Influence of neostigmine treatment on embryonic development of acetylcholine receptors and neuromuscular junctions.

The postulated role of the acetylcholine receptor in the formation of neuromuscular synapses during the course of embryonic development was investigated in the superior oblique muscle of white Peking duck embryos. The possibility that the number of receptors could be experimentally lowered by chronic injections of the anticholinesterase agent, neostigmine methylsulfate, was determined using 125I-alpha-bungarotoxin. The total number of acetylcholine receptors on incubation day 12, 2 d subsequent to the onset of treatment, was reducted 45% as compared to saline-treated controls. A similar reduction in total receptor content (49%) was also observed on day 19. Radioautographic preparations showed that clusters of acetylcholine receptors were rare and that the grain density of extrajunctional receptors was also reduced. Hence, chronic treatment with neostigimine during development was observed to exert an effect on both the number and distribution of receptors in the developing superior oblique muscle. These changes occurred in the absence of any apparent effect on muscle differentiation in general. Myoblasts and myotubes were present on day 14 and further differentiated into myofibers by day 18 in both neostigmine and saline-treated muscles. The cytology of the develop;ing muscle cells also appeared normal. This is in contradistinction to the striking morphological changes that take place in adult mammalian and avian muscle after anticholinesterase treatment. More significantly, the decreased total receptor content and sparsity of clusters had no apparent effect on the formation of developing neuromuscular junctions at the electron microscopic level. The frequency of neuromuscular junctions in neostigmine-treated muscles was similar to that of the controls. It is concluded that acetylcholine receptor clusters are not required for the events leading to the morphological formation of neuromuscular junctions during in vivo development.

The effect of the floor plate on pattern and polarity in the developing central nervous system.

The effect of floor plate on cellular differentiation in the neural tube of quail embryos was examined. In the developing neural tube the floor plate, which consists of specialized neuroepithelial cells, is located in the ventral midline of the neural tube. When Hensen's node was extirpated the floor plate and notochord did not develop, and the normal differentiation of the ventral horn motor neurons and dorsal and ventral roots did not occur. When one side of the neural tube was deprived of notochord, the ventro-dorsal differentiation took place on both sides. However, when one side of the neural tube was deprived of the floor plate, the ventral horn motor neurons and dorsal and ventral roots did not develop on that side. These observations suggest that the floor plate influences motor neuron differentiation and acts as an intrinsic organizer to establish pattern and polarity in the developing nervous system.

Development of the trochlear nucleus in quail and comparative study of the trochlear nucleus, nerve, and innervation of the superior oblique muscle in quail, chick, and duck.

The present study was undertaken to examine the development of the trochlear nucleus in quail and to compare the mature trochlear nucleus, nerve, and their sole target of innervation, the superior oblique muscle, in quail, chick, and duck. Study of the trochlear nucleus in quail from embryonic day 5 through hatching shows a maximum of 1,248 neurons on embryonic day 10 followed by spontaneous degeneration of 40% of the neurons between days 10 and 16. Previous studies have shown that although the initial and final number of neurons is different in chick and duck, the magnitude of trochlear cell loss in both species is about 40%. This study shows the average number of neurons in the nucleus of quail, chick, and duck, 2 weeks post-hatching, to be 658, 743 and 1,459, respectively. Fiber counts in the trochlear nerve from electron micrograph montages at the same period indicated a ratio of about 1:1 between neurons and axons. While a majority of the fibers in these nerves are myelinated, an average of 3-6% of the fibers are unmyelinated. The nucleus in the quail not only contains the smallest number of neurons but it also innervates the smallest muscle in terms of total number of muscle cells and endplates. However, the opposite relationship does not hold true. The nucleus in duck contains the largest number of neurons, yet the largest number of muscle cells and endplates were found in the chick. The ratios between the neurons and muscle cells as well as between neurons and endplates are about the same in quail and duck. These ratios are much higher in the chick, reflecting the relatively small neuron pool destined for a relatively large target. In spite of variations in the number of neurons, muscle fibers, and endplates the average number of endplates per muscle fiber is relatively constant among the three species.

Influence of reduced neuron pool on the magnitude of naturally occurring motor neuron death.

The present investigation was undertaken to examine the role of peripheral competition in survival of motor neurons during development. A loss of approximately half of the trochlear motor neurons in duck and quail occurs during the course of normal embryogenesis. The number of motor neurons in the nucleus of quail prior to the onset of cell death is identical to the final number of survivors in the nucleus of duck embryos (about 1,300 neurons). In the present study competition at the peripheral target was decreased by reducing the number of trochlear motor neurons initially projecting to their target muscle. This was accomplished by substituting the midbrain of duck embryos with the same neural tissue from quail embryos. Midbrain transplantation was performed before motor axon outgrowth and normal cell death begin. The development of the motor neurons and their sole target of innervation, the superior oblique muscle, was examined by using a variety of techniques. The source of the grafted motor neurons and of a reduction in the size of the motor neuron pool was confirmed from histological sections and cell counts. The grafted motor neurons projected their axons into the appropriate peripheral target, which was determined by the use of HRP tracing technique. Counts of muscle fibers, motor endplates, and acetylcholine receptors and measurement of total muscle protein indicated that the size of the superior oblique muscle in the chimera embryos was similar to that of the normal duck but significantly larger than the muscle in quail embryos. Electrophysiological observations indicated that the grafted trochlear motor neurons made functional connections with the superior oblique muscle. Counts of the trochlear motor neurons after the period of cell death indicated an average of 1,310 neurons in the nucleus of duck, 772 in quail, and 690 in the chimera embryos. The number of motor neurons in the chimera embryos is not significantly different from that in the normal quail. In other words, in spite of reduced peripheral competition trochlear motor neuron death of normal magnitude occurred. Lack of increased cell survival in our study suggests that trochlear motor neurons do not compete for survival at the peripheral target.

Influence of grafting a smaller target muscle on the magnitude of naturally occurring trochlear motor neuron death during development.

About half of the motor neurons produced by some neural centers die during the course of normal development. It is thought that the size of the target muscle determines the number of surviving motor neurons. Previously, we tested the role of target size in limiting the number of survivors by forcing neurons to innervate a larger target (Sohal et al., '86). Results did not support the size-matching hypothesis because quail trochlear motor neurons innervating duck superior oblique muscle were not rescued. We have now performed the opposite experiment, i.e., forcing neurons to innervate a smaller target. By substituting the embryonic forebrain region of the duck with the same region of the quail before cell death begins, chimera embryos were produced that had a smaller quail superior oblique muscle successfully innervated by the trochlear motor neurons of the duck. The number of surviving trochlear motor neurons in chimeras was significantly higher than in the normal quail but less than in the normal duck. The smaller target resulted in some additional loss of neurons, suggesting that the target size may regulate neuron survival to a limited extent. Failure to achieve neuron loss corresponding to the reduction in target size suggests that there must be other factors that regulate neuron numbers during development.

A comparison of lectin binding in rat and human peripheral nerve.

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.

Gonadotropin receptor occupancy and stimulation of cAMP and testosterone production by purified Leydig cells: critical dependence on cell concentration.

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 X 10(-10) M) was linear as a function of cell concentration up to 7.0 X 10(6) cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 X 10(6) cells/1.25 ml. The average value for cAMP production by hCG was 133.8 +/- 8.5 pmol cAMP/2 X 10(6) cells. The production of testosterone was biphasic, increasing linearly up to 5 X 10(6) cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 X 10(-10) M hCG, produced an average of 24.2 +/- 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 +/- 0.6 ng of testosterone in 250 microliters. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25, 2.0, and 2.5 X 10(6) cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells.

Peripheral neuronal changes in growth-retarded neonates: an ultrastructural study.

To study the effect of intrauterine growth retardation (IUGR) on fetal peripheral neuronal elements, we examined preputial skin tissues of ten growth-retarded neonates (mean gestational age 36 +/- 2.3 weeks) and six normal control neonates (mean gestational age 35 +/- 3.0 weeks) by transmission electron microscopy. The myelinated and unmyelinated nerve fibers of growth-retarded neonates contained significant ultrastructural alterations, consisting of 1) local aggregation of mitochondria, 2) loss of mitochondrial cristae, 3) the presence of large vacuoles within the axoplasm, and 4) myelinated fiber degeneration. The Schwann cells of these neonates exhibited accumulation of glycogen, disruption of cytoplasm, dilatation of rough endoplasmic reticulum, and extensive development of polyribosomes. None of the above changes were observed in the control infants, those appropriate for gestational age. We conclude that IUGR may cause significant ultrastructural changes in developing peripheral neuronal tissue.

Development of the oculomotor nucleus, with special reference to the time of cell origin and cell death.

The developmental pattern of the oculomotor nucleus from day 7 of incubation through two weeks after hatching was studied in white Peking duck embryos. The neuroblasts comprising the nucleus complete their last phase of DNA synthesis on days 4 and 5 and the anlage first appears on day 7. The various subnuclei become identifiable as distinct cell groups on day 8 or 9. There is a cell migration between the ventral-most portions of the two ventromedial nuclei on days 9 through 11, and as a result a well-developed oculomotor commissure is established between these two subnuclei. The maximum number of cells in the nucleus is present on day 11. There is a normally occurring overall loss of approximately 43% of the cells during ontogenesis. Cell death appears to be random, without any gradient, and virtually all of it occurs between days 11 and 15. Although the duration of cell death is essentially similar in all subnuclei, great variations exist in its magnitude. For example, there is a cell loss of approximately 61% in the accessory nucleus, 38% in the dorsolateral nucleus, 40% in the dorsomedial nucleus and 33% in the ventromedial nucleus. Cell loss in the oculomotor nucleus is compared with that observed in the other two eye-muscle nuclei.

Effects of reciprocal forebrain transplantation on motility and hatching in chick and duck embryos.

Effects of reciprocal forebrain transplantation on the embryonic motility and the hatching behavior in the chick and the duck embryos were studied. The forebrains were transplanted before the establishment of circulation. The grafted tissue formed the telencephalon, diencephalon, eyes, upper beak and part of the cranium. Data indicate that the size of the forebrain has no significant influence on the early embryonic motility (Type I and II) in the chick and the duck embryos. The initiation and maintenance of the pre-hatching behavior (Type III motility) does not reside in the forebrain. The final stage of hatching (climax) on the other hand may be controlled by the forebrain.

Grafting of additional periphery reduces embryonic loss of neurons.

Effects of increased peripheral field of innervation on the magnitude of the spontaneously occurring embryonic cell death in the chick trochlear and isthmo-optic nuclei were examined. Grafting of an additional optic primordium and the surrounding mesoderm (which forms extraocular muscles) was performed at stage 11 (HH stage series). The grafted tissue is innervated by the appropriate neuron pool as revealed by the retrograde axonal flow of HRP. Cell counts of the trochlear nucleus on day 19 indicate a mean increase of 37% (range 19--62%) in the number of cells on the experimental side (contralateral to the graft) as compared to the ipsilateral control nucleus of the same embryos. Cell counts of the isthmo-optic nucleus on day 19 show an average increase of 35% (range 27--41%) on the experimental (contralateral) side over the ipsilateral control side. This increase in cell survival is not due to a stimulatory effect of grafting on cellular proliferation as revealed by the cell counts of trochlear and isthmo-optic nuclei on day 9 and 11 respectively. Thus, the increased cell survival is attributed solely to the reduction in the magnitude of the embryonic cell death. Whether this reduction in embryonic cell death is due to increased number of synaptic sites or increased amounts of trophic substances remains uncertain.

Development of the trochlear nerve: loss of axons during normal ontogen.

Development of the trochlear nerve from day 11 of incubation through hatching was studied in white Peking duck embryos. Counts of fibers from the electron micrograph montages indicate that initially there is an abundant collateral sprouting which roughly coincides with the time of neuromuscular contacts, suggesting some sort of interaction between the developing nerve and the periphery. The maximum number of trochlear cells and fibers is present on day 12. Average cell and fiber counts on this day are 2325 and 47,386 respectively. Assuming all cells send their axons into the nerve and that all cell bodies are present within the trochlear nucleus, the ration of cells to fibers is 1:20. Average cell and fiber counts at hatching are 1338 and 1506 respectively. Thus, losses of approximately half the trochlear cells and of 97% of the fibers occur during normal development. Degenerating cells and fibers are first observed on day 13. Degeneration involves both the myelinated and the unmyelinated axons. The actual number of degenerating fibers which were observed, however, was very small compared to the number of fibers lost during development; thus, it is suggested that, in the majority of cases, fiber loss is perhaps via retraction of axon collaterals. In general, cell death slightly precedes axon loss, which suggests that the direction of the degeneration is from cell body to the axon. A cell/fiber ratio of approximately 1:1 is first observed on day 18 and remains so thereafter. Indirect evidence is discussed, suggesting that at least some cells which die during normal devleopment had sent their axon into the nerve prior to their death. Whether these axons make meaningful connections with the muscle is uncertain.

Development of postsynaptic-like specializations of the neuromuscular synapse in the absence of motor nerve.

It was previously reported that the acetylcholine receptor clusters and acetylcholinesterase appear on embryonic superior oblique muscle cells developing in vivo without motor nerve contacts. The objective of this study was to examine whether some other components of neuromuscular junction also form on muscle cells developing in vivo in the absence of motor neurons. In the present study, postsynaptic specializations such as junctional folds, postsynaptic density and basal lamina were studied in normal and aneural muscles. The superior oblique muscle of duck embryos was made aneural by permanent destruction of trochlear motor neurons by cauterizing midbrain on embryonic day 7; 3 days before the motor neurons normally project their axons into the muscle. Normal and aneural muscles from embryonic days 10 to 25 were processed for electron microscopy. The results indicate that morphological specializations such as junction-like folds, postsynaptic-like density, and basal lamina also develop in the absence of motor neuron contacts. Whether the differentiation of specialized synaptic basal lamina is dependent on the presence of motor neurons was examined by utilizing a monoclonal antibody against heparan sulfate proteoglycan. Immunohistochemical studies indicate that specialized synaptic basal lamina differentiates in the absence of motor neurons. Thus, the mechanism of development of postsynaptic components of neuromuscular junction in this muscle is not dependent on motor neuron contacts. These results also suggest that the postsynaptic cell plays a more active role in synapse formation than previously realized. The results are discussed in relation to the control of synapse numbers by the postsynaptic cell.

Development of trochlear motor neurons, superior oblique muscle, and neuromuscular junctions following prevention of cell death by myasthenia gravis immunoglobulin.

Paralysis of embryo during the period of naturally occurring motor neuron death produces an increase in the number of surviving neurons while the progressive differentiation and maturation of target muscle is severely retarded. Application of immunoglobulin G from patients with acquired myasthenia gravis to duck embryos during the period of trochlear motor neuron death also prevents this neuron loss but without paralyzing the embryo. The present study was conducted to investigate whether or not the differentiation and maturation of motor neurons, target muscle, and neuromuscular junctions were retarded following prevention of cell loss with myasthenic immunoglobulin. Immunoglobulin concentrates from myasthenics and normal human volunteers were applied daily to the chorioallantoic membrane of duck embryos from day 10 onward. The development of the trochlear neurons and the superior oblique muscle was examined with light and electron microscopy on embryonic days 12, 16 and 20. The motor neurons at the light and electron microscopic level were cytologically indistinguishable between the myasthenic and normal immunoglobulin-treated embryos. Myoblasts fused to form myotubes. which further differentiated into mature myofibers at the same time in both groups. Numerous neuromuscular junctions of normal ultrastructure and nerve fibers with myelin wrappings were observed in both cases. It is concluded that the increased neuron survival following myasthenic immunoglobulin treatment does not accompany retardation in differentiation and maturation of the target muscle which is contrary to the results obtained from studies utilizing neuromuscular blocking agents producing increased cell survival.

Synapse formation on trochlear motor neurons in relation to naturally occurring cell death during development.

About half of the trochlear motor neurons die during the course of normal development. The present study was undertaken to determine whether the afferent synapses form before the onset of motor neuron death and also to determine whether the number of synapses differs between the healthy and degenerating trochlear motor neurons. Brains of duck embryos from days 10 to 20 were prepared for quantitative electron microscopical observations on synaptogenesis. Results indicate that synapses form on the trochlear motor neuron soma before cell death begins suggesting that afferent input is in a position to exert an influence on survival or death of motor neurons. There were no significant differences in the number of synapses between the healthy and dying neurons during the period of cell death. This observation suggests that the mechanism by which afferent synapses could be involved in neuron survival or death is not related to the number of synapses on the cell soma. The number of synapses on the cell process, synaptic transmission and/or molecules released at the synapses are likely candidates for the mechanism of action of afferent input.

Synapse formation on trochlear motor neurons under conditions of increased and decreased cell death during development.

There is a normally occurring death of about half of the trochlear motor neurons during development. Early removal of the target muscle results in death of almost all neurons whereas neuromuscular blockade prevents neuron death. The present investigation was undertaken to determine whether the number of central afferent synapses on motor neurons is altered under conditions which either accentuate cell loss or rescue neurons. The sole peripheral target of innervation of the trochlear motor neurons, the superior oblique muscle, was extirpated in duck embryos before the motor axon outgrowth begins. The neuromuscular blockade was achieved by application of paralyzing dosages of alpha bungarotoxin on to the vascularized chorioallantoic membrane. This treatment began prior to the onset of cell death and embryos were treated daily throughout the period of cell death. Brains were processed for electron microscopy and quantitative observations were made on synapses at the onset, during the period of, and at the end of cell death. It was found that there was no significant difference in the number of synapses on neurons following target removal, following neuromuscular blockade, and those developing normally. This observation indicates that the number of central afferent synapses on cell soma is not altered under conditions which either decrease or increase neuron survival. These results suggest that the synapse number per se may not be directly involved in the process of naturally occurring cell death. The results also suggest that the number of synapses on trochlear motor neurons is independent of interactions with the target.

Dependence of cranial motor neuron formation on ventromedial brain stem.

The formation of motor neurons in the spinal cord is dependent on inductive signals from the floor plate and notochord. Motor neurons in the brain stem, on the other hand, develop in the absence of both structures. This suggests that either the germinal epithelium is specified intrinsically to form specific cranial motor nuclei or that the inductive signals for the formation of cranial motor neurons arise from some other structure. These possibilities were investigated experimentally by using the formation of trochlear motor neurons in the midbrain of duck embryos as a model system. The trochlear motor neurons, which form the nucleus of the fourth cranial nerve, developed normally after early damage to the prospective germinal epithelium, suggesting that it is unlikely to be specified intrinsically to form these cranial motor neurons. Instead, their development was found to be dependent on the cells within, or associated with, the ventromedial region of the brain stem, as the extirpation of this region results in the absence of motor neuron formation. These results show that structures other than the floor plate and notochord provide inductive signals for the cellular differentiation and patterning of the developing central nervous system. The raise the possibility that the inductive signals for motor neuron differentiation in the spinal cord and the brain stem may not be necessarily identical.

A second source of precursor cells for the developing enteric nervous system and interstitial cells of Cajal.

The enteric nervous system is believed to be derived solely from the neural crest cells. This is partly based on the belief that the neural crest cells are the sole neural tube-derived cells colonizing the gastrointestinal tract. However, recent studies have shown that after the emigration of neural crest cells an additional population of cells emigrate from the cranial neural tube. These cells originate in the ventral part of the hindbrain, emigrate through the site of attachment of the cranial nerves, and colonize a variety of developing structures including the gastrointestinal tract. This cell population has been named the ventrally emigrating neural tube (VENT) cells. We followed the fate of these cells in the gastrointestinal tract. Ventral hindbrain neural tube cells of chick embryos were tagged with replication-deficient retroviral vectors containing the LacZ gene, after the emigration of neural crest from this region. In control embryos, the viral concentrate was dropped on the dorsal part of the neural tube. Embryos were sacrificed from embryonic days 3-12 and processed for the detection of LacZ positive ventrally emigrating neural tube cells. These cells colonized only the foregut, specifically the duodenum and stomach. Immunostaining with the neural crest cell marker HNK-1 showed that they were HNK-1 negative, indicating that they were not derived from neural crest. Cells were detected in three locations: (1). the myenteric and submucosal plexus of the enteric nervous system; (2). circular smooth muscle cell layer; and (3). mucosal lining of the lumen. A variety of specific markers were used to identify their fate. Some ventrally emigrating neural tube cells differentiated into neurons and glial cells, indicating that the enteric nervous system in the foregut develops from an additional source of precursor cells. It was also found that some of these cells differentiated into interstitial cells of Cajal, which mediate impulses between the enteric nervous system and smooth muscle cells, whereas others differentiated into epithelium. Altogether, these results indicate that the ventrally emigrating neural tube cells are multipotential. More importantly, they reveal a novel source of precursor cells for the neurons and glial cells of the enteric nervous system. The developmental and functional significance of the heterogeneous origin of the cell types remains to be established.

Ventrally emigrating neural tube cells migrate into the developing vestibulocochlear nerve and otic vesicle.

Virtually all cell types in the inner ear develop from the cells of the otic vesicle. The otic vesicle is formed by the invagination of non-neural ectodermal cells known as the otic placode. We investigated whether a recently described cell population, originating from the ventral part of the hindbrain neural tube known as the ventrally emigrating neural tube (VENT) cells, also contributes cells to the otic vesicle. The ventral hindbrain neural tube cells were labeled with the fluorescent vital dye DiI or replication-deficient retroviruses containing the LacZ gene in chick embryos on embryonic day 2, after the emigration of neural crest from this region. One day later, the labeled cells were detected only in the hindbrain neural tube. Shortly thereafter, the labeled cells began to appear in the eighth (vestibulocochlear) cranial nerve and otic vesicle. From embryonic day 3.5-5, the labeled cells were detected in the major derivatives of the otic vesicle, i.e. the endolymphatic duct, semicircular canals, utricle, saccule, cochlea, and vestibulocochlear ganglion. That the emigrated cells originated from the ventral part of the hindbrain neural tube was confirmed by focal application of DiI impregnated filter paper and with quail chimeras. It is concluded that, in addition to the otic placode cells, the otic vesicle also contains the ventrally emigrating neural tube cells, and that both cell populations contribute to the structures and cell types in the inner ear. It is well known that inductive signals from the hindbrain are required for the morphogenesis of the inner ear. The migration of the hindbrain neural tube cells into the otic vesicle raises the possibility that the inductive effect of the hindbrain might be mediated, at least in part, by the ventrally emigrating neural tube cells and that, therefore, a mechanism exists that involves cells rather than diffusible molecules only.

Formation of the cranial motor neurons in the absence of the floor plate.

The inductive signals for the differentiation of motor neurons in the spinal cord have been experimentally shown to arise from cells in the midventral region of the neural tube, often referred to as the floor plate, and from the notochord. Although the prevailing view is that a similar mechanism accounts for the differentiation of motor neurons in the brain stem, supporting experimental evidence is lacking. Here, using the formation of the trochlear nucleus in the midbrain of duck embryos as a model system, we report that the floor plate and the notochord are not necessary for the development of these motor neurons in the brain stem. Early damage to the floor plate or extirpation of the floor plate and notochord does not prevent the development of these cranial motor neurons. Thus, either the inductive signals for the formation of these cranial motor neurons arise from some other structure or the germinal epithelium of the cranial neural tube is intrinsically programmed to form specific cranial motor nuclei.