Spark of Life
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Previously, developmental biologists thought that left and right must be determined later in development, when organs are being formed. But a postdoc in Tabin's lab discovered that a gene known as Sonic hedgehog (so called because it gives fruit fly embryos a spiky appearance) was expressed in a tiny spot only on the left side very early on. Levin volunteered to look into the phenomenon. Indeed, he found a number of consistently left-sided and right-sided genes and showed that the cascade in which they progressively turned each other on and off ultimately determined the position of the heart, stomach, and other internal organs.
In his postdoctoral work with Mark Mercola, in the cell biology department of Harvard Medical School, Levin traced the process back to an even earlier stage of development, discovering that the genes themselves know which side to express themselves on because of flows of electric charge across the embryo. Cells were known to have "gap junctions," little submarine hatches that line up to allow small molecules to pass from one cell to another. What Levin found is that these gap junctions line up in a continuous path across the entire early embryo. They form a conveyor belt that passes signals all the way across. Interrupt this long-range communication path—as Levin did—and organs grow on either side of the body at random.
Levin formed a hypothesis: this path along which the left and right sides communicated was the equivalent of an open electrical circuit, pointing the way to the right side of the animal as surely as a neon arrow. Remarkably, this process occurred when the embryo had just sixteen cells—long before the cascade of genes that ultimately directs the organs of the developing animal, and much sooner than biologists had previously thought possible.
Like Susan Ernst at Tufts, Levin's advisers at Harvard were impressed by his willingness to follow a few vague clues down the road of a particularly risky hypothesis. "He is one of the most creative people I have ever had in my lab," says Tabin. "He enjoys working on the frontiers of science." But, Tabin adds, the frontiers are inherently risky. Levin "is more likely than most people at his stage to do things that are fundamentally important and have a real breakthrough. He is also someone who could slave away at a problem that turns out to be less significant than he had hoped." Lucky for Levin, his experiments have mostly been the former—though he admits there have been blind alleys that haven't led to publication.
After his success in explaining the electrical flows behind left-right patterning, Levin went in for a closer look at the battery itself. He began with a simple question: If the movement of charges could determine the shape and position of organs during early development, what would happen if you artificially changed the pattern of these electrical signals after injury? This round of experiments used the African frog Xenopus, a muck-dwelling amphibian that, like most frogs, can regenerate its tail and legs while it's a tadpole but not as an adult frog.
On a tour of his lab, Levin ushers me into a swampy-smelling room he calls the "Frog Farm." Inside, a couple of hundred frogs laze in tanks filled with greenish water. "They probably just got fed," Levin says, pointing out a shelf full of granular frog chow. "They are just chilling out." A room next door is piled with Tupperware containers holding the frog's progeny—transparent tadpoles that are the real focus of the experiments.
For years, Levin and his fellow researchers have been inserting RNA into cells of the developing frog embryo. The RNA produces proteins called ion channels, or similar structures known as proton pumps, which flick ions across the cell membrane, creating an excess of positive or negative ions in the cell. This dramatically alters the cells' electrical properties. Cutting off a tadpole's tail, Levin and his fellow researchers found that they could stop it from growing back if they inserted RNA that produced a mutant protein, one that prevented a particular enzyme— V-ATPase—from pumping hydrogen ions.
In a cluster of cells that make up an early frog embryo (top left), signals travel across membranes to control the embryo's development (here, blue denotes areas of higher voltage; red, lower voltage; green, medium voltage). By inducing similar signals in the severed tail of a developing frog (middle left), Levin stimulated the tail to regrow. The frog at right grew extra limbs through the same process.
More impressively, Levin's lab also showed that by inserting a proton pump into the cells surrounding a wound, they could spark regeneration later in development, past the stage where tadpoles can normally sprout new appendages. This was the first demonstration that muscle, spinal cord, and blood vessels could be completely regenerated—arranged in the right pattern in a normal appendage—by the molecular tweaking of bioelectric properties.
Not stopping there, the researchers experimented with other ion transporters, and hit pay dirt with a particular potassium ion channel that allowed the tadpole to grow extra legs or eyes. Outside Michael Levin's sunny office near the Medford/Somerville campus is a large photo of a six-legged frog, just one of the creatures the lab has created by inserting these channels in multiple cells of the developing organism. Determining exactly which transporters cause the development of which organs will require further research. "Right now we don't have a good handle on how we tell it to grow an eye rather than a leg or a tail," Levin says. "But ion flows and electrical properties seem to induce different shape-changing events."
Frogs aren't the only animals that Levin has successfully manipulated. In another room in his lab, he opens a refrigerator to reveal more stacks of Tupperware, which seem to contain grains of dirty rice, but are actually full of flatworms called planaria, wriggling in water. "Poland Spring, in fact," he deadpans. "I tried to get money out of Poland Spring because it turns out the flatworms refuse to live in any other kind of spring water." These worms can grow back their heads and tails after being cut into pieces, but how they "know" which structure—head or tail—to grow at each end has been a mystery. As with the frog embryos, Levin was able to show that regeneration depends on physiological signals moving long distances to tell the wound what to do. Once the tail is cut off, signals flow through gap junctions from the wound to the head to ensure that it is still there. Block that flow, and the worm will grow another head. To grow a tail, you block another ion transporter. Just by making these electrical changes, Levin's group has been able to grow worms with tails on both ends, heads on both ends, or even four-headed worms from multiple cuts. (continued)
MICHAEL BLANDING is an award-winning magazine writer whose work has appeared in The Nation, The New Republic, Boston Magazine, and The Boston Globe Magazine. This is his second cover story for Tufts Magazine.
Top photo by John Soares. Additional photos by Alonso Nichols, University Photography
This story originally appeared in the Spring 2009 edition of Tufts Magazine. It ran online on July 20, 2009.