In history texts, Columbus, de León, and Magellan are European explorers of old. In Samuel Pfaff's lab, however, they are the names of mutant mice that are providing critical insights into how motor axons—the slender projections from nerve cells—travel from the brain to their targets throughout the body.
"We were looking for genes that were involved in neuron development and axon navigation," Pfaff says. "So we chose to give the mice shorthand names based on people who were known for heroic navigations."
Pfaff is using these mice to identify and understand the molecular and genetic components controlling the development of motor neurons, which are responsible for movement. The research is an outgrowth of his postdoctoral work in HHMI investigator Thomas Jessell's lab at Columbia University.
"I became interested in science because I was fascinated with development," Pfaff says. He grew up in the shadow of the Mayo Clinic in Minnesota, where as a teenager he had the opportunity to volunteer in a lab and study peripheral neuropathy. But developmental biology became his passion at Carleton College, when he watched a fertilized frog develop into a tadpole during an undergraduate laboratory experience. "I spent 24 hours in the lab watching this embryo become a swimming tadpole and wondering how its different cell types were specified," he laughs. "It really was a eureka moment for my career."
Pfaff entered graduate school at the University of California, Berkeley eager to learn more about molecular biology in relation to development. His adviser, Peter Duesberg, was studying how cells transform from normal to cancerous. "I thought it would be interesting to study how cancer subverts normal development," he says.
After graduate school, Pfaff went on for a postdoctoral fellowship studying frog development in the lab of William Taylor at Vanderbilt University before he finally found his way to studying the development of the nervous system during a second postdoc in Jessell's lab. "I was so impressed with Tom, and his lab was so interesting and exciting that if he had been studying the development of the endocrine system, I'd probably be working on that right now," he says. "Some of the hardest and most exciting questions about development come from the nervous system, so it ended up being a great fit." Those big questions include the genetic and biochemical processes that connect the nervous system's cells or cause them to specialize as a particular type of nerve cell.
While in Jessell's lab, Pfaff began to work on the question of how the cells that ultimately become motor neurons choose from among many possible developmental fates. When an egg is fertilized and grows into an embryo, the cells formed very early in development can become any type of cell in the organism: skin cells, muscle cells, sensory neurons, or motor neurons. Pfaff focused on studying proteins called transcription factors that turn genes on or off. These genetic "switches" help shape the fate of growing cells. While in Jessell's lab, Pfaff discovered a group of proteins, called LIM transcription factors, that direct cells to become motor neurons.
At Columbia and in his own lab at the Salk Institute, Pfaff showed that the LIM transcription factors act in combination. "We were asking whether the LIM factors combine to target the same genes or entirely new and different genes," Pfaff says. "It turns out they target entirely new genes when they are in combination. It's a problem we're still pecking away at, but we are moving on to bigger questions."
In Pfaff's lab, the Magellan mouse may help answer one of those bigger questions. Pfaff developed a method of genetically engineering mice so that all of their motor neurons are tagged with green fluorescent protein. This fluorescence allows him to visually identify mutants that have errors in motor neuron development and function. The Magellan mouse has an error in a gene called Phr1 that is so severe that the axons of motor neurons wander and grow in all directions. "It is absolutely clear that a byproduct of losing Phr1 activity is that motor neurons cannot find their way to their targets," Pfaff says, noting that one of the activities of the Phr1 protein appears to be the degradation of other cellular proteins.
Pfaff's group is also exploring the DNA changes that can cause a cell to become a motor neuron. As an embryonic cell goes down a specific developmental pathway, its DNA is marked with small chemical modifications that turn off certain genes and developmental avenues. This chemical marking is known as epigenetic regulation, an important mechanism for controlling the activation or repression of genes. "We can add transcription factors to embryonic stems cells and turn them into motor neurons," Pfaff says. "But we can't convert every type of cell into a motor neuron. We are hoping to discover a way to relax the constraints on the cell so that we turn a skin cell into, for example, a motor neuron."
As an HHMI investigator, Pfaff will step out of his comfort zone to explore a neuronal network in mice that controls rhythmic movements of the hind limbs. The network, called a central pattern generator (CPG), resides in the lower spinal cord and controls gait. Like other CPGs, such as those responsible for breathing, it operates rhythmically—oscillating between right and left—and automatically, without input from the brain. Scientists have known about the CPG related to limb movement for nearly a century, but the identity and function of the cells in the circuit and the developmental mechanisms responsible for their wiring are poorly understood.
Undertaking something as complex as studying the CPG forced Pfaff to start out slowly and assemble a lab group with many areas of expertise, including imaging, physiology, and molecular genetics. "Some members of the group have inherent tinkering skills that allow them to solve technical problems as they arise," he says, noting the lab often ends up looking like an auto shop, with all of the sophisticated electronics and microscopes its members use to monitor cell physiology. "It's really interesting to see [the team] struggle to communicate with one another and see them come together."
The work will provide fundamental insights into how vertebrate locomotion is achieved, which could one day translate into new treatments for people with spinal cord injuries. "I would consider myself a very fortunate scientist if I could make discoveries that would improve people's lives," Pfaff says. "I really see that as such an important part of our work as scientists. If we lose sight of that, the public may lose interest in our work."