Preparation and biological properties of native and recombinant ciliary neurotrophic factor

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons. © 1992 John Wiley & Sons, Inc.     

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo.

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.     

Purification and characterization of a trophic factor for embryonic peripheral neurons: comparison with fibroblast growth factors

The validation of NGF as a physiologically important neurotrophic factor has led to intense efforts to identify novel polypeptide growth factors for neurons. We report here the details of a greater than 80,000-fold purification of a neurotrophic molecule, referred to as growth-promoting activity (GPA), from chicken sciatic nerves. The final product of the purification migrated as a protein band of 21.5 kd, its apparent pI was approximately 4.8, and the ED50 of the most active preparation was approximately 10 pg/ml. Amino acid sequence of a proteolytic digestion fragment of GPA revealed homology with the recently published sequences for rabbit and rat sciatic nerve CNTF. Thus this molecule may be the chicken form of CNTF. Analysis of the specificity of action of GPA showed that, in addition to E8 ciliary ganglion neurons, the factor was able to support short-term survival of E8 dorsal root ganglion and E12 sympathetic neurons. This range of specificities of biological action was also seen with both acidic and basic FGF in the presence of heparin. The biological activity of GPA differed from that of FGF in that it was not potentiated by heparin and did not stimulate mitogenesis in chick fibroblasts.     

Isolation and characterization of ciliary neurotrophic factor from rabbit sciatic nerves

Ciliary neurotrophic factor (CNTF) has been purified 35,000-fold to homogeneity from rabbit sciatic nerves using its ability to promote the survival of chick embryo ciliary ganglion neurons as the bioassay. The purification involved a combination of acid treatment, ammonium sulfate fractionation, hydrophobic interaction chromatography, chromatofocusing, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high performance liquid chromatography. Overlapping peptide sequences were obtained which accounted for 49% of the primary structure of the molecule. This information was used to prepare synthetic peptides in order to elicit antibodies. Purified CNTF exhibited two major and several minor bands between 24 and 22 kDa on silver-stained sodium dodecyl sulfate-polyacrylamide electrophoresis gels. All of the molecular forms were immunostained in Western blots by antiserum to synthetic peptides. The peptide sequences also provided a basis for cloning and expression of the rabbit CNTF gene (Lin, L-F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., III, Vannic, J. L., and Collins, F. (1989) Science 246, 1023-1025) confirming that the protein purified as reported here is CNTF.     

Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are molecules which regulate the development and maintenance of specific functions in different populations of peripheral and central neurons, amongst them sensory neurons of neural crest and placode origin. Under physiological conditions NGF is synthesized by peripheral target tissues, whereas BDNF synthesis is highest in the CNS. This situation changes dramatically after lesion of peripheral nerves. As previously shown, there is a marked rapid increase in NGF mRNA in the nonneuronal cells of the damaged nerve. The prolonged elevation of NGF mRNA levels is related to the immigration of activated macrophages, interleukin-1 being the most essential mediator of this effect. Here we show that transsection of the rat sciatic nerve also leads to a very marked increase in BDNF mRNA, the final levels being even ten times higher than those of NGF mRNA. However, the time-course and spatial pattern of BDNF mRNA expression are distinctly different. There is a continuous slow increase of BDNF mRNA starting after day 3 post-lesion and reaching maximal levels 3-4 wk later. These distinct differences suggest different mechanisms of regulation of NGF and BDNF synthesis in non-neuronal cells of the nerve. This was substantiated by the demonstration of differential regulation of these mRNAs in organ culture of rat sciatic nerve and Schwann cell culture. Furthermore, using bioassays and specific antibodies we showed that cultured Schwann cells are a rich source of BDNF- and ciliary neurotrophic factor (CNTF)-like neurotrophic activity in addition to NGF. Antisera raised against a BDNF-peptide demonstrated BDNF-immunoreactivity in pure cultured Schwann cells, but not in fibroblasts derived from sciatic nerve.     

Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration

Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to less than 5% in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration.     

Multiple cholinergic differentiation factors are present in footpad extracts: comparison with known cholinergic factors.

Sweat glands in rat footpads contain a neuronal differentiation activity that switches the phenotype of sympathetic neurons from noradrenergic to cholinergic during normal development in vivo. Extracts of developing and adult sweat glands induce changes in neurotransmitter properties in cultured sympathetic neurons that mimic those observed in vivo. We have characterized further the factors present in the extract and compared their properties to those of known cholinergic factors. When assayed on cultured rat sympathetic neurons, the major activities in footpad extracts from postnatal day 21 rat pups that induce choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP) and reduce catecholamines and neuropeptide Y (NPY) are associated with a soluble protein of 22-26 x 10(3) M(r) and a pI of 5.0. These properties are similar to those of ciliary neurotrophic factor (CNTF). Moreover, the purified fraction from footpads has ciliary neurotrophic activity. Antibodies to CNTF that immunoprecipitate all differentiation activity from sciatic nerve extracts, a rich source of CNTF, immunoprecipitate 80% of the cholinergic activity in the footpad extracts, 50% of the VIP and 20% of the NPY activities. Neither CNTF protein nor CNTF mRNA, however, can be detected in immunoblot and northern analysis of footpads even though both CNTF protein and mRNA are evident in sciatic nerve. CNTF-immunoreactivity is associated with a sparse plexus of sensory fibers in the footpad but not with sweat glands or the Schwann cells associated with them. In addition, in situ hybridization studies with oligonucleotide probes failed to reveal CNTF mRNA in sweat glands. Comparison of the sweat gland differentiation activity with the cholinergic differentiation factor from heart cells (CDF; also known as leukemia inhibitory factor or LIF) suggests that most of the cholinergic activity in foot pads is biochemically distinct from CDF/LIF. Further, antibodies that block the activity of CDF/LIF purified from heart-cell-conditioned medium do not block the ChAT-inducing activity present in footpad extracts of postnatal day 8 animals. A differentiation factor isolated from skeletal muscle did not induce cholinergic properties in sympathetic neuron cultures and therefore is unlikely to be the cholinergic differentiation factor produced by sweat glands. Taken together, our data suggest that there are at least two differentiation molecules present in the extracts and that the major cholinergic activity obtained from footpads is related to, but distinct from, CNTF. The second factor remains to be characterized. In addition, CNTF associated with sensory fibers may make a minor contribution to the cholinergic inducing activity present in the extract.     

Ciliary Neurotrophic Factor (CNTF) Genotypes and CNTF Contents in Human Sciatic Nerves as Measured by a Sensitive Enzyme-Linked Immunoassay

Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme-linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.     

Cholinergic neuronal differentiation factors: evidence for the presence of both CNTF-like and non-CNTF-like factors in developing rat footpad.

Catecholaminergic sympathetic neurons are able to change their transmitter phenotype during development and to acquire cholinergic properties. Cholinergic sympathetic differentiation is only observed in fibers innervating specific targets like the sweat glands in the rat footpad. A function for ciliary neurotrophic factor (CNTF) in this process has been implied as it is able to induce cholinergic properties (ChAT, VIP) in cultured chick and rat neurons. We show here that a CNTF-like, VIP-inducing activity is present in rat footpads and that its increases 6-fold during the period of cholinergic sympathetic differentiation. Immunohistochemical analysis of P21 rat footpads demonstrated CNTF-like immunoreactivity in Schwann cells but not in sweat glands, the target tissue of cholinergic sympathetic neurons. The expression of this factor in footpads seems to be dependent on the presence of intact nerve axons, as nerve transection results in a loss of CNTF-like cholinergic activity and immunoreactivity. Immunoprecipitation experiments with rat footpad extracts provided evidence for the presence of ChAT-inducing factors other than CNTF, which may independently or together with CNTF be involved in the determination of sympathetic neuron phenotype.     

Purification of the Chick Eye Ciliary Neuronotrophic Factor

Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.     

The cholinergic neuronal differentiation factor from heart cell conditioned medium is different from the cholinergic factors in sciatic nerve and spinal cord

Environmental cues play an important role in determining the transmitter phenotype of developing sympathetic neurons. Several factors have been described which can induce cholinergic function in cultured sympathetic neurons. We have compared certain biological and immunological properties of three of them, cholinergic differentiation factor (CDF), membrane-associated neurotransmitter-stimulating factor (MANS), and ciliary neurotrophic factor (CNTF), to determine whether they are different. As previously reported, all three increased acetylcholine synthesis in cultured sympathetic neurons. In addition, MANS as well as CNTF and CDF decreased catecholamine synthesis. CNTF and MANS, but not CDF, promoted the survival of embryonic chick ciliary neurons. Affinity-purified antibodies raised against a synthetic peptide corresponding to the N-terminal sequence of CDF immunoprecipitated CDF, but not MANS or CNTF. These results indicate that although CDF, MANS, and CNTF have similar effects on transmitter synthesis by cultured sympathetic neurons, CDF lacks the ciliary neurotrophic activity of MANS and CNTF. Further, CDF possesses an N-terminal epitope which is absent from both MANS and CNTF. Thus, CDF is distinct from MANS and CNTF, and at least two factors exist which can alter the transmitter phenotype of sympathetic neurons in vitro.     

Does oligodendrocyte survival depend on axons?

BACKGROUND: We have shown previously that oligodendrocytes and their precursors require signals from other cells in order to survive in culture. In addition, we have shown that about 50% of the oligodendrocytes produced in the developing rat optic nerve normally die, apparently in a competition for the limiting amounts of survival factors. We have hypothesized that axons may control the levels of such oligodendrocyte survival factors and that the competition-dependent death of oligodendrocytes serves to match their numbers to the number of axons that they myelinate. Here we test one prediction of this hypothesis - that the survival of developing oligodendrocytes depends on axons. RESULTS: We show that oligodendrocyte death occurs selectively in transected nerves in which the axons degenerate. This cell death is prevented by the delivery of exogenous ciliary neurotrophic factor (CNTF) or insulin-like growth factor I (IGF-1), both of which have been shown to promote oligodendrocyte survival in vitro. We also show that purified neurons promote the survival of purified oligodendrocytes in vitro. CONCLUSION: These results strongly suggest that oligodendrocyte survival depends upon the presence of axons; they also support the hypothesis that a competition for axon-dependent survival signals normally helps adjust the number of oligodendrocytes to the number of axons that require myelination. The identities of these signals remain to be determined.     

Immunohistochemical,Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF,CNTF or NT3

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB₄ or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.     

Membrane-associated neurotransmitter stimulating factor is very similar to ciliary neurotrophic factor.

Membrane-associated neurotransmitter stimulating factor (MANS) can modulate sympathetic neurotransmitter expression and promote ciliary neuron survival in cell culture. Previous studies have shown that its biological effects and biochemical properties are similar to those of ciliary neurotrophic factor (CNTF). In addition, CNTF is present in spinal cord, the source of MANS. These observations raised the possibility that MANS preparations contain CNTF. We find that partially purified MANS fractions contain a 24-kD protein that is recognized in Western blots by an antiserum generated against recombinant rat CNTF (rCNTF). This antiserum immunoprecipitates virtually all the cholinergic-inducing and the ciliary neurotrophic activities present in MANS preparations. When iodinated rCNTF is incubated with spinal cord membranes, a significant proportion of the labeled CNTF segregates with the membrane pellet. The membrane-associated exogenous CNTF can be eluted from the membrane fraction by treatment with high-salt solutions, similar to that used to solubilize MANS from spinal cord membranes. Our data suggest that a substantial portion of the cholinergic differentiation and ciliary neurotrophic activities present in MANS preparations can be attributed to CNTF or a CNTF-like molecule.     

The Protective Effects of Achyranthes bidentata Polypeptides on Rat Sciatic Nerve Crush Injury Causes Modulation of Neurotrophic Factors

Pharmacological treatment is a therapeutic approach to improving nerve regeneration and functional recovery after peripheral nerve crush injury. The objective of the present study was to investigate the effects of the polypeptides isolated from Achyranthes bidentata Blume (abbreviated as ABPP) on rat sciatic crush injury and to test the possible involvement of neurotrophic factors. After surgical crush injury, rats received daily intraperitoneal injection of 0.2 ml saline containing 2 mg ABPP, 1 μg nerve growth factor (NGF) or no additive. The results from walking track analysis, electrophysiological assessment and histological evaluation indicated that the repair outcomes by ABPP treatment were close to those by NGF treatment, but better than those by treatment with saline alone. The quantitative real-time RT-PCR was used to monitor the mRNA expression of growth associated protein in the crush nerves and the mRNA expression of NGF, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine kinase (Trk)A and TrkB in the dorsal root ganglia (DRGs) at L4–L6. The mRNA expression of these genes in the crush nerve sample and DRGs sample was higher after treatment with ABPP or NGF than after treatment with saline alone. Our findings suggest that ABPP might protect peripheral nerve against crush injury through stimulating release of neurotrophic factors and the other cytokines.     

A ciliary neuronotrophic factor from peripheral nerve and smooth muscle which is not retrogradely transported

We have found that a CNTF-like molecule which supports ciliary and sympathetic neurons is not retrogradely transported in either sympathetic or parasympathetic nerves. The factor has an apparent M_r of 21 kDa, a pI of 4.9, and is present in peripheral nerves and smooth muscle of the chick. Our experiments indicate that CNTF-like activity does not accumulate on the distal side of ligated chickexpansor nerves. In contrast, there is a clear accumulation of NGF. The activity further differs from NGF in that it is not removed from a smooth muscle of the chick wing by innervating sympathetic fibers. Transection of these fibers does not lead to an accumulation of ciliary activity in theexpansor secundariorum muscle, suggesting that neurons do not actively deplete the muscle of factor by retrograde transport. Finally, recombinant CNTF or semi-purified preparations of CNTF-like activity labelled with¹²⁵I were not transported to the ciliary ganglion of chicks following injection of biologically active material into the eye. Our results suggest either that endogenous CNTF does not act as a survival factorin vivo, or that retrograde transport is not a property inherent to all neuronotrophic molecules.Special issue dedicated to Dr. Lawrence Austin     

BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system. motor neurons; neuromuscular development; neurotrophins     

Comparison of the effects of HGF, BDNF, CT-1, CNTF, and the branchial arches on the growth of embryonic cranial motor neurons

In the developing embryo, axon growth and guidance depend on cues that include diffusible molecules. We have shown previously that the branchial arches and hepatocyte growth factor (HGF) are growth-promoting and chemoattractant for young embryonic cranial motor axons. HGF is produced in the branchial arches of the embryo, but a number of lines of evidence suggest that HGF is unlikely to be the only factor involved in the growth and guidance of these axons. Here we investigate whether other neurotrophic factors could be involved in the growth of young cranial motor neurons in explant cultures. We find that brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) all promote the outgrowth of embryonic cranial motor neurons, while glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) fail to affect outgrowth. We next examined whether HGF and the branchial arches had similar effects on motor neuron subpopulations at different axial levels. Our results show that HGF acts as a generalized rather than a specific neurotrophic factor and guidance cue for cranial motor neurons. Although the branchial arches also had general growth-promoting effects on all motor neuron subpopulations, they chemoattracted different axial levels differentially, with motor neurons from the caudal hindbrain showing the most striking response.