Lose a finger, and you’d better hope you can get it on ice. Lose a fingertip, and it might not matter. Fingertips are one of the few parts of the human body that can match the regenerative abilities of a starfish or a salamander. Children and some adults can grow new fingertips in a few weeks after amputation. The replacement fingertips wouldn’t pass for the originals; they lack fingerprints and their nails have flattened, square ends. Still, they have the full complement of bone, nerves and nails seen in regular fingertips.
This regeneration will occur only if enough of the fingertip is left intact. If any of the nail remains, even a sliver, the tip can regrow. Cut off the nail entirely, and neither it nor the fingertip will come back. Makoto Takeo of the New York University School of Medicine and colleagues investigated why nails were so crucial for regeneration, publishing their results online in Nature in June 2013. The key wasn’t the hard part of the nail, the nail plate, but instead the cells that produce it, the nail matrix. Looking at the tips of mice’s toes, the researchers found that the nail matrix gave off chemical signals that induced the regrowth of nerves and bone. Mice lacking these signaling molecules were unable to regenerate. Only the nail matrix cells closer to the tip, the ones below the nail plate, released the chemicals that enabled regeneration.
Ever wondered what that whitish crescent on your thumbnail is? It’s the nail matrix, or at least the part we can see. The rest extends underneath the skin at the base of the nail. If the matrix as a whole were what enabled regeneration, then it shouldn’t matter for amputees whether they have any nail left. An injury that removed the entire nail could still preserve some of the nail matrix, and the fingertip could still regenerate. Since this didn’t occur, it suggested that only certain cells in the nail matrix were involved in regeneration.
One possibility was that regeneration was governed by the nail stem cells. These slowly dividing cells produce the rest of the nail matrix, which goes on to form the nail plate and the nail bed. No one was sure where in the matrix the stem cells were located. To find out, the researchers examined genetically engineered mice known as reporter mice. These mice were designed to express reporter genes, genes that produce identifying chemical markers in particular cells. When a substance called tamoxifen was administered, cells making a protein involved in nail production expressed the gene for an enzyme called LacZ.
The researchers injected the mice with the synthetic chemical tamoxifen and examined their toes over the following months. There were streaks of LacZ activity underneath and behind the nail, but these faded as the cells divided. The distal portion of these streaks, the part closer to the tip of the toe, faded more quickly than the proximal portion toward the base of the toe. This indicated that the proximal cells were the slowly dividing nail stem cells. The distal ones were the more rapidly dividing cells that create the rest of the nail.
No Bones without Wnt
These distal cells appeared to be the best candidates for affecting regeneration. Was there anything in their biochemistry to support this? The researchers analyzed the patterns of gene expression in cells from different parts of the nail matrix. They found that the distal cells expressed genes involved in a set of chemical interactions called the Wnt signaling pathway. In embryos, Wnt signaling coordinates many aspects of development, including the formation of limbs and nails.
In the nail matrix, Wnt probably caused the distal cells to differentiate, changing them into the different types of cells in the nail. To verify this, the researchers prevented expression of β-catenin, a protein involved in the Wnt pathway. Since mice couldn’t develop normally without this vital protein, the researchers used another genetically engineered strain of mice called a conditional knockout. Just as the reporter mice produced an enzyme only under specific conditions, these knockout mice were genetically engineered to stop producing β-catenin in nail cells when the same chemical, tamoxifen, was administered. Two months after tamoxifen was given to these mice, they had stopped creating nails. In their place, the mice had a layer of cells resembling undifferentiated nail stem cells.
If Wnt signaling caused nail stem cells to differentiate into new nails, maybe it also caused tissue to differentiate into a new fingertip. The researchers took mice from the same strain of conditional knockouts and amputated the tips of their toes, so that only a small amount of nail remained. Unlike humans, normal adult mice will always regrow the tips of their digits. Some of the mice were treated with tamoxifen after amputation, stopping β-catenin expression and the Wnt signaling pathway. Others didn’t receive the tamoxifen injection. These control mice regenerated completely, growing new nails and toe bones in five weeks. The ones that had been given tamoxifen didn’t regenerate their nails or their bones.
Guiding Nerves, Growing Bone
How did Wnt signaling enable the mice to regenerate? A clue came from their nerves. As the control mice healed from their amputations, the nerves in their toes extended toward the tip of the regrown digit. The nerves made contact with the mesenchyme, the cells from which new bone forms. The tamoxifen-treated mice didn’t show this kind of nerve growth. New cells formed over the wound, but the mice’s nerves didn’t grow past the initial site of injury. The nerves never reached the mesenchyme, which consequently never differentiated into bone.
It’s well known that nerves are important for the regeneration of both rodent digits and salamander limbs. The researchers investigated what effect a lack of nerves in the tip of a digit would have on regeneration. In another group of mice, they severed the nerves leading to one foot and then amputated the tips of toes on that foot and the opposite one. The amputated toes on the feet with intact nerves would act as a control. As in the conditional knockout mice, the toes on the feet with severed nerves didn’t regenerate. The team found that these toes had low levels of FGF2 (fibroblast growth factor 2), one of a set of proteins that aid in wound healing and are essential for limb regeneration in amphibians. The conditional knockout mice with the amputated tips had lacked FGF2 as well. The researchers found other evidence for the importance of FGF2 when they grew cultures of mouse mesenchymal cells. When they administered FGF2, the cells divided more rapidly and began to turn into bone.
Restoring Wnt and Regeneration
If nail matrix cells could induce regeneration, then the researchers might be able to induce it artificially. The team repeated the severed nerve experiment. This time, after the tips of the mice’s toes were amputated, the researchers inserted beads soaked in FGF2 into the wounds. As FGF2 leached off of the beads, it spread into the mice’s tissues. After three weeks, they had begun to regrow bone.
Was it possible to give mice an improved ability to regenerate? The researchers amputated the tips of mice’s toes so that only the proximal part of the nail matrix remained. This meant that the injury was just past the point where the tip can normally grow back. The mice were genetically engineered so that, when treated with tamoxifen, their remaining nail stem cells produced β-catenin and initiated Wnt signaling. After the amputation and the tamoxifen injection, these mice made a full recovery, regrowing their nails along with new bone.
Regeneration is a complex process, but the researchers had identified two of its major elements. Wnt signaling from the nail matrix cells was necessary both to produce new nails and to make nerves grow toward the tip of the healing digit. These nerves triggered expression of FGF2, inducing the growth of bone. The skin, the nail, the nerves and the bone could all come into place and form a new fingertip.
Besides restoring missing digits, what other medical applications could come out of this discovery?
Unlike humans, normal mice can regenerate the tips of their digits throughout their lives. How might this affect efforts to apply this research to human patients?