Learning About Limb Regeneration from Fingernails


Fingertip amputation in mammals results in regeneration of the nail, the attendant nerves, and even the damaged bone. Humans can also regenerate a fingertip in as little as two months. This seemingly simple regenerative event remains poorly understood.

However work from the NYU Langone Medical Center has provided a greater understanding of this somewhat opaque event. By using genetically engineered mice, the NYU team was able to elucidate a chain of events that unfolds after finger amputation.

This may seem like a terribly small thing, but understanding the regeneration of a finger tip can lead to augmentation of this process so that eventually entire fingers can be regenerated and even entire limbs.

“Everyone knows that fingernails keep growing, but no one really knows why,” said lead author Mayumi Ito, assistant professor of dermatology in the Ronald O. Perelman Department of Dermatology at NYU School of Medicine. Also, the connection between the regenerative ability of the bone and surrounding to the growth and/or regeneration of the nail is equally poorly understood.

Ito and others have discovered an important clue, and that is a population of stem cells in the nail matrix. The nail matrix contains a bed that is rich in nerve termini and blood vessels that stimulate nail growth.

To review the structure of the nail, the nail plate consists of the hard visible part of the nail. The nail plate is composed of hard, keratinized, squamous cells that are loosely attached to the germinal matrix but strongly attached to the sterile matrix. The nail matrix is the tissue that a nail [nail plate] protects. It lies beneath the nail and contains nerves, lymph and blood vessels. The matrix is responsible for producing cells that become the nail plate. It has two parts: the sterile matrix and the germinal matrix.

anatomy_nail

The stem cell population lies within the nail matrix, and these stem cells depend on a family of signaling proteins known as “Wnt” proteins. Wnt proteins are secreted glycoproteins that bind to Frizzled receptors. The Frizzled receptors bind Wnts and cause the polymerization of the Dsh or Disheveled protein at the cell membrane, and this inhibits GSK-3, a protein kinase. GSK-3 places phosphate groups on beta-catenin, and this marks beta-catenin for destruction. Once GSK-3 is inhibited, beta-catenin levels increase and it moves into the nucleus where it combines with Tcf proteins to activate the transcription of target genes.

Wnt signaling pathway

Wnt proteins play a crucial role in hair and tissue regeneration, and now they appear to play a truly vital role in bone regeneration as well.

Ito recounted her experiments: “When we blocked the Wnt-signaling pathway in mice with amputate fingertips, the nail and bone did not grow back as they normally would.”

a, Experimental scheme. Three-week-old K14–Cre-ER;β-catenin conditional knockout (cKO) mice and littermates were treated with Tam for 7 days immediately after distal-tip amputation, and analysed at the indicated time points. b, Whole-mount transparent specimen of a regenerated digit 5 weeks after amputation. c, Whole-mount alizarin red analysis. d, Trichrome staining. e, f, Quantification analyses of the nail length and the bone length 5 weeks after amputation. g, Analysis of Wnt activation in regenerating nail epithelium using TOPGAL at 3 weeks after amputation. The lower panel is a schematic illustration of the upper panel. h, Quantitative analyses of the distance between nerve tip and wound epidermis and the innervations at 3 weeks after amputation. i, Proliferation analyses by Ki67 immunohystochemistry at 3 weeks after amputation. Red bars in h, right panel, indicate the averages. Dashed lines indicate the border between nail epithelium and connective tissue. Asterisks in part h, bottom panel, indicate autofluorescence from blood cells. Data are presented as the mean ± s.d. Scale bars, 500 μm (b–d); and 100 μm (h).
a, Experimental scheme. Three-week-old K14–Cre-ER;β-catenin conditional knockout (cKO) mice and littermates were treated with Tam for 7 days immediately after distal-tip amputation, and analysed at the indicated time points. b, Whole-mount transparent specimen of a regenerated digit 5 weeks after amputation. c, Whole-mount alizarin red analysis. d, Trichrome staining. e, f, Quantification analyses of the nail length and the bone length 5 weeks after amputation. g, Analysis of Wnt activation in regenerating nail epithelium using TOPGAL at 3 weeks after amputation. The lower panel is a schematic illustration of the upper panel. h, Quantitative analyses of the distance between nerve tip and wound epidermis and the innervations at 3 weeks after amputation. i, Proliferation analyses by Ki67 immunohystochemistry at 3 weeks after amputation. Red bars in h, right panel, indicate the averages. Dashed lines indicate the border between nail epithelium and connective tissue. Asterisks in part h, bottom panel, indicate autofluorescence from blood cells. Data are presented as the mean ± s.d. Scale bars, 500 μm (b–d); and 100 μm (h).

When Ito and her team manipulated the Wnt pathway they discovered that they could stimulate regeneration of bone and tissue just beyond the fingernail. “Amputations of this magnitude ordinarily do not grow back,” noted Ito.

a, Experimental scheme. Three-week-old K14–Cre-ER;β-cateninfl/ex3 (mutant) mice and littermate controls were treated with Tam for 7 days starting from 2 weeks after amputation at the proximal level. b–f, Immunohistochemical analyses with indicated markers 3 weeks after amputation. g, Whole-mount transparent specimen of regenerated digits. h, Whole-mount alizarin red analysis. i, j, Quantification analyses of the nail (i) and bone length (j) 4 weeks after amputation. Red bars in d show the averages. Arrowheads in c and e, bottom panels, indicate TCF1− proximal matrix and FGF2+ epidermis, respectively. Arrowheads in d point to nerves. Fine dotted lines in b and h indicate the amputation plane. Dashed lines indicate the border between epidermis and connective tissue. Quantified data are presented as the mean ± s.d. Scale bars, 100 μm (b–f); and 500 μm (g and h).
a, Experimental scheme. Three-week-old K14–Cre-ER;β-cateninfl/ex3 (mutant) mice and littermate controls were treated with Tam for 7 days starting from 2 weeks after amputation at the proximal level. b–f, Immunohistochemical analyses with indicated markers 3 weeks after amputation. g, Whole-mount transparent specimen of regenerated digits. h, Whole-mount alizarin red analysis. i, j, Quantification analyses of the nail (i) and bone length (j) 4 weeks after amputation. Red bars in d show the averages. Arrowheads in c and e, bottom panels, indicate TCF1− proximal matrix and FGF2+ epidermis, respectively. Arrowheads in d point to nerves. Fine dotted lines in b and h indicate the amputation plane. Dashed lines indicate the border between epidermis and connective tissue. Quantified data are presented as the mean ± s.d. Scale bars, 100 μm (b–f); and 500 μm (g and h).

These findings suggest that Wnt signaling is essential for fingertip regeneration, and indicate that the way to develop therapies for regenerating lost limbs is to more deeply understand Wnt signaling and its role in limb regeneration. Some 1.7 million people in the US alone live with amputations. Therefore, research of this type could prove remarkably useful.