Classic Profile

In the fall of 2007, 2 years shy of her 100th birthday, Nobel laureate and National Academy of Sciences member Rita Levi-Montalcini yearned to start a new research project. Her idea stemmed from studies she had carried out more than half a century earlier, through which she unveiled and characterized the function of NGF. Time had taken its toll on her sight and hearing, but her keen observation skills and scientific insight were as strong as ever. To embark on this project, she solicited the help of her colleague Antonino Cattaneo, a neuroscientist at the European Brain Research Institute (EBRI) in Rome, which Levi-Montalcini founded in 2002. “Although she’s a person who looks forward, she still goes over and over what she did in the past and looks at it from different angles,” Cattaneo says. “She said, ‘When I did my experiments I showed that sympathetic and sensory neurons were dependent on NGF at relatively advanced stages of development. Yet, it is known in the literature that NGF and its receptors are expressed much earlier in development,’” Cattaneo continued. “She was well aware that this data was present in the literature, and during one of our discussions she remarked, ‘There is a question here. If the receptors are present much earlier in the embryo, is it possible that NGF, acting through these receptors, has some effects in early development that are different from the effects of NGF I had discovered years ago?’” To find out, Cattaneo assembled a group of scientists from his laboratory, each with different areas of expertise. Levi-Montalcini proposed that the group use the technique she first described in the 1960 PNAS Classic Article, “Destruction of the sympathetic ganglia in mammals by an antiserum to a nervegrowth protein” by Levi-Montalcini and Barbara Booker (1). In this work, LeviMontalcini uncovered convincing evidence of NGF’s function in vivo by injecting newborn animals with antibodies against NGF to eliminate its activity. This pioneering and creative use of antibodies to probe the function of a protein—a type of phenotypic knockout—revealed a nearly complete destruction of the animals’ sympathetic nerves, which regulate blood pressure, heart rate, and myriad other bodily functions. The finding not only led to a better understanding of the function of the peripheral nervous system but also solidified the conceptual basis for the now widely studied group of growthregulating chemical messengers known as growth factors. The work furthermore provided an experimental blueprint for subsequent studies of growth factors and other proteins by the use of antibodies to block protein function. Levi-Montalcini knew that Cattaneo’s group possessed the techniques and reagents to inject anti-NGF antibodies into the early chicken embryo but lacked experience working with chicken embryos. So she demonstrated how to do the injections, as early as possible in the living embryo, and during her daily visits to the institute to discuss the forthcoming data, taught the researchers what to look for under the microscope (Fig. 1). “Her enthusiasm was very, very contagious,” says Annalisa Manca, a postdoctoral fellow in Cattaneo’s laboratory and one of the researchers who worked on the project. “What was also impressive was that almost every day she was here in the Institute, in the lab, and every day she had a new idea, a new insight. She was very curious about new technologies, new techniques, and of everything we did. She wanted to come into the lab to see the experiments with the embryos using the microscope.” The team started out by scanning the embryos for signs of massive nerve cell death, an effect that Levi-Montalcini observed in the 1950s when cells are deprived of NGF. “This was something that was worrying me in the analysis, because it was not there, but it was something that Professor Levi-Montalcini was looking for,” says Simona Capsoni, a former EBRI postdoctoral fellow and now a neurobiology professor at Scuola Normale Superiore in Pisa. To her surprise, Capsoni observed localized cell death at the level of somites, but also noticed a more significant effect: the embryos injected with anti-NGF antibodies displayed an abnormal 3D shape within 48 hours of injection, which the team determined was caused by a defect in a crucial rotation process that the embryo undergoes during development (Fig. 2) (2). This rotation is the result of a coordinated wave of proliferation that seems to be regulated by NGF. “We were all surprised about these results,” Capsoni says. “We were not thinking about an effect on rotation.” “Rotation is actually important for the final fate and development of nervous system, and so it is not just a matter of geometry,” explains Francesca Paoletti, a postdoctoral fellow in Cattaneo’s laboratory and coauthor of the paper. “It’s really important for the