1 Center for Hearing and Deafness and
2 Department of Anatomy and Cell Biology, State University of New York at Buffalo, Buffalo, NY 14214, USA
3 Max-Planck-Institut für Hirnforschung, Deutschordenstrasse 46, D-60628 Frankfurt, Germany
4 Departments of Pathology and Internal Medicine,
5 Departments of Neurology, Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA
*Author for correspondence (e-mail: hashino{at}acsu.buffalo.edu)
Accepted July 12, 2001
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SUMMARY |
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Key words: GDNF, Neurturin, CNTF, GFR, Ret, Ciliary ganglion, Axon, Chicken
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INTRODUCTION |
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Recent evidence has pointed to another class of neurotrophic factors that is required for the normal development of several parasympathetic neuron populations. Mice carrying a null mutant allele for neurturin (NRTN) (Kotzbauer et al., 1996) or its high-affinity receptor, GFR2, displayed a similar phenotype, showing a substantial reduction in parasympathetic innervations to the lacrimal and salivary glands (Heuckeroth et al., 1999; Rossi et al., 1999). In addition, NRTN as well as glial cell line-derived neurotrophic factor (GDNF) (Lin et al., 1993) has been shown to promote the survival of developing chick ciliary ganglion neurons in vitro (Buj-Bello et al., 1995; Hashino et al., 1999b). On the basis of these lines of evidence, we hypothesized that one or more members of the GDNF family ligands (GDNF, NRTN, Artemin (ARTN)(Baloh et al., 1998), and Persephin (PSPN) (Milbrandt et al., 1998)) are secreted from target tissues of ciliary ganglion neurons during innervation and that these proteins provide a long-distance cue for growing ciliary ganglion axons. To test this hypothesis, we analyzed proteins synthesized in target eye tissues and determined their identity. In addition, functional biological assays were conducted to assess diffusible components of eye proteins.
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MATERIALS AND METHODS |
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Conditioned medium and function-blocking experiments
Eye-conditioned medium was prepared by modifications of the previously described procedure (Hashino et al., 1999a). Briefly, eyes with attached connective tissues were removed from E5 or E12 chick embryos. Ten E5 eyes or two E12 eyes were placed in a well of 24-well culture dishes that contained 800 µl of serum-free medium. The eyes were incubated for 2 days at 37°C and the culture medium was collected and filtered through a low protein-binding PVDF membrane filter. The conditioned medium was diluted 1:1 in defined DMEM/F12 culture medium. Function-blocking antibodies to GDNF (R&D Systems) or NRTN (PeproTech, Rocky Hill, NJ) at 0.1, 0.2 or 0.5 µg/ml were added to either eye-conditioned medium or defined culture medium containing GDNF, NRTN or CNTF (10 ng/ml, each) prior to the start of an incubation period. E9 ciliary ganglion explants were cultured for 48 hours, after which the mean neurite outgrowth was measured as described above.
Dissociated neuron culture
Ciliary ganglion neurons from E9 or E16 chick embryos were dissociated as previously described (Hashino et al., 1999b). Dissociated neurons were plated on 24-well tissue culture dishes coated with poly-D-lysine (200 µg/ml) and laminin (50 µg/ml, Gibco), and incubated in serum-free medium supplemented with GDNF, NRTN, ARTN, PSPN, CNTF, BDNF (50 ng/ml, each), or no factor (control). Neuronal survival was determined 2 days after the start of incubation by counting phase-bright cells with a neuronal morphology (Hashino et al., 1999b).
Western blot analysis and production of an anti-GFR2 antibody
Freshly dissected chick embryonic ciliary ganglia or eyes were frozen on dry ice and kept at 80°C until processed. Approximately 120 (E6), 60 (E9), 30 (E12) and 20 (E16) ciliary ganglia were collected to equalize the volume of tissues from different embryonic stages. Tissues were homogenized by several passages through a 23-gauge syringe needle in boiling lysis buffer (10 mM Tris, pH 7.4, 1% SDS, 1 mM Na3VO4). Samples were centrifuged at 14,000 g for 5 minutes at 4°C, the resulting supernatant was added to an equal volume of sample buffer (100 mM Tris, pH 6.8, 4% SDS, 0.2% Bromophenol Blue, 2% ß-mercaptoethanol (for eye lysates only) and 20% glycerol). The protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA). In each lane, equal amount of protein was run on a 7.5%, 10% or 12% SDS-polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad). The blots were incubated sequentially in blocking buffer (Tropix, Bedford, MA) supplemented with 0.1% Tween 20 overnight at 4°C, a primary antibody overnight at 4°C and alkaline phosphatase-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA). Primary antibodies used in this study were anti-Ret (1:300, Santa Cruz Biotechnology, Santa Cruz, CA), anti-GFR1 (1:3000, Transduction Laboratories, Lexington, KY), anti-CNTFR
(1:1000) (von Holst et al., 1997), anti-gp130 (1:500, Santa Cruz Biotechnology), anti-NRTN (1:1000, PeproTech), anti-GDNF (1:500, Santa Cruz Biotechnology). A polyclonal GFR
2 antibody was produced by immunizing rabbits with a synthetic peptide corresponding to an extracellular domain of chicken GFR
2 (aa 375-389: NH2-KVEKSPALPDDINDS-CONH2). The resultant antiserum was purified with immobilized protein G. This antibody was used for western blot analysis at the concentration of 0.4-0.8 µg/ml. The blots were visualized with the Western-Star chemiluminescent detection system (Tropix) and exposed to X-ray film.
Quantitative RT-PCR
The expression of GFR1, GFR
2, Ret and L27 mRNAs in E5 and E15 ciliary ganglion neurons were analyzed by real-time RT-PCR. Poly(A)+ RNA was isolated from freshly dissected ciliary ganglia using the Oligotex Direct mRNA purification kit (QIAGEN Inc., Valencia, CA) according to the manufactures instructions. Single-stranded cDNA was then synthesized using the Omniscript reverse transcriptase (QIAGEN Inc.) and Oligo(dT) primers. The resultant cDNA was amplified on the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) using TaqMan Universal PCR Master Mix (PE Applied Biosystems) that contains dNTPs with dUTP, AmpliTaq Gold DNA polymerase, AmpErase UNG, optimized buffer and the passive reference dye. For each PCR reaction, a mixture containing cDNA template (5 ng), Master Mix (1x), forward and reverse primers (400 nM) and TaqMan probe (200 nM) was placed in a 96-well MicroAmp Optical Reaction Plate with Optical Caps (PE Applied Biosystems). In most cases, the PCR reaction was run under the following conditions: 1x, 50°C, 2 minutes; 1x, 95°C, 10 minutes; 55x, 95°C, 15 seconds, 60°C, 1 minute; 1x, 4°C, hold. After amplification, the PCR products were analyzed with the ABI PRISM sequence detection software. To check if amplification yields PCR products of a single molecular mass, the PCR products were electrophoresed on 2.5% agarose gels containing ethidium bromide. To check for DNA contamination, purified mRNA was run for the PCR reaction. To check the linearity of the detection system, a cDNA dilution series (1, 1:10, 1:100, 1:1000) was amplified with a primer pairs and TaqMan probe and a correlation coefficient was calculated from the standard curve that displays threshold cycles (CT) as a function of log10 cDNA concentrations. The mRNA level for each probe (x) relative to L27 mRNA (internal control) was calculated and described as follows: mRNA(x)=2Ct (L27)-Ct(x)x100. The primer sets and TaqMan probes used were: GFR
1 [forward, 5'-AAC TCT GTC TTA ATG AGA ATG CTA TTG G-3', reverse, 5'-GAA TGA GTG GTG TGC TTG GAT AGA-3' and probe, 5'FAM (6-carboxy fluorescein)-TCT CCA CCA GCC ACA TAT CCT CGG A-3'TAMRA (6-carboxy-tetramethlrhodamine)], GFR
2 [forward, 5'-CCC TCA CCT CCC ATC ACA AT-3', reverse, 5'-TTC TGT CCG TGC TCC TGG AT-3' and probe, 5'FAM-CAA GGT GGA GAA AAG TCC TGC CTT GCC-3'TAMRA], Ret [forward, 5'-GCC AAG AGA GAG GAA CTC AAC-3', reverse, 5'-AAA TTC TGT GTC GTT CAC CAA-3' and probe, 5' FAM-CAG TCA CAG AGC ATC GTT CCT TCC A-3'TAMRA] and L27 [forward, 5'-AAG GCT GTC ATC GTG AAG AAC AT-3', reverse, 5'-TCG ATG CCT GCC ACC AA-3' and probe, 5'FAM-CTG ATC GGC CCT ACA GCC ACG C-3'TAMRA].
Immunohistochemistry
E7 chick embryos were fixed in 4% paraformaldehyde for 4 hours and embedded in paraffin. Transverse sections (10 µm) of the head were incubated with blocking solution [0.3% Triton X-100, 5% BSA, 10% normal horse serum in hi-salt (1.8% NaCl) PBS], and then either with anti-GDNF (1:100, Santa Cruz Biotechnology), anti-NRTN (1:100, PeproTech) or anti-Ret (1:100, Santa Cruz Biotechnology) in blocking solution at 4°C overnight. Thereafter, the sections were incubated with biotinylated secondary antibody (Vector Lab, 5 µl/ml) and the reaction product was visualized by the biotin-avidin-HRP detection system with the Vector VIP substrate kit. To confirm the specificity of the staining, sections were processed with omission of incubation with a primary antibody.
In vivo axon collapse assay
The function-blocking GDNF antibody (200 µg/ml in PBS, 150 µl) or PBS (150 µl) was injected into the chorioallantoic membrane through a window cut in the shell of E3.5 embryos. The eggs were incubated for a further 4 days, harvested and fixed in 4% paraformaldehyde. After dissecting an eye with an attached ciliary ganglion, crystals of DiI (Molecular Probes, Eugene, OR) were implanted into the ciliary ganglion and the tissue was incubated at 37°C for 3-5 days. At the end of the incubation, the frontal part of the eye as well as the lens was removed, thereby flattening the lateral-posterior surface of the eye. DiI-labeled processes emerging from the ciliary ganglion were examined using a fluorescence microscope. Some of the embryos grown in the presence of the GDNF antibody were fixed and embedded in paraffin. Transverse serial sections of the whole heads were collected and stained with Toluidine Blue.
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RESULTS |
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GFR1 and Ret are down-regulated, whereas GFR
2, CNTFR
and gp130 levels are constant in ciliary ganglion neurons during development
To test if the differences in the stages at which GDNF/NRTN and CNTF exert positive effects on the ciliary ganglion neurons are attributable to differences in their receptor expression, we evaluated the expression level of GDNF/NRTN receptors and CNTF receptors at different embryonic stages by western blot analysis. We first characterized an anti-chicken GFR2 antiserum that we generated. Western blot analysis with E9 ciliary ganglion lysates indicated that this antiserum specifically recognized a protein species that migrates at an apparent molecular mass of 55 kDa (Fig. 2A). The size of the recognized protein is consistent with a predicted molecular mass for human GFR
2 (approx. 55 kDa). In addition, the 55 kDa immunoreactive band was undetectable when primary antibody was preincubated with antigen peptide (data not shown), verifying the specificity of the GFR
2 antibody. The cross-reactivity of the anti-GFR
1 and anti-Ret antibodies used in this study were also tested by western blot analysis. These antibodies specifically recognized protein species in E9 chick ciliary ganglion lysates of 55-65 kDa and 170 kDa, respectively, consistent with the size of human counterparts (Fig. 2A).
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To test whether developmental changes in the expression of GFR1 and Ret in ciliary ganglion neurons occur at a transcriptional level, we performed quantitative RT-PCR. We first checked the linearity and the reliability of the detection system that we used (Fig. 3A). Real-time PCR was run with E5 ciliary ganglion neuron cDNA and a primer pair and TaqMan probe for L27, a housekeeping ribosomal gene. At each PCR cycle, the fluorescence intensity (Rn) was measured from quadruplicate samples per probe. At the end of a PCR run (55 cycles), a graph showing the change in Rn as a function of the cycle number was generated. From this graph, the Ct value was calculated as the cycle number at which Rn first exceeds a threshold. The correlation coefficient calculated from 4 different cDNA concentrations was 0.999, demonstrating the linearity of the detection system over the range of 104. Average PCR reaction curves for GFR
1, GFR
2, Ret and L27 for E5 and E15 ciliary ganglion neuron cDNA samples are presented in Fig. 3B,C. Comparison of relative mRNA levels between E5 and E15 cDNA samples showed that the mRNA level of GFR
1 and Ret was significantly higher in E5 ciliary ganglion neurons than in E15 ciliary ganglion neurons (Fig. 3D,F). The GFR
1 and Ret mRNAs in E5 cDNA sample were 19 times and 1.5 times higher, respectively, than in E15 cDNA samples. In contrast, an equivalent level of GFR
2 mRNA was detected in both E5 and E15 ciliary ganglion neurons (Fig. 3E).
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GDNF and NRTN are secreted as diffusible proteins from eyes during development
To test whether GDNF or NRTN is released as a diffusible protein, we prepared eye-conditioned medium from E5 or E12 chick embryos. In control experiments, we first tested the specificity of a function-blocking antibody to GDNF that we used. The anti-GDNF antibody suppressed GDNF-induced neurite outgrowth in a dose-dependent manner, reducing approximately 80% of neurite outgrowth at 0.5 µg/ml (Fig. 5A,B,E). In contrast, this antibody had little effect on NRTN- or CNTF-induced neurite outgrowth (Fig. 5C,E). E5 eye conditioned medium (ECM) promoted neurite outgrowth of E9 ciliary ganglion neurons to a degree comparable to that of 10 ng/ml GDNF (Fig. 5F), whereas E5 otocyst conditioned medium had no effect on the ciliary neuron outgrowth (data not shown). When the anti-GDNF antibody was applied to a culture medium containing E5 ECM, the neurite outgrowth usually seen with E5 ECM was suppressed (Fig. 5D,F). E12 ECM also promoted the outgrowth of E9 ciliary ganglion neurons, but to a lesser extent than E5 ECM (Fig. 5F). The neurite outgrowth promoting effects of E12 ECM, however, were not suppressed by the GDNF antibody.
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GDNF is required for peripheral target innervation of ciliary ganglion neurons
Because the function-blocking antibody to GDNF almost entirely suppressed E5 ECM-induced neurite extending activities in vitro, we wanted to test whether blocking of exogenous GDNF in vivo would have effects on ciliary ganglion neuron innervation patterns. We injected the function-blocking GDNF antibody used in the in vitro experiments into live chick embryos at E3.5, a stage before ciliary ganglion neurons start to extend their axons toward their peripheral targets. The embryos were then allowed to grow in the presence of the antibody for the next 4 days, after which axonal processes of ciliary ganglion neurons were traced with DiI. In control embryos injected with PBS solution, DiI-labeled axon arbors extending toward the frontal direction of the eye were clearly seen (Fig. 6C). In contrast, virtually no processes except for thin short fibers around the ganglion were observed in embryos injected with the anti-GDNF antibody (Fig. 6D). The average length of ciliary ganglion axons in the GDNF antibody-treated group was only 227 µm, compared with 1590 µm in the PBS-treated control group (t=7.839, df=13, P<0.001; Table 1). In some of the ciliary ganglia treated with the GDNF antibody, a small proportion of neurons had shrunken cell bodies, leaving empty spaces in sections (Fig. 6E). In contrast, other cranial ganglia, such as vestibular and trigeminal ganglia, contained normal complements of neuron cell bodies and extended axons into their peripheral and central target tissues (Fig. 6F).
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DISCUSSION |
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In contrast to the results obtained with E5 ECM, the function-blocking GDNF antibody failed to suppress neurite extending activities of E9 ciliary ganglion neurons that were induced by E12 ECM (Fig. 5F). This is consistent with our western blot analysis, in which GDNF protein was not detected in E12 eye lysate (Fig. 4A), confirming that virtually no GDNF is secreted from eyes at this developmental stage. However, the function-blocking antibody to NRTN was able to suppress E12 ECM-induced neurite outgrowth, albeit to a lesser extent than we expected (Fig. 5H). As GDNF was absent in E12 eye lysate, it is not clear why the NRTN antibody did not totally inhibit E12 ECM-induced neurite outgrowth. Since cDNA for chicken NRTN has not been isolated, we do not know the sequence homology between chicken NRTN and its human counterpart. Thus, one might suggest that the anti-human NRTN antibody used in this study is not able to neutralize chicken NRTN. An alternative possibility is that E12 ECM may contain another neurotrophic factor in addition to NRTN. Support for this hypothesis comes from evidence that detectable levels of CNTF mRNA were expressed in chick eyes at E10 and later stages and that chicken CNTF is a diffusible protein (Leung et al., 1992). Since the CNTF protein level in eyes increased more than 10-fold from E12 to E18 (Finn and Nishi, 1996), we assume that the CNTF protein level at E12 may be very low and that CNTF is not synthesized until most, if not all, of the growing ciliary ganglion axons reached their peripheral targets. Collectively, NRTN may be a major diffusible protein present in eye tissues during the cell death period (E9-E13) (Landmesser and Pilar, 1974).
The significance of the differential regulation of GDNF and NRTN in ciliary ganglion neuron development is unclear. However, NT-3 and BDNF, two closely related neurotrophins, have also been shown to be expressed in distinct regions in pathways of trigeminal ganglion axons and to act cooperatively to facilitate axon outgrowth toward their peripheral target (OConnor and Tessier-Lavigne, 1999). It is thus plausible to hypothesize that two developmental programs independently control the expression and, consequently, synthesis of two neurotrophic factors that signal through common receptors, resulting in similar effects on the developing nervous system. The redundancy in the production of factors that are essential for neuron survival and innervation would guarantee the accomplishment of normal development, and protect the developmental program against potential gene mutation. Alternatively, it is possible that a spatial and temporal distribution of multiple neurotrophic factors may generate subtle concentration gradients throughout the trajectory pathways of developing neurons.
Expression of GDNF receptors is developmentally regulated in ciliary ganglion neurons
A considerable body of evidence has been accumulated to indicate that GFR1 and Ret constitute a functional-receptor complex for GDNF, while GFR
2 and Ret are the high-affinity coreceptors for NRTN (Jing et al., 1996; Jing et al., 1997; Treanor et al., 1996; Baloh et al., 1997; Creedon et al., 1997; Klein et al., 1997). GFR
1 and GFR
2 are also able to bind NRTN and GDNF, respectively, albeit with less efficacy (Baloh et al., 1997; Buj-Bello et al., 1997; Klein et al., 1997). We found in this study that a sharp decline in GDNF synthesis in the eye is accompanied by a down-regulation of its high-affinity coreceptors in ciliary ganglion neurons. The levels of GFR
1 and Ret proteins were high at E6 and E9, after which they declined through E12 and E16 (Fig. 2). The number of Ret-positive neurons in the ciliary ganglion also declined concomitantly from E9 to E16 (Hashino et al., 1999b). In contrast, the levels of GFR
2, CNTFR
and gp130 proteins remained constant between E6 and E16 (Fig. 2). Consistent with these data, the level of GFR
1 and Ret mRNA in E5 ciliary ganglion neurons was significantly higher than that in E15 ciliary ganglion neurons (Fig. 3). A stable expression of GFR
2 mRNA and protein, however, correlate with a sustained level of NRTN. Together, these results suggest that a decrease in GDNF released from pre-synaptic target cells may trigger a chain of molecular events in post-synaptic neurons, thereby down-regulating the expression of GFR
1 and Ret. Support for this hypothesis comes from a previous study where an increase in NGF in target tissues was shown to induce up-regulation of NGF receptor mRNA in sympathetic neurons at the transcriptional level (Miller et al., 1991). Likewise, exogenously applied NGF increased NGF receptor mRNA in adult sensory neurons in vitro (Lindsay et al., 1990).
What is the molecular basis of such reciprocal ligand-receptor interactions? Several transcription factors have been implicated to act as upstream effectors that directly or indirectly regulate the expression of the neurotrophic factor receptors. Among the candidate transcription factors, retinoic acid regulates the expression of Ret receptors both in vitro and in vivo (Tahira et al., 1991; Hashino et al., 1999b; Batourina et al., 2001). Interestingly, retinoic acid, alone or together with bone morphogenetic proteins, was shown to induce a substantial increase in GFR1 in cultured rat sympathetic neurons, without changing the expression of GFR
2 and Ret mRNAs (Thang et al., 2000). Phox2a and Phox2b, the paired-homeodomain transcription factors, also control Ret expression (Morin et al., 1997; Pattyn et al., 1999; Stanke et al., 1999). Verification of involvement of these transcription factors in the GDNF-mediated regulation of GFR
1 and Ret transcription, however, awaits further investigations.
A critical role of GFR2 in parasympathetic neuron development has been implicated since a strong parasympathetic phenotype was observed in GFR
2 null mutant mice (Rossi et al., 1999). One such phenotype is dry eyes due to a lack of parasympathetic innervation to the lacrimal gland. In these mutant mice, a reduction of parasympathetic innervation to the salivary gland was also detected. In addition, GFR
2 is predominantly expressed in several cranial parasympathetic ganglia, including ciliary, otic, sphenopalatine and submandibular ganglia (Forgie et al., 1999; Enomoto et al., 2000). Our RT-PCR analysis essentially supports these previous studies by showing that GFR
2 mRNA is expressed in ciliary ganglion neurons at a level higher than GFR
1 mRNA (Fig. 3). However, our data also reveal that at an early embryonic age the GFR
1 mRNA level is unexpectedly high, approximately 60% of the GFR
2 mRNA level. Thus, in ciliary ganglion neurons, both GFR
1 and GFR
2 are likely to participate GDNF- and NRTN-mediated signaling during a period of axon growth, after which GFR
2 becomes a major receptor in these neurons. The reduction in the survival responsiveness (ED50) to GDNF, but not to NRTN, occurs between E9 and E12 in chick ciliary ganglion neurons (Forge et al., 1999), supporting our assumption.
Conclusions
We have shown that GDNF and NRTN are synthesized in and secreted from ciliary muscle and striated muscle in the eye at a stage that precisely coincides with that at which ciliary ganglion neurons extend their axon processes toward their peripheral targets. We have also demonstrated that the synthesis of GDNF declines markedly at a time when ciliary ganglion axons reach their peripheral targets, while the synthesis of NRTN is maintained throughout the cell death period. Finally, we have presented evidence that the expression of Ret and GFR1, with the exception of GFR
2, decreases concomitantly with the reduction of GDNF, suggesting that target-derived GDNF regulates the expression of its high-affinity coreceptors in innervating neurons. On the basis of these lines of evidence, we conclude that GDNF is a classically defined target-derived neurotrophic factor that plays an essential role in the ciliary axon outgrowth during the period of target innervation. The dramatic down-regulation of GDNF and GFR
1 in contrast to a stable level of NRTN and GFR
2 suggests a novel mechanism by which multiple neurotrophic factors and their high-affinity receptors contribute to neural differentiation and survival.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Adler, R., Landa, K. B., Manthorpe, M. and Varon, S. (1979). Cholinergic neurotrophic factors: intraocular distribution of trophic activity for ciliary neurons. Science 204, 1434-1436.[Medline]
Baloh, R. H., Tansey, M. G., Golden, J. P., Creedon, D. J., Heuckeroth, R. O., Keck, C. L., Zimonjic, D. B., Popescu, N. C., Johnson, E. M. Jr. and Milbrandt, J. (1997). TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret. Neuron 18, 793-802.[Medline]
Baloh, R. H., Tansey, M. G., Lampe, P. A., Fahner, T. J., Enomoto, H., Simburger, K. S., Leitner, M. L., Araki, T., Johnson, E. M. Jr. and Milbrandt, J. (1998). Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFR3-RET receptor complex. Neuron 21, 1291-1302.[Medline]
Batourina, E., Gim, S., Bello, N., Shy, M., Clagett-Dame, M., Srinivas, S., Constantini, F. and Mendelsohn, C. (2001). Vitamin A controls epithelial/mesenchymal interactions through Ret expression. Nat. Genet. 27, 74-78.[Medline]
Buj-Bello, A., Buchman, V. L., Horton, A., Rosenthal, A. and Davies, A. M. (1995). GDNF is an age-specific survival factor for sensory and autonomic neurons. Neuron 15, 821-828.[Medline]
Buj-Bello, A., Adu, J., Pinon, L. G. P., Horton, A., Thompson, J., Rosenthal, A., Chinchetru, M., Buchman, V. L. and Davies, A. M. (1997). Neurturin responsiveness requires a GPI-linked receptor and the Ret receptor tyrosine kinase. Nature 387, 721-724.[Medline]
Creedon, D. J., Tansey, M. G., Baloh, R. H., Osborne, P. A., Lampe, P. A., Fahrner, T. J., Heuckeroth, R. O., Milbrandt, J. and Johnson, E. M. Jr. (1997). Neurturin shares receptors and signal transduction pathways with glial cell line-derived neurotrophic factor in sympathetic neurons. Proc. Natl. Acad. Sci. USA 94, 7018-7023.
Enomoto, H., Heuckeroth, R. O., Golden, J. P., Johnson, E. M. Jr. and Milbrandt, J. (2000). Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin. Development 127, 4877-4889.
Finn, T. P. and Nishi, R. (1996). Expression of a chicken ciliary neurotrophic factor in targets of ciliay ganglion neurons during and after the cell death phase. J. Comp. Neurol. 366, 559-571.[Medline]
Forgie, A., Doxakis, E., Buj-Bello, A., Wyatt, S. and Davies, A. M. (1999). Difference and developmental changes in the responsiveness of PNS neurons to GDNF and neurturin. Mol. Cell. Neurosci. 13, 430-440.[Medline]
Fuhrmann, S., Kirsch, M., Heller, S., Rohrer, H. and Hofmann, H. D. (1998). Differential regulation of ciliary neurotrophic factor receptor- expression in all major neuronal cell classes during development of the chick retina. J. Comp. Neurol. 400, 244-254.[Medline]
Hashino, E., Dolnick, R. Y. and Cohan, C. S. (1999a). Developing vestibular ganglion neurons switch trophic sensitivity from BDNF to GDNF after target innervation. J. Neurobiol. 38, 414-427.[Medline]
Hashino, E., Johnson, E. M. Jr., Milbrandt, J., Shero, M., Salvi, R. J. and Cohan, C. S. (1999b). Multiple actions of neurturin correlate with spatiotemporal patterns of Ret expression in developing chick cranial ganglion neurons. J. Neurosci. 19, 8476-8486.
Heuckeroth, R. O., Enomoto, H., Grider, J. R., Golden, J. P., Hanke, J. A., Jackman, A., Molliver, D. C., Bardgett, M. E., Snider, W. D., Johnson, E. M. Jr. and Milbrandt, J. (1999). Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory and parasympathetic neurons. Neuron 22, 253-263.[Medline]
Jing, S., Wen, D., Yu, Y., Holst, P. L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R., Louis, J. C., Hu, S., Altrock, B. W. and Fox, G. M. (1996) GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-, a novel receptor for GDNF. Cell 85, 1113-1124.[Medline]
Jing, S., Yu, Y., Fang, M., Hu, Z., Holst, P. L., Boone, T., Delaney, J., Scultz, H., Zhou, R. and Fox, G. M. (1997) GFRalpha-2 and GFR alpha-3 are two new receptors for ligands of the GDNF family. J. Biol. Chem. 272, 33111-33117.
Klein, R. D., Sherman, D., Ho, W. H., Stone, D., Bennett, G. L., Moffat, B., Vandlen, R., Simmons, L., Gu, Q., Hongo, J. A., Devaux, B., Poulsen, K., Armanini, A., Nozaki, C., Asai, N., Goddard, A., Phillips, H., Henderson, C. E., Takahashi, M. and Rosenthal, A. (1997). A GPI-linked protein that interacts with ret to form a candidate neurturin receptor. Nature 387, 717-721.[Medline]
Kotzbauer, P. T., Lampe, P. A., Heuckeroth, R. O., Golden, J. P., Creedon, D. J., Johnson, E. M. Jr. and Milbrandt, J. (1996). Neurturin, a relative of glial cell line derived neurotrophic factor. Nature 384, 467-470.[Medline]
Landmesser, L. and Pilar, G. (1974). Synaptic transmission and cell death during normal ganglionic development. J. Physiol. 241, 737-749.[Medline]
Leung, D. W., Parent, A. S., Cachianes, G., Esch, F., Coulombe, J. N., Nikolics, K., Eckenstein, F. P. and Nishi, R. (1992). Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons. Neuron 8, 1045-1053.[Medline]
Lin, L. F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., Vannice, J. L. and Collins, F. (1989). Purification, cloning and expression of ciliary neurotrophic factor (CNTF). Science 246, 1023-1025.[Medline]
Lin, L. H., Doherty, D. H., Lile, J. D., Bektesh, S. and Collins, F. (1993). GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260, 1130-1132.[Medline]
Lindsay, R. M., Shooter, E. M., Radeke, M. J., Misko, T. P., Dechant, G., Thoenen, H. and Lindholm, D. (1990). Nerve growth factor regulates expression of the nerve growth factor receptor gene in adult sensory neurons. Eur. J. Neurosci. 2, 389-396.[Medline]
Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G. and Thoenen, H. (1993). Disruption of the CNTF gene results in motor neuron degeneration. Nature 365, 27-32.[Medline]
Milbrandt, J., de Sauvage, F. J., Fahrner, T. J., Baloh, R. H., Leitner, M. L., Tansey, M. G., Lampe, P. A., Heuckeroth, R. O., Kotzbauer, P. T., Simburger, K. S., Golden, J. P., Davies, J. A., Vejsada, R., Kato, A. C., Hynes, M., Sherman, D., Nishimura, M., Wang, L. C., Vandlen, R., Moffat, B., Klein, R. D., Poulsen, K., Gray, C., Garces, A. and Johnson, E. M. Jr. (1998). Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron 20, 245-253.[Medline]
Miller, F. D., Mathew, T. C. and Toma, J. G. (1991). Regulation of nerve growth factor receptor gene expression by nerve growth factor in the developing peripheral nervous system. J. Cell Biol. 112, 303-312.[Abstract]
Morin, X., Cremer, H., Hirsch, M., Kapur, R. P., Goridis. C. and Brunet, J.-F. (1997). Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a. Neuron 18, 411-423.[Medline]
Nishi, R. and Berg, D. K. (1981). Two components from eye tissue that differentially stimulate the growth and development of ciliary ganglion neurons in cell culture. J. Neurosci. 1, 505-513.[Abstract]
OConnor, R. and Tessier-Lavigne, M. (1999). Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons. Neuron 24, 165-178.[Medline]
Pattyn, A., Morin, X., Cremer, H., Goridis, C. and Brunet, J.-F. (1999). The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399, 366-370.[Medline]
Pilar, G., Landmesser, L. and Burstein, L. (1980). Competition for survival among developing ciliary ganglion cells. J. Neurophysiol. 43, 233-254.
Rende, M., Muir, D., Ruoslahti, E., Hagg, T., Varon, S. and Manthrope, M. (1992). Immunolocalization of ciliary neurotrophic factor in adult rat sciatic nerve. Glia 5, 25-32.[Medline]
Rossi, J., Luukko, K., Poteryaev, D., Laurikainen, A., Sun, Y. F., Laakso, T., Eerikainen, S., Tuominen, R., Lasko, M., Rauvala, H., Arumae, U., Pasternack, M., Saarma, M. and Airaksinen, M. S. (1999) Retarded growth and deficits in the enteric and parasympathetic nervous system in mice lacking GFR2, a functional neurturin receptor. Neuron 22, 243-252.[Medline]
Sendtner, M., Kreutzberg, G. W. and Thoenen, H. (1990). Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy. Nature 345, 440-441.[Medline]
Sendtner, M., Stockli, K. A. and Thoenen, H. (1992). Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration. J. Cell Biol. 118, 139-148.[Abstract]
Sendtner, M., Carroll, P., Holtmann, B., Hughes, R. A. and Thoenen, H. (1994). Ciliary neurotrophic factor. J. Neurobiol. 25, 1436-1453.[Medline]
Stanke, M., Junghans, D., Geissen, M., Goridis, C., Ernsberger, U. and Rohrer, H. (1999). The Phox2 homeodomain proteins are sufficient to promote the development of sympathetic neurons. Development 126, 4087-4094.
Tahira, T., Ishizuka, Y., Itoh, F., Nakayasu M., Sugimura, T. and Nagano, M. (1991). Expression of the ret proto-oncogene in human neuroblastoma cell lines and its increase during neuronal differentiation induced by retinoic acid. Oncogene 6, 2333-2338.[Medline]
Takahashi, R., Yokoji, H., Misawa, H., Hayashi, M., Hu, J. and Deguchi, T. (1994). A null mutation in the human CNTF gene is not casually related to neurological disease. Nat. Genet. 7, 79-84.[Medline]
Thang, S. H., Kobayashi, M. and Matsuoka, I. (2000). Regulation of glial cell line-derived neurotrophic factor responsiveness in developing rat sympathetic neurons by retinoic acid and bone morphogenetic protein-2. J. Neurosci. 20, 2917-2925.
Treanor, J. J. S., Goodman, L., Sauvage, F., Stone, D. M., Poulsen, K. T., Beck, C. D., Gray, C., Armanini, M. P., Pollock, R. A., Hefti, F., Phillips, H. S., Goddard, A., Moore, M. W., Buj-Bello, A., Davies, A. M., Asai, N., Takahashi, M., Vandlen, R., Henderson, C. E. and Rosenthal, A. (1996). Characterization of a multicomponent receptor for GDNF. Nature 382, 80-83.[Medline]
von Holst, A., Heller, S., Junghans, D., Geissen, M., Ernsberger, U. and Rohrer, H. (1997). Onset of CNTFR expression and signal transduction during neurogenesis in chick sensory dorsal root ganglia. Dev. Biol. 191, 1-13.[Medline]
Winter, C. G., Saotome, Y., Levison. S. W. and Hirsh, D. (1995). A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury. Proc. Natl. Acad. Sci. USA 92, 5865-5869.