the DP. When the DP was viewed as a static lineage compartment, depletion of that restricted population could be invoked as the cause of reduction in follicle size. The observation of de novo generation of DP cells has important implications for the way we view the consistency of hair growth across hair cycles and its decay during aging or pathological hair loss (Tobin et al., 2003aTobin D.J. Gunin A. Magerl M. et al.Plasticity and cytokinetic dynamics of the hair follicle mesenchyme: implications for hair growth control.J Invest Dermatol. 2003; 120: 895-904Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). It emphasizes that regulation of DP size is an active process and that conservation of hair size is not the passive result of a fixed DP compartment, nor is decay in follicle size an inevitable consequence of damage within this compartment. Although it is reasonable to infer, based on close proximity and shared properties that the CTS is a likely source for new DP cells, identifying the pool of potential DP progenitors and the mechanisms that regulate DP cell number remain important steps toward improved management of hair loss. We are grateful for expert technical assistance from E Wu and B Czyzewski. This work was supported by grants 5R01AR055256 and 5R01AR052339 from NIAMS to BAM. WC was supported in part by training grant 5T32AR007098-35 from NIAMS.