Viktor Hamburger of Washington University extended Harrison's work but chose to study the chick embryo because its nervous system, although more complex than that of an amphibian, lends itself better to experimental analysis: its nerve centers are more clearly delineated and their strong affinity for silver stain enables the experimenter to visually examine the nerve structures more easily. Hamburger grafted limb buds onto chick embryos at very early stages of development and observed how the modified peripheral field was innervated by sensory and sympathetic fibers. Unfortunately the responses were often so complex that they defied interpretation,
In 1948, in an effort to get more straightforward results, Elmer D. Bueker of Georgetown University modified Hamburger's experimental approach. He had the ingenious idea of replacing one limb bud of a chick embryo with a fragment of a bird or mammalian tumor. The tumor cells were all undifferentiated and hence provided a homogeneous peripheral field, in contrast to the cells of the normal limb bud, which were destined to differentiate into many types of tissue. Bueker's experiment was therefore expected to reveal how a homogeneous but fast-growing peripheral field would be innervated.
Of three different tumors implanted in the body wall of three-day-old chick embryos only one, a mouse tumor of connective-tissue cells called sarcoma 180, grew vigorously and was invaded by nerve fibers growing out from adjacent sensory ganglia. In embryos sacrificed after five days the sensory ganglia innervating the tumor were 33 percent larger than those innervating the normal limb bud on the opposite side of the embryo. At first these findings seemed to suggest that the size of a sensory ganglion depends on the size and rate of growth of its field of innervation. According to this hypothesis the rapidly growing tumor provided a more favorable peripheral field for the innervating sensory fibers than the slowly growing limb bud did.
A reexamination of Bueker's findings by our group at Washington University revealed new aspects of the phenomenon that obliged us to revise his conclusions. We found not only that the growth of the sensory ganglia innervating the sarcoma-180 tumors increased but also that the sympathetic ganglia increased enormously in volume, becoming five to six times larger than they were in control animals. This increase in size of the sympathetic ganglia was considerably greater than that exhibited by the sensory ganglia. Together with the sensory fibers the sympathetic fibers branched all over the peripheral field provided by the tumor but did not form any synapses with the tumor cells.
In addition, and indicating an even more striking departure from normality, the viscera of the embryos with the transplanted tumors were flooded with excessive numbers of sympathetic fibers long before the embryos of the control animals were even sparsely innervated. The sympathetic fibers forced their way into large and small veins, impeding and sometimes completely obstructing the flow of blood. These extraordinary effects suggested that the overgrowth of the sympathetic ganglia was more than simply a response to the rapidly growing peripheral field of innervation provided by the tumor, as Bueker had proposed. Rather it seemed the tumor was releasing some chemical factor that was in turn inducing the remarkable growth of the sympathetic ganglia and the exuberant branching of their nerve fibers.
This hypothesis was tested by transplanting sarcoma-180 tumors into the respiratory membranes of the chick egg, which are permeated with blood vessels from the embryo. The tumor and the developing chick embryo therefore shared the same blood supply, although they were not in direct contact. We found that when the tumor was transplanted into the respiratory membranes, it elicited the same growth-promoting effects on the sympathetic ganglia as it did when it was implanted in the embryo itself, providing convincing proof that the tumor was releasing a soluble factor that was carried in the bloodstream to the embryo.