Cell death in the development of the lateral motor column of the chick embryo.

Cell counts were made in the lumbar lateral motor column (l.m.c.) of chick embryos of 5.5, 6, 7, 8, 9, 12, 18 days of incubation and five days posthatching (n equal 68). Only nuclei with nucleoli were counted and corrections were made for double counting (Abercrombie, '46). The population attains a peak value of over 20,000 cells (corrected figure: over 17,000) at 5.5-6.5 days equal stages 28 and 29 (Hamburger and Hamilton, '51). The l.m.c. loses between 7,000 and 8,000 cells between days 6.5 and 9.5, (between stages 29 and 36). In other words, 60% of the population survive. A plateau of approximately 12,300 cells (corrected figure: 10,300) is maintained through five days posthatching. Massive cell degeneration was observed in 7- and 8-day embryos. Counts of distinctly pyknotic cells indicate that at least 5-6% of the total population is in the process of degeneration at any particular time. This figure is probably an underestimation; hence it is virtually certain that the depletion of the l.m.c. is due entirely to cell death. Arguments are preue to the failure of their axons to survive in a competition process at the periphery. Observations of the time pattern of muscle differentiation and their neurotization in the leg further endorse this hypothesis. However, it is not clear whether the axons compete for contact sites on muscle fibers or for a "trophic" agent.

Reduction of the naturally occurring motor neuron loss by enlargement of the periphery.

Motor hyperplasia following the enlargement of the periphery by implantation of a supernumerary leg is not due to "remote control" of proliferation, as shown by motor neuron counts in 6-day chick embryos. We have tested the alternative hypothesis that we are dealing with reduction of the naturally occurring cell death. In normal development, the lumbar lateral motor column (l.m.c.) undergoes motor neuron degeneration resulting in a cell loss of at least 40%, which occurs between six and one-half and nine and one-half days. Following transplantation of supernumerary legs, cases selected for vigorous motility showed a numerical difference between experimental and contralateral (control) sides amounting to +11.0% to +27.5%. The transplants were innervated by varying combinations of thoracic and rostral lumbar nerves. We interpret our data in terms of survival of motor neurons which normally would have failed in a competition at the periphery but which were sustained by the enlarged peripheral fields. Our data do not permit a decision between the two alternatives: competition for synaptic sites or for a trophic agent. The surviving motor neurons are not limited to the rostral segments of the motor column but in most instances distributed along its entire rostro-caudal extent, implying a redistribution of all l.m.c. axons. The term "hyperplasia" is no longer appropriate for the phenomenon under consideration and should be replaced by the term "hypothanasia.""

An autoradiographic study of the formation of the lateral motor column in the chick embryo.

An autoradiographic determination of the time of origin of the lateral motor columns (LMC) of the chick embryo has been made. The first motor neurons of the brachial LMC are born at stage 15; the earliest birthdates of lumbar LMC neurons are at stage 17. At least 95% of the motor neurons of both brachial and lumbar columns are produced by stage 23 (4 days). The remaining 5% of the motor neurons are produced during the next two days. A clear rostrocaudal gradient of motor neuron production is seen beoth between the brachial and lumbar LMCs and within the LMCs themselves. The LMCs are assembled in a mediolateral sequence: the early-born motor neurons settle medially, the later-born motor neurons settle more laterally. Observations were made of other large early-born neurons which remain permanently in the dorsal gray of the spinal cord.

The history of the discovery of the nerve growth factor.

The Nerve Growth Factor (NGF) is the progenitor of a family of growth factors which is still expanding. The history of its discovery is very colorful; it is a rare combination of scientific reasoning, intuition, fortuities, and good luck. In addition, I believe that the collaboration of three scientists with very different backgrounds contributed to the success: I had grown up in a laboratory of experimental embryology, Dr. Levi-Montalcini came from neurology, and Dr. Stanley Cohen was from biochemistry. The decision where to begin the history of a discovery is always arbitrary. I shall give my reasons why I begin this story with my wing bud extirpations on chick embryos and the analysis of the effects of the operation on the development of spinal nerve centers, published in 1934. Of course, I am aware of the fact that the analysis of neurogenesis had been pioneered by Dr. R. G. Harrison and his students at Yale University since the beginning of this century. It should be mentioned that their experiments had been done on amphibian embryos. My own interest in problems of neurogenesis dates back to my Ph.D. thesis in the Zoology Department of Professor H. Spemann at the University of Freiburg in (the Federal Republic of) Germany; it dealt with the influence of the nervous system on the development of limbs in frog embryos. After I had obtained some inconclusive results I did the crucial experiment of producing nerveless legs. I removed the lumbar part of the spinal cord and the spinal ganglia before the outgrowth of nerve fibers. The nerveless legs developed normally in every respect, but the muscles atrophied eventually.

History of the discovery of neuronal death in embryos.

The German anatomists, M. Ernst and A. Glücksmann, deserve credit for the discovery of widespread cell death in embryonic tissues, including the nervous tissue. In 1934, V. Hamburger described a significant hypoplasia in dorsal root ganglia (DGR) and lateral motor columns, following the extirpation of limb buds in chick embryos. In the early 1940s, Dr. Rita Levi-Montalcini in Turin (Italy) repeated the experiment and suggested that the hypoplasia might result from the death of young differentiated neurons. In a joint reinvestigation, published in 1949, large numbers of degenerating neurons were described in brachial DRG, following wing bud extirpations. In the same embryos, Dr. Levi-Montalcini observed massive neuronal death in cervical and thoracic DRG which had not been affected by the operation. This was the discovery of naturally occurring neuronal death. Long after the discovery of Nerve Growth Factor (NGF) it was recognized that NGF and natural neuronal death are two sides of the same coin: the latter results from an insufficient supply of the former by the target tissues.

Reduction of experimentally induced neuronal death in spinal ganglia of the chick embryo by nerve growth factor.

Extirpation of the wing bud in 2-day chick embryos results in a conspicuous degeneration of neurons in both populations of brachial dorsal root ganglia (DRG). Daily injections of 1 to 6 micrograms of nerve growth factor (NGF), beginning at 4 1/2 days of incubation, rescued all small, late differentiating (DM) neurons and approximately 50% of large, early differentiating (VL) neurons, which would have died otherwise. The fact that NGF is an effective substitute for the hypothetical trophic maintenance factor for DRG which is normally produced by limb tissues strengthens our belief that NGF is identical with this factor. The control experiment, i.e., wing extirpation without NGF injections, revealed an inconsistency with previous data. Experiments on a number of different neuronal units had shown rather consistently that the period of experimentally induced neuron degeneration, caused by removal of the target, is synchronous with the period of normally occurring neuronal death in the same neuronal unit. This synchrony rule is violated by the VL population of brachial DRG. In this unit, the peak of degeneration resulting from wing bud extirpation occurs considerably earlier than the peak of normally occurring neuronal death. The competition hypothesis for the explanation of neuronal death had been based, in part, on the synchrony rule. We discuss the question of whether the deviation from the synchrony rule observed in our material represents a serious challenge to the competition hypothesis.

Retrograde transport of nerve growth factor in chicken embryo.

We have demonstrated retrograde transport of nerve growth factor to the dorsal root ganglia in the chicken embryo. Polyacrylamide gel pellets impregnated with 125I-labeled nerve growth factor were implanted subcutaneously in the leg of stage 36 (10-day) chicken embryos. The embryos were killed 8 hr after implantation and were processed for routine autoradiography. Examination revealed significant labeling of all lumbar dorsal root ganglia on the side of the implanted pellet. The lateral motor columns and ventral roots as well as the dorsal roots on both sides were unlabeled. These observations show that in the chicken embryo, during the period of sensory cell sensitivity to nerve growth factor, there is a selective uptake and retrograde transport of nerve growth factor from the target area to the dorsal root ganglia. These observations supplement reports from other laboratories in which selective retrograde transport of nerve growth factor in neonatal and adult rodents has been demonstrated.

Localization of motor neuron pools supplying identified muscles in normal and supernumerary legs of chick embryo.

Neuromuscular specificity has been investigated in chick embryos with a grafted supernumerary leg. The nerves of the lumbar plexus are divided between the two legs so that rostral nerves innervate the grafted leg and the caudal nerves supply the host's original leg. The basic topographic organization of the histologically definable motor neuron clusters of the lateral motor columns remains unchanged by the addition of a supernumerary leg. Intramuscular injections of identified leg muscles have been used to map the intraspinal location of specific motor pools in stage 38 (12-day) embryos. In the normal embryo, the gastrocnemius muscle is innervated by neurons in a central dorsal cluster of motor neurons in segments 26-29. In six experimental cases, the motor neurons supplying the gastrocnemius muscle of a rostrally placed grafted leg were consistently located in a specific medial cluster of neurons in segments 23-25. Motor neurons in this location never normally innervate a gastrocnemius muscle, even in the very young embryos during the period of naturally occurring cell death. This observation of a systematic mismatch between a particular motor cluster and an abnormally innervated muscle indicates the operation of a selective developmental process. A hierarchy of selective chemoaffinities may best explain our experimental results.

Coordinated motor output in the hindlimb of the 7-day chick embryo.

Electromyographic recordings from individual identified ankle muscles of the 7-day chick embryo (stage 31) were used to determine the organization of motor output at a developmental stage shortly after the onset of spontaneous motility in the leg. During spontaneous motility of the embryo, the electromyographic recordings from the gastrocnemius, peroneus, and tibialis muscles displayed bursts of motor unit activity which alternated with periods of little or no activity. Since the control of skeletal muscle in the chick embryo is neurogenic rather than myogenic, these findings imply that the motoneurons to a given muscle are driven by a common source. Since flexor and extensor muscles are attivated at different times, different central connections to flexor and extensor motoneurons must be present in the central nerbous system of the 7-day embryo. Moreover, since inhibition is known to play an important role in the selective activation of agonist and antagonist muscles, the present results suggest that functional inhibitory synapses may be present in the lumbosacral central nervous system at this stage of development. The basic pattern of muscle activation observed in the 7-day embryo is similar to that seen in older embryos. Since these patterns appear prior to the time at which motor responses to sensory stimulation of the leg can be demonstrated, it is likely that the neural pattern generating circuits for selective activation of muscles are established in the central nervous system without reliance on functional reflexes.

Unit activity in the isolated spinal cord of chick embryo, in situ.

Unit electrical activity was recorded from single neurons in the isolated lumbo-sacral spinal cord of 14- to 19-day chick embryos, in situ. Spinal cord transection was combined with transection of all lumbo-sacral dorsal roots. The spontaneous discharge of cells is confined, for the most part, to the lower two thirds of the cord. A quasilinear reduction in the number of spontaneously active units was found during the developmental period studied. Comparable results were obtained in decentralized cord with sensory inputs blocked by xylocaine.

Electrical activity in the spinal cord of the chick embryo, in situ.

Unit electrical activity was recorded from single neurons in the lumbo-sacral spinal cord of 15-, 17-, and 19-day chick embryos, in situ. The dorsal columns showed relatively continuous single-unit activity. Below this lies an area of relative quiet 100-200mu deep. The ventral two thirds of the cord was the most active region, being characterized by polyneuronal bursts and intermittently active single units.