Lessons learned from lizards and newts
by Bess Andrews
As a child, did you ever wonder how a newt or lizard regrew a new leg or tail when it lost one? And why humans couldn't? Newts and lizards can also regrow organs and eyes, and zebrafish can regrow their fins. According to Mark Keating, MD, senior associate in Cardiology and a Howard Hughes Medical Institute investigator, newts, lizards and zebrafish can regenerate virtually every tissue and organ, yet humans have very limited powers of regeneration. Humans can regenerate liver tissue, but have little ability to do so elsewhere in the body—presenting a major medical problem, but an interesting scientific question.
Keating started out in the field of medicine expecting to pursue the clinical side of things, working directly with patients. Before long, however, he found himself migrating toward basic science. "I almost immediately recognized how little we understood about nearly all diseases and how much of therapy was based on trial and error. So I decided that I wanted to be a physician who looked at the basic science behind the disease. I ultimately arrived at the question of regeneration. What are the triggers for regeneration? Why don't humans regenerate? And what can we do about it?"
Keating, who trained as a cardiologist, came to Children's in September 2000 from the University of Utah, where his lab was looking at the genetic basis of congenital and acquired heart disease. In 1993, he was trying to unravel the origin and development of supravalvular aortic stenosis, a vascular disease caused by an elastin defect. It was then that it occurred to him that the disease process was really a recurrent "injury-and-repair model," a common mechanism in many diseases. "In thinking about it more deeply, I realized that the real problem was that instead of regenerating when we are injured, humans scar," says Keating.
It appears that newt-like regeneration requires a special ability known as cellular de-differentiation. Cells begin as stem cells, which are the raw material or building blocks of all cells. Differentiated cells are those that have developed from their earliest stage as stem cells to a final cell type, for example, muscle, bone or cartilage. Also called "terminally differentiated," these cells remain in that state, doing their muscle, bone or cartilage job, until they are sloughed off or die through damage. De-differentiation refers to the process where cells, when confronted with the right molecular triggers, regress to the earlier stem cell stage, and then regrow into the various types of cells needed to recreate the missing tissue or organ.
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This process in amphibians has been observed for years. When a newt's leg is lost, a layer of cells forms to create the epidermis to cover the wound. Then, a blastema, or a mass of stem cells, arises. No one yet knows if these cells are pre-existing stem cells called in from elsewhere in the body or are created spontaneously. The blastema cells then begin to proliferate, and those nearest the wound surface begin to differentiate into the cell types needed to recreate the extremity—cartilage, nerve, blood vessel, fat and skin. Finally, the cells finish the progression and terminate, leaving a limb identical to the original and with no visible scarring.
Whether this gift of nature was lost to humans during evolution, or if it is a power newts and other amphibians somehow acquired, is not known. The bottom line is that humans scar instead of repair. When we lose a limb, epithelial cells cover the wound and then scar tissue grows over it. End of story. Or so people used to think.
In experiments conducted over the last four years, Keating demonstrated in vitro that when placed in the right molecular milieu, mouse skeletal muscle cells can be coaxed back into blastema, and then re-specialized into cells resembling bone, fat and cartilage. Keating, with his University of Utah colleagues, published this work in the December 22, 2000, issue of Cell. The paper challenged the reigning theory on the inability of terminally differentiated mammalian cells to reverse their development and revert to stem cells.
From previous in vitro experiments, Keating knew that the gene msx1 was able to prevent precursor muscle cells from progressing to their terminally differentiated state. This led Keating to question whether msx1 was one of the genes involved in de-differentiation of newt cells. With this clue in the back of his mind, Keating decided to see if msx1 might be a trigger of regeneration in mammalian cells. He began by stimulating mouse muscle cells to turn on the msx1 gene. As msx1 turned on, levels of certain specialized muscle proteins decreased. Some of the muscle cells then broke apart to create stem cells, which began to multiply. Keating's team then placed the stem cells in culture with growth factors conducive for specialization of other types of cells. Results showed that in the manipulated environment, the once-specialized muscle cells took on characteristics of bone, fat and cartilage.
Although this study was done with mouse skeletal muscle, Keating believes that there is no reason to expect it will not work with all types of mammalian cells. "Clearly there are molecular triggers that lead to the recruitment and/or creation of stem cells. And because we know that humans have some very limited regenerative capacity, it is likely that there is nothing structurally in humans that will keep them from regenerating," says Keating.
He speculates that there will be hundreds to thousands of genes involved in complex regeneration. To find the genes in humans, the Keating lab is starting with zebrafish mutants that have been bred to not regenerate. The zebrafish is a valuable model since its genome is markedly similar to that of humans, and a great scientific push to sequence the zebrafish genome is now under way. By sifting through the tens of thousands of genes in the mutant genotype and comparing them to the normal zebrafish genome, they hope to identify the genes involved in regeneration. Once they are known, scientists will be able to study the correlating region of the human genome for a similar gene.
According to Keating, the time when physicians will be able to call into action all the molecular mechanisms needed to trigger regeneration in their human patients is still in the far distant—but foreseeable—future. "The idea that a human will someday regrow a limb is pretty far out, but that's the essence of research’Äîto be at the cutting edge," says Keating.
