From Neuroblastoma to Homeobox Genes, 1976-1992
With the success of his neuroblastoma research, Nirenberg became firmly established as a leader in the field of neurobiology during the 1970s and 1980s. He participated in major international conferences and symposia and received countless requests for advice and access to his patented cell lines from researchers at prominent universities worldwide. Nirenberg also received numerous letters from cancer patients, medical schools, and hospitals, suggesting that his work became a symbol of widespread hope that the disease could be fought on the frontlines of science. In addition to awards of merit from the National Institutes of Health and honorary degrees in science from various universities, Nirenberg also gained recognition for his contributions to cancer research and received honorary doctorates from schools of public health.
Nirenberg's research using neuroblastoma and embryonic cells brought the promise of many biomedical applications. Children's hospitals, for example, used Nirenberg's tissue cultures to study neuromuscular connections, hormonal regulation, and neuronal growth. The cultures of identical cloned cells provided an experimental alternative to the complex mixture of cell types found in the normal nervous system. The cell lines were sensitive to environmental conditions so that researchers could study the impact of various factors on cell development. As early as 1975, Nirenberg and his team used these cells to develop a technique for diagnosing and analyzing neuromuscular disorders by defining what conditions were necessary for the transmission of information from cell to cell.
In addition to neuroblastoma research, Nirenberg established a project to study the formation of neural synapses in the retinas of chickens. In the 1976 article, "Localization of Acetylcholine Receptors during Synaptogenesis in Retina," Nirenberg found that retina cells could be dissociated (separated), then reassociated, and still produce synapses. Normal neurons are nondividing and cannot produce synapses after being dissociated. Like neuroblastoma cells, retina cells provided an important model for explaining the process of synapse formation. Working with chick retina also offered a chance to apply in a new medium the knowledge he gained from working with neuroblastoma cells.
As Nirenberg's research on neuroblastoma and chick retina developed in the mid-1970s, he became interested in a different kind of problem. Intrigued by a theory developed in the 1960s by the grandfather of neurobiology, Roger Sperry, Nirenberg embarked on a new project at the Biochemical Genetics laboratory. Sperry predicted that scientists could determine the molecular basis for the location of each cell in the retina. In order to transmit a cohesive picture of the outside world it seemed clear that the cells making up the neural pathways were somehow directed to end up in a specific location. Nirenberg wanted to explain the molecular basis of this precise "molecular address" for retinal cells.
Using chick retina, Nirenberg developed a way to test Sperry's prediction that there is a molecular topographic map that exists in the retina. Nirenberg used genetically identical proteins that were cloned in the laboratory, called monoclonal antibodies, that bind to foreign molecules to fight off infection. By exposing monoclonal antibodies to antigens from different parts of the retina, he showed that the antibody recognized a specific antigen molecule distributed in a unique pattern across the retina. This validated Sperry's prediction by demonstrating that proteins are concentrated in specific areas. These proteins are instrumental in directing cellular development in particular locations with great precision. Nirenberg published his findings in a 1981 article in the Proceedings of the National Academy of Sciences, "A Topographic Gradient of Molecules in Retina Can be Used to Identify Neuron Position." He subsequently purified the protein molecule that the antibody recognized and published the results in the 1986 essay, "Purification of a Membrane Protein Distributed in a Topographic Gradient in Chicken Retina."
While this represented an important advance in neurobiology, Nirenberg's next major research success would stem from failure. As Nirenberg explained, "we failed to clone DNA for this protein [the antigen that he purified from chick retina] and that was an important question because if we could have cloned it we could have identified the amino acid sequence of the protein. It would have given us a tool to use, a very important tool, for further studies. For some reason we were not successful in cloning it." In 1987, researchers discovered proteins in the embryo of Drosophila melanogaster, the common fruit fly, which were similar in distribution to the proteins Nirenberg mapped in chick retina. As one of the most valuable and studied organisms in the history of biological research, Drosophila presented Nirenberg with an opportunity to tie up the loose ends of his chick retina work. Nirenberg remembered, "I thought that to really understand this problem you have to go to a simpler system where you have genetics you can use. You can use genetics as a tool. Drosophila has been studied for a hundred years almost, ninety years, and there is a tremendous amount of genetic information that is known and wonderful genetic tools that can be used with Drosophila. And that's the reason I switched."
Annual reports from the Laboratory of Biochemical Genetics show that even before Nirenberg began his Drosophila work the focus of his research had gradually shifted in the 1980s toward genetic explanation for nervous system development. His work with neuroblastoma and chick retina capitalized on new laboratory tools made available by the accelerated pace of genetic technologies. In 1983, for example, Kary B. Mullis, a scientist with the Cetus Corporation in California, developed a technique that revolutionized the work of molecular biologists. Mullis developed a procedure for amplifying DNA, the polymerase chain reaction (PCR), which made it possible to read the sequence of virtually any DNA fragment. PCR has been used to detect DNA sequences, diagnose genetic diseases, carry out DNA fingerprinting, detect bacteria and viruses, and research human evolution. It even has been used to clone the DNA of an Egyptian mummy! By 1985, Nirenberg's Biochemical Genetics laboratory utilized these new techniques to develop massive collections of DNA nucleotide sequences known as DNA libraries. This allowed Nirenberg to compare the genetic composition of human cells to those of other animals. His laboratory notebooks show that this information also allowed him to begin working out the relationship between gene sequences, viruses, and neurological disease.
Nirenberg's move toward genetic explanations for nervous system function and development coincided with a growing atmosphere of excitement that pervaded the biomedical research community in the 1980s. A series of experiments demonstrated that cancer, whatever its ultimate cause, was the result of activating a family of genes called oncogenes. These genes, involved in the control of cell division, could cause cancer when modified or "over-expressed." Early reports suggested that over-expressed oncogenes were "switched on" by biological or environmental forces, causing them to synthesize more RNA from a sequence of DNA than they normally would. This hypothesis paralleled conclusions drawn from Nirenberg's neurobiology research. Key articles such as "Synapse Formation by Neuroblastoma Hybrid Cells," published in 1983, showed that biological and environmental factors can both influence gene expression in the nervous system. Cancer researchers also believed oncogenes might control the action of other genes in a cascade until the cancer was finally initiated. This closely reflected the role of a new group of genes that caught Nirenberg's attention. Homeobox genes, discovered by Walter Gehring of the University of Basel in 1983, influence the expression of other genes important in physical development. As gene regulators, they recognize the sequences in DNA that turn genes on or off. For Nirenberg, research on homeobox genes offered a forum for answering one of the questions remaining in his work on nervous system growth: what was the relationship between genes themselves and the development of the nervous system as a whole?
After reading a paper by Michael Levine of Oxford University in 1987, Nirenberg found the perfect opportunity to bring his experience in genetics and neurobiology together. Since homeobox genes influence the process by which hereditary information is converted into physical characteristics during development, Nirenberg recognized that understanding their function could provide new avenues for research. He recalled that, "Levine found a homeobox protein that was distributed quite remarkably in some neurons in the developing embryo and not in other neurons... [Homeobox genes are] an important class of genes and to find them quite specifically distributed in specific sets of neurons was quite a remarkable observation. At that time, there were seventeen homeobox genes that were known, that had been found in Drosophila, and it was a burgeoning field of study." The relationship between homeobox genes and neural development presented an ideal opportunity for new discoveries.
Nirenberg would have to face the same risks involved with his earlier transition from genetics to neurobiology. Once again he had to branch out into unfamiliar territory. Nirenberg explained, "I had never worked with Drosophila before, but when Yongsok Kim came to my lab as a postdoctoral fellow immediately after he got his Ph.D. in Korea, I suggested to him that we look for new homeobox genes in Drosophila." By comparing the base sequences of genes with the seventeen homeobox genes already known, Kim soon discovered four new homeobox genes which he named NK-1, NK-2, NK-3, and NK-4.
In the annual report of the Laboratory of Biochemical Genetics for 1987-88, Nirenberg revealed the importance of this work. For Nirenberg, the genes provided an "experimental system" that could be used to define the relationship between specific genes and the physical development of an organism--the hope being that lessons learned from Drosophila could be applied to humans. Discovering the sequence of nucleotides in DNA allowed for the possibility of genetic therapy for diseases. A handwritten draft for a research project report from 1992 reveals that studies on the NK-2 homeobox genes enabled his team "to predict with a high degree of certainty" the relationship between genetic instructions and the development of part of the central nervous system of Drosophila. Since one of the "major goals in neurobiology" was to "understand the developmental program for the assembly of the nervous system," identifying the homeobox genes and defining their developmental role was an important advance. Nirenberg predicted that a "similar but slightly modified strategy" could be used to explain the assembly of the human nervous system.
Following the mapping of the entire genetic sequence of Drosophila in 2000, Roger Hoskins, of the Berkeley Drosophila Genome Project, confirmed Nirenberg's optimism by revealing that in a set of 289 human genes implicated in diseases, 177 are closely similar to fruit fly genes. Knowledge of homeobox genes brings the promise of understanding their role in development and may eventually prove to be beneficial in combating cancers, neurological diseases, and metabolic and immune-system disorders. Until his death in 2010, Dr. Nirenberg continued this project by using advanced digital scanning technology to study the genetic development of neural networks in the brains of Drosophila embryos.