Neural development

I first became aware of Viktor Hamburger at the seminar series that Max Cowan held every Saturday morning. Most of the hard-core neuroscientists at Washington University from both the medical school and the undergraduate campus made a point of attending these sessions, and Hamburger (Figure 5.1) was a regular. Initially, he was simply the old guy from the Biology Department who usually sat toward the back of the room. (He was then 73.) When he asked a question or made a comment in precise but heavily accented English, however, it was always authoritative and smart. Eventually, I asked around about him. After I figured out that he was a major figure in the field of neural development and not the neuroanatomist I had confused him with, and he figured out that I was a new hire in Hunt's department working on a problem he knew something about, we struck up an acquaintance. It grew into an important scientific and personal friendship that lasted until his death in 2001, more than 27 years later.

Hamburger had been a faculty member in the Biology Department across the park from the medical school since 1935 and was an enormously knowledgeable and accomplished neuroscientist, in the same league with Hodgkin, Huxley, Katz, and Kuffler. But he was working in a different area that did not overlap with research on neural signaling. Although it soon became clear that his work was pertinent to the research that I was starting, Hamburger's fundamental studies of how the nervous system develops had not been part of the traditional neurophysiology and neuroanatomy that I had been exposed to at Harvard or University College.

Figure 5.1 Viktor Hamburger in his office in the Biology Department at Washington University in the late 1970s.

Figure 5.1 Viktor Hamburger in his office in the Biology Department at Washington University in the late 1970s.

Hamburger was born in 1900 and grew up in a rural town in Silesia, then a province of Germany (and now part of Poland). He attended the universities in Breslau, Heidelberg, and Munich before getting his Ph.D. under Hans Spemann at the University of Freiburg in 1925 (moving around academically was not usual in that era). Spemann, whose work was equally unknown to me, came from a pedigree of German embryologists and zoologists as distinguished as the British physiologists and émigrés such as Katz and Kuffler who had joined them in the 1930s. Spemann had won a Nobel Prize in 1935 for his studies of embryonic development and had trained a generation of leading embryologists, Hamburger preeminent among them. After a few years as a junior faculty member in Germany, Hamburger had come to the United States in 1932 under the auspices of a Rockefeller fellowship (the same fellowship that had sponsored Katz's emigration to England a few years later) for what was to have been a year working with Frank Lillie, an American embryologist who was studying the development of chicks at the University of Chicago. During that year, Hamburger received a letter dismissing him from his position at the University of Freiburg because he was Jewish. As a result, he stayed in Chicago for three years until he was hired at Washington University. In St. Louis, he had continued turning out a series of key studies on the development of the nervous system of chicks, a preparation he continued until he quit doing experimental work more than 50 years later.

As I gained some familiarity with Hamburger's work, it dawned on me that his expertise was especially relevant to what I was doing in the autonomic system, or at least to the part that involved looking at the failure of synaptic maintenance when neurons were cut off from their peripheral targets (see Chapter 4). Hamburger had used the embryonic transplantation techniques he learned from Spemann to either add or remove limb buds (the forerunners of the chicken's wings and legs) in embryonic chicks, assessing what happened to the spinal neurons that would have innervated the ablated limbs, or that innervated the extra peripheral targets ( Figure 5.2). By making a window in the egg, he could carry out these operations in embryos at nearly any stage of development with the skillful use of fine glass needles. Hamburger found that when he examined the spinal cord microscopically after the chicks had hatched, the number of corresponding motor neurons in the spinal cord was much diminished if the progenitor of the hind limb had been amputated at an early embryonic stage ( Figure 5.2A). Conversely, if he implanted an extra limb bud, the hatched chicks had more motor neurons than normal in the related part of the spinal cord ( Figure 5.2B). Evidently the amount of peripheral target tissue the axons of spinal neurons encountered when they grew out into the limb bud somehow regulated the number of developing motor neurons in the spinal cord. This work begun in the 1930s established the phenomenon of target-dependent neuronal death or survival. By the 1970s, neuronal death regulated in this way during development was known to be a general phenomenon in the peripheral nervous system and in some parts of the central nervous system.

Figure 5.2 Hamburger's experiments showing how the presence or absence of peripheral targets affects the number of related motor neurons in the developing spinal cord of chicks. A, B) Amputation of the limb bud in an early embryo depletes motor neurons in the relevant region of the cord examined microscopically after the chick has hatched. C, D) Conversely, implanting an extra limb bud augments the amount of muscle and other tissue in the periphery, increasing the number of related spinal motor neurons. (After Purves, Augustine, et al., 2008)

Figure 5.2 Hamburger's experiments showing how the presence or absence of peripheral targets affects the number of related motor neurons in the developing spinal cord of chicks. A, B) Amputation of the limb bud in an early embryo depletes motor neurons in the relevant region of the cord examined microscopically after the chick has hatched. C, D) Conversely, implanting an extra limb bud augments the amount of muscle and other tissue in the periphery, increasing the number of related spinal motor neurons. (After Purves, Augustine, et al., 2008)

Hamburger's early results implied the presence of some agent in the targets of the axons growing into the limb bud that instructed the axons and their parent neurons about how much tissue existed and the degree to which they had been successful in making contact with it. A corollary was that the developing neurons in the spinal cord were competing for acquisition of the postulated material, dying off if they didn't get enough of this trophic agent ( trophic refers to nourishment, and this phrase described what was evidently happening). As in most science, recognition of these implications occurred more slowly than I am suggesting here: Not until the 1950s, when much more work had been done, was the picture as clear as I am making it seem. But by the time I met Hamburger, these discoveries had long since been accepted. Although nobody in my Department of Physiology and Biophysics was very interested in any of this, Cowan was thoroughly familiar with Hamburger's work and had based many of his own studies of the developing chick brain on Hamburger's work.

Understanding how trophic interactions regulate neuronal numbers in early development had been greatly advanced by a collaboration that Hamburger began with Rita Levi-Montalcini in the late 1940s. Their work together lasted for about eight years and led to the discovery of nerve growth factor, a trophic molecule derived from smooth muscle that is the peripheral "nourishing" agent for at least two types of neurons, one of which was the nerve cell type in the ganglia of the sympathetic nervous system I had been working on. Nerve growth factor has served as a paradigm for the interaction between nerve cells and their targets ever since, and it remains the central example of trophic interactions in neurobiology (ironically, the factor that mediates the trophic effects of skeletal muscle on the spinal neurons shown in Figure 5.2 has, despite much effort, never been identified). Among other reasons for the intense ongoing interest in trophic factors is the possibility that neurological diseases that entail neuronal death (such as Alzheimer's disease and amyotrophic lateral sclerosis) are disorders of trophic interactions, and that better understanding this aspect of neurobiology could provide useful treatments. Many millions of research and biotech dollars have been invested in this idea during the last 30 or 40 years, although so far without clinical success. And although other growth factors are clearly important in brain development and function, that story has turned out much more complex.

The collaboration began in 1946 when Hamburger wrote to Levi-Montalcini in Turin to ask her if she would be interested in working in his lab for a year. The invitation made good sense because Levi-Montalcini had also been studying cell death and the support of developing neurons by peripheral targets. She accepted Hamburger's invitation and remained in the Department of Biology at Washington University for more than 25 years, a period that she described as the "happiest and most productive of my life." She returned to Rome in 1974 to continue her research there, but was a frequent visitor to Washington University, where she retained a part-time appointment. By any criterion, Levi-Montalcini was (and still is, at the age of 100) a remarkable scientist and a heroic figure. When she graduated from medical school in Turin in 1936, she had intended to pursue a clinical career in neurology and psychiatry, but that plan had to be put aside in 1938 by Mussolini's prohibition of "non-Aryans" from academic positions. Although Levi-Montalcini could have emigrated, she chose to remain in Italy, carrying out research in a small laboratory she set up in her home in collaboration with neuroanatomist Guiseppe Levi, her professor and mentor when she was a medical student (they were not related). Despite these conditions, she and Levi, who had been dismissed from his post because he was Jewish, managed to turn out several important papers during the war, stimulated in part by Hamburger's earlier studies on cell death. Hamburger's admiration of this work led to the invitation he tendered.

Hamburger and Levi-Montalcini's discovery of nerve growth factor and the subsequent isolation of the molecule by Levi-Montalcini and biochemist Stanley Cohen introduced a whole new perspective about how nerve cells interact with each other and with non-neural targets. The long path to this discovery began with one of Hamburger's students in the 1940s, a little-known figure in the history of neuroscience named Elmer Bueker. As illustrated in Figure 5.2B, one of the issues that had interested Hamburger was augmentation of the periphery as a means of salvaging developing neurons that would otherwise die during the normal course of development. Shortly after getting his Ph.D. with Hamburger, Bueker (who had taken a position at Georgetown University) had the unusual idea of implanting tumor tissue into a chick limb bud as a means of augmenting the periphery in a simpler and more dramatic way. Although the tumor that worked best had little or no effect on the neurons in the spinal cord, he noted that the sensory and autonomic ganglia (see Figure 4.1) were obviously enlarged on the side of the chick embryos in which the tumor was implanted. Bueker showed this result to Hamburger and Levi-Montalcini in 1948. They quickly appreciated its potential significance, and although Bueker went on to an unremarkable career as an anatomist at Georgetown and then New York University, Hamburger and Levi-Montalcini began to pursue his observation with a vengeance.

Their enthusiasm was fueled by the possibility of extracting and ultimately identifying the active agent from Bueker's tumor tissue. While spending a few months at the Carlos Chagas Institute in Brazil in a lab that specialized in tissue culture, Levi-Montalcini devised a way of assaying the presumptive agent in the tumor by measuring the outgrowth of nerve cell processes (axons and dendrites) from embryonic chick ganglia placed in tissue culture medium laced with tumor extract (Figure 5.3). This exuberant outgrowth of neuronal processes had caused the gross enlargement of the ganglia Bueker had first observed. Stanley Cohen, then a postdoctoral fellow in the Department of Biochemistry at the school of medicine, was invited to join the Hamburger lab to help solve the problem. Although they initially worked together in the early 1950s, Cohen and Levi-Montalcini continued without Hamburger, using this assay during the mid-1950s to isolate and eventually identify what they called nerve growth stimulating factor. (The molecule was eventually sequenced by another group at Washington University in 1971 and for decades has simply been called nerve growth factor, or NGF.)

Figure 5.3 The bioassay that enabled identification of nerve growth factor. A) A chick ganglion grown in tissue culture for several days without nerve growth factor. B) Another ganglion grown for several days with the factor, showing the extraordinary outgrowth of nerve cell processes stimulated by this agent. (After Purves and Lichtman, 1985)

Figure 5.3 The bioassay that enabled identification of nerve growth factor. A) A chick ganglion grown in tissue culture for several days without nerve growth factor. B) Another ganglion grown for several days with the factor, showing the extraordinary outgrowth of nerve cell processes stimulated by this agent. (After Purves and Lichtman, 1985)

Despite its importance in understanding how neural circuitry is normally established and maintained (Levi-Montalcini and Cohen went on to win the Nobel Prize in Physiology or Medicine for this work in 1986), the larger community of neuroscientists did not immediately appreciate this work. I first encountered Levi-Montalcini in 1970 when she visited Kuffler's department at Harvard to give a lunchtime seminar. She talked about studies she had been carrying out on the effects of nerve growth factor on cockroach neurons, a tangent she undertook for reasons I don't remember. When she continued speaking well beyond the allotted hour and was only halfway through a second carousel of slides, Kuffler surreptitiously motioned his lab technician, who was running the projector, to advance the carousel. The talk finished promptly, apparently without Levi-Montalcini noticing the elision of 20 or so remaining slides. The talk didn't seem to make much of an impression on anyone, least of all me. She didn't mention the importance of Hamburger's work in her exposition, explaining in part why he was unknown to me when I got to St. Louis a few years later.

Although Hamburger described her as having been "mousey" when she arrived in St. Louis in 1947, by the time I came in 1973, Levi-Montalcini was a distinctly regal figure who contrasted sharply with Hamburger's description of her 25 years earlier (Figure 5.4). (Having been long divorced, Hamburger was a man who noticed such things, and he had devoted girlfriends until he outlived them all well into his nineties.) Although Hamburger had opted out of the effort to isolate and identify nerve growth factor in the 1950s when it became clear that the enterprise had become largely biochemical, the Nobel Committee unfairly excluded him from the award presented to Cohen and Levi-Montalcini in 1986. Levi-Montalcini was largely responsible for the exclusion. The ambition and determination that had enabled her to pursue research during the war and accomplish so much in the face of long odds also led her to politic incessantly for recognition as a preeminent figure in neuroscience, which she certainly was. In this quest, she diminished Hamburger's importance in their collaboration, even after she had triumphed in Stockholm. After the prize had been awarded to her and Cohen in 1986, my colleague Josh Sanes and I wrote an article praising their accomplishment, but pointing out that Hamburger had been treated shabbily because his earlier work, his invitation to Levi-Montalcini, the results of his student Bueker, and Hamburger's collaboration through the early 1950s had obviously been critical to their success. As a matter of routine, the journal in which the article was to appear had shown the proof to Levi-Montalcini to check the facts. The next day, I was awakened by an angry phone call from Levi-Montalcini in Rome complaining that the article gave Hamburger credit that he did not deserve and asked us to revise it. Sanes and I did not change the article, but the call made her conflicted feelings abundantly clear. Hamburger never understood this behavior toward him, and although they continued a polite and superficially cordial relationship, he never forgave her.

Was this article helpful?

0 0

Post a comment