A New Spin on a Salamander’s Old Tricks

Although not often the subject of respect and admiration, the lowly flatworm is capable of at least one skill that the average human is not: regeneration. If you cut a flatworm into two precise halves, each segment will grow into a new flatworm! Many amphibians also possess this special skill. For example, if a tiger salamander has its tail or limb removed, within a few months, a new limb has replaced it. Salamanders can even regenerate parts of their brains—a talent any Yalie would find useful.

The seeming prevalence of limb regeneration leads to the age-old question: if worms can do it, why can’t we?

Dr. Craig Crews, Yale Professor of Molecular, Cellular, and Developmental Biology, Chemistry, and Pharmacology, is perhaps more interested in this question than anyone else. Focusing on cell dedifferentiation and limb regeneration in amphibian models, the Crews lab has made ground-breaking discoveries by taking a unique molecular-level approach to examining an age-old question: why can some animals regenerate?

While many in the field prefer to focus on issues such as patterning, Crews approaches regeneration as an offshoot of developmental biology. Crews in particular studies limb regeneration in axolotls. Also known as Ambystoma mexicanum, the axolotl is a Mexican salamander popular among researchers for its remarkable regenerating capabilities. The salamander is capable of regenerating entire lost appendages in mere months and also easily accepts transplants from other individuals.

When an axolotl loses a limb, the wound is rapidly closed within twenty-four hours. Cells from the surrounding epidermis migrate and cover the stump. Over the course of the next few days this monolayer of keratinocytes starts to change, ultimately forming a structure known as the wound epidermis.

By the time the wound epidermis is completely formed, it overlies a mass of cells called the blastema. A blastema is a set of cells that has reverted to a stem cell-like state and is assembled when formerly fully differentiated cells (bone cells, muscle cells, blood vessels, and cartilage) “travel back in time” and dedifferentiate. The bulk of the regenerating process depends on the blastemal cells, which are capable of redifferentiating after they have grown to maturity to form the complex absent limb structure.

“Taking a fully committed cell and going backwards in the developmental timeline to make it more stem cell-like is not a normal event,” explained Crews, “That was my original question, how do you turn a cell backwards?”

The Wound Epidermis

The key to understanding the cell dedifferentiation patterns that are so critical to regeneration, Crews believed, was the wound epidermis.

Crews claims, “The question I’ve always been interested in is a very focused question, and it has to do with the wound epidermis. If you were to remove a piece of skin from an arm, it will heal…nothing happens. But if you amputate [the limb] in a transverse way, you get this unique [wound epidermis]. This wound epidermis is truly different from the epidermis that you get here.”

Crews found not only morphological but also gene expression differences between the wound epidermis and regular epidermis, suggesting that the wound epidermis supports and possibly even induces dedifferentiation and regeneration. Experimental evidence further hinted at the role of the wound epidermis in inducing cell dedifferentiation.

The limb does not regenerate if the wound epidermis is removed, showing that the contact between the epidermis layer and the mesenchymal layer is essential for regeneration. Crews is studying how the wound epidermis has the unique capacity to induce regeneration.

Crews hypothesized why a wound epidermis would form for a transverse amputation but not for a lateral wound. When a wound is inflicted, the body closes the wound by having cells from neighboring epidermis migrate to the open area. On a lateral wound, all of the cells that migrate to seal the opening are from the same “neighborhood.” When a limb is amputated however, cells from four different “neighborhoods” (dorsal, ventral, anterior, and posterior) migrate radially inwards towards each other, coming into contact with each other over the surface of the stump. Crews believes that the positional discontinuity of contact between skin cells from different areas around the limb circumference is the trigger for wound epidermis formation.

To test this idea, he performed an experiment that involved removing a cuff of skin from a salamander limb. The skin was then rolled out and cut into pie wedges with the tips of each pie wedge corresponding to different positions around the circumference of the limb. Then, the skin was removed from a different animal in a lateral fashion. The pie wedges were then arranged in a pie-like shape so that the tips of each pie wedge were in close proximity to the others. This arrangement both closed the wound and artificially brought skin cells from different parts of the amphibian limb together without amputation.

The result was a limb bud and an encouraging first discovery that inspired Crews to continue his work on limb regeneration. “It was a hypothesis I thought was testable, and we got this induced limb bud,” Crews stated. “What this tells us is that there’s nothing unique about the amputation, but it’s more about how this wound epidermis forms over the stump. That has now focused us on the wound epidermis in particular.”

It’s All in the Genes

The Crews lab is mostly interested in studying dedifferentiation, specifically how the blastemal cells form and how the wound epidermis influences this formation. The answer to these questions lies in the genetic code.

Now there are ways to coax stem cells to go back in time. Induced pluripotent stem cells (IPS) was done empirically with Japan,” Crews said, referring to Shinya Yamanaka’s 2006 successful transformation of adult mice somatic cells into stem cells. “But [limb regeneration] occurs naturally. And I want to know how nature solves this problem.”

The genes upon which regenerative capabilities are hinged seem to operate by a kind of dual-purpose switch. When the switch is “off,” the genes are repressed and cells perform their specialized functions normally. When the switch is turned “on” however, dedifferentiation is induced and the cells become blastema.

On a molecular level, the switches consist of chemical modifications to DNA—specifically to DNA in its wrapped, chromatin form. Certain regulatory proteins have the power to control the expression of these genes.

Crews hypothesized that the wound epidermis is actually inducing regeneration by sending a signal to the differentiated cells underneath it, possibly by secreting some kind of protein to the underlying stump. Only when these cells receive this signal can they become blastema.

Then what is the wound epidermis secreting? To determine this, Crews isolated RNA from a lateral wound and mRNA from the wound epidermis, labeled the two populations, and subjected the samples to microarray analysis. By mixing the two pools of RNA together and allowing them to hybridize to a gene chip, he was able to compare the two sequences and find differential gene expression. What he found was about 100 genes that are significantly more expressed in the wound epidermis than in the lateral epidermis—among them, genes that govern the secretion of proteins on the wound epidermis cell surface that prompt dedifferentiation when in immediate contact with the underlying stump cells.

Crews feels “that by first identifying which genes are uniquely expressed in the wound epidermis, we could now take those wound epidermis-specific genes and express them in the lateral epidermis—an epidermis that doesn’t have those genes. If we force those genes, can we get a regenerative response? Can we get a limb bud? …What is the minimal genetic code to induce regeneration?”

Like Shinya Yamanaka did in Kyoto University, Crews hopes to identify this “minimal genetic code” by systematically forcing the expression of wound epidermis-specific genes in the lateral epidermis and looking for molecular-level regenerative responses from the stump cells to gauge the effectiveness of a particular combination of genes

Crews seeks to determine what can be added to a cell to make it go backwards in time and dedifferentiate. A protein? A gene? A hormone?

In the Future

The ability to regenerate does not belong to axolotls alone. Asides from simple creatures like planarians, starfish, and sponges, there are many more animals that regenerate parts. Lizards and their tails. Deer and their antlers. Sharks and their teeth.

Even humans can regenerate—to a limited extent. We are capable of regenerating our fingertips, but only if they are allowed to heal on their own. There is debate as to whether human beings possess a latent capability to regenerate, but it is not impossible to think that it may someday be possible, by forcing the expression of some gene in our systems, to regenerate human and mammal limbs. Even partial regeneration could significantly impact quality of life—particularly for amputees, for whom physical attachment of prosthetics is difficult.

The potential implications for limb regeneration research are immense. Induced, controlled tissue regeneration would give humans the ability not only to heal wounds without scaring, but also to effectively combat tissue degenerative diseases like Parkinson’s Disease and arthritis. The day the results of this research change the medical landscape is still far off, but Professor Crews and his lab have brought us that much closer.

Further Reading:

  1. Campbell LJ and C.M.Crews (2008). Wound Epidermis Formation and Function in Urodele Amphibian Limb Regeneration. Cellular and Molecular Life Sciences,65:73-79
  2. Takahashi K and Yamanaka Shinya (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell – 25 August 2006 (Vol. 126, Issue 4, pp. 663-676)
  3. Alvarado A (2009). A Cellular View of Regeneration. Nature, Vol. 460 No. 7250, June 25, 2009.

About the Author: Linda Wang is a freshman prospective Molecular Biophysics & Biochemistry major in Jonathan Edwards College. She is the Copy Editor at Yale Scientific Magazine and a big fan of amphibians. Especially the baby axolotls in the Crews lab. She once tried to release captive toads into the wild, only to learn three hours later that fresh-water animals do not belong in the sea.

Acknowledgements: The author would like to thank Professor Crews for showing her what true passion for research looks like, as well as Leah Campbell for her time and dedication.