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. 2013 Jul 11;499(7457):228-32.
doi: 10.1038/nature12214. Epub 2013 Jun 12.

Wnt activation in nail epithelium couples nail growth to digit regeneration

Makoto Takeo  1 , Wei Chin Chou, Qi Sun, Wendy Lee, Piul Rabbani, Cynthia Loomis, M Mark Taketo, Mayumi Ito
Affiliations

Wnt activation in nail epithelium couples nail growth to digit regeneration

Makoto Takeo et al. Nature. .

Abstract

The tips of mammalian digits can regenerate after amputation, like those of amphibians. It is unknown why this capacity is limited to the area associated with the nail. Here we show that nail stem cells (NSCs) reside in the proximal nail matrix and that the mechanisms governing NSC differentiation are coupled directly with their ability to orchestrate digit regeneration. Early nail progenitors undergo Wnt-dependent differentiation into the nail. After amputation, this Wnt activation is required for nail regeneration and also for attracting nerves that promote mesenchymal blastema growth, leading to the regeneration of the digit. Amputations proximal to the Wnt-active nail progenitors result in failure to regenerate the nail or digit. Nevertheless, β-catenin stabilization in the NSC region induced their regeneration. These results establish a link between NSC differentiation and digit regeneration, and suggest that NSCs may have the potential to contribute to the development of novel treatments for amputees.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. NSCs are harbored in the proximal nail matrix
a. Experimental scheme. B, c. Whole mount (b) and sectioned (c) specimens of K14-CreER;R26R reporter mice. LacZ expression was detected at indicated times after TAM treatment. Inset in (b) shows top view of the nail. d, Quantitative analysis of LacZ positive streaks. e, Tissue section analysis of a LacZ+ colony at 5 mo after chase and schematic representation, to illustrate cell lineages from proximal matrix cells. f. A typical nail sample used for microdissection to obtain proximal, distal and bed fragments. g, Immunocytochemistry for K14 and K17 using single cell suspensions from each compartment. h–j, In vitro colony forming assay with single cell suspensions obtained from indicated fragments. Visualization of colonies by Rhodamine B staining (h) and quantification of colonies >3 mm2 (i). Brightfield images of proximal and distal nail epithelial colonies (j). Arrowheads indicate LacZ+ cell or colony. Dashed lines delineate the boundary between nail epithelium and underlying connective tissue (ct). Asterisk indicates nonspecific background. Data are presented as the mean ± SD. Scale bars, 500 μm in (b and f); 100 μm in (c and e). dm, distal matrix, kz, keratogenous zone, nb, nail bed, np, nail plate, pm, proximal matrix.
Fig. 2
Fig. 2. Epithelial β-catenin is required for nail differentiation
a. Experimental scheme. Three-week-old K14-CreER; β-catenin cKO mice and littermates were treated with TAM for 7 d, and analyzed at 2 mo after TAM treatment. b–e. Gross appearance (b and d) and H&E staining (c and e) of control (b and c) and cKO digit (d and e). f–h. Immunofluorescence for indicated markers at 2 mo after TAM treatment. i. Summary of immunohistochemistry in (f–h). Dashed lines indicate the border between nail basal layer and connective tissue. Lines indicate the outline of nail plate in (f–h). Asterisks show nonspecific background. Scale bars, 500 μm in (b, c and f).
Fig. 3
Fig. 3. Nail epithelial β-catenin is required for blastema growth and digit regeneration
a. Experimental scheme. Three-week-old old K14-CreER;β-catenin cKO mice and littermates were treated with TAM for 7 d immediately after distal tip amputation, and analyzed at the indicated time points. b, Gross appearance of regenerated digit at 5 w after amputation. c. Whole mount alizarin red analysis. d. Trichrome staining. e and f. Quantification analyses of the nail length (h) and the bone length (i) at 5 w after amputation. g. Analysis of Wnt activation in regenerating nail epithelium using TOPGAL at 3 w after amputation. Lower panel is the schematic illustration of the upper panel. h. Quantitative analyses of the distance between nerve tip and wound epidermis and the innervations at 3w after amputation. i. Proliferation analyses by Ki67 immunohystochemistry at 3w after amputation. Red bar in o indicates the average. Dashed lines, border between nail epithelium and connective tissue. Arrows Asterisks in (n, o and r) indicate autofluorescence from blood cells. Data are presented as the mean ± SD. Scale bars, 500 μm in (b–d); 100 μm in (h).
Fig. 4
Fig. 4. Forced Wnt activation in wound epidermis can overcome the limitation of regeneration following proximal amputation
a. Experimental scheme. Three-week-old K14-CreER;β-cateninfl/ex3 (mutant) mice and littermate controls were treated with TAM for 7 days starting from 2 w after amputation at the proximal level. b–f, Immunohistochemical analyses with indicated markers at 3 w after amputation. g and, Gross appearance of regenerated digits. h, Whole mount alizarin red analysis. i and j, Quantification analyses of the nail (i) and bone length (j) at 4 w after amputation. Red bars in n show the meal values. Arrows in (c) and (e) indicate Tcf1- proximal matrix and FGF2+ epidermis, respectively. Arrowheads in d point nerves. Dotted lines in b, c, g and h indicates amputation plane. Dashed lines indicate the border between epidermis and connective tissue. Dotted lines in (b) indicate amputation planes. Quantified data are presented as the mean ± SD. Scale bar, 100 μm in (b–f), 500 μm in (g and h).
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