Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA
Present address: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705-2222, USA
*Author for correspondence (e-mail: jorgensen{at}biology.utah.edu)
Accepted July 24, 2001
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SUMMARY |
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Key words: Growth cone, Axon branching, Sprouting, Neuron stabilization, unc-119, Caenorhabditis elegans
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INTRODUCTION |
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We have discovered that a new molecule, UNC-119, suppresses abnormal axon branching. unc-119 was originally identified as a mutation affecting nematode locomotion (Maduro and Pilgrim, 1995). The unc-119 transcript is expressed primarily in neurons early in development and throughout adulthood (Maduro and Pilgrim, 1995). UNC-119 does not contain any well-defined structural motifs, although several proteins that are similar to UNC-119 have been identified in C. elegans, Drosophila melanogaster and vertebrates (Maduro et al., 2000). Two related vertebrate proteins, HRG4 (human UNC119) and RRG4 (rat UNC119), were recently identified based on their high level of gene expression in the retina (Higashide, 1996; Swanson et al., 1998). Specifically, these proteins are localized to the presynaptic zone of the retinal ribbon synapses (Higashide, 1998). The human homolog, HRG4 was recently mapped to 17q11.2 (Swanson et al., 1998; Higashide and Inana, 1999). The function of the UNC-119 protein family members has not yet been determined.
Here we demonstrate that GABA motor neuron axons are severely branched in adult unc-119 mutants and synapses are inappropriately localized. Axon branching in unc-119 mutants could result from defects that occur before, during or after outgrowth. Time-lapse analyses of unc-119 growth cones demonstrated that, despite timing defects, the behaviors exhibited by GABA motor neuron axons during migration are normal. Instead, motor axon branching occurred after axon outgrowth and was the result of supernumerary growth cone activity. Transient expression of UNC-119 protein after outgrowth rescued the unc-119 phenotype. Together these results suggest that the UNC-119 protein maintains the differentiated morphology of the neuron by suppressing supernumerary axon branching and restricting the distribution of synapses.
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MATERIALS AND METHODS |
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Scoring D-type motor neuron axon defects
Wild-type (EG1285) and unc-119(ed3) (EG1322) worms were collected within 30 minutes of hatching and raised at 20°C to particular stages during larval development. At 1 hour or 48 hours after hatching, larvae were mounted on agarose pads containing 10 mM NaN3 (Knobel et al., 1999) and the morphology of the D-type GABA motor neurons was scored using fluorescence microscopy. At both timepoints we could distinguish between the DD and VD neurons because of the location of the cell bodies and axons along the ventral nerve cord. Moreover, DD axons expressing Punc-47::GFP (oxIs12) were brighter under fluorescence illumination than VD axons. The morphologies of DD axons were evaluated after outgrowth of the primary growth cone was complete. Axon morphologies were categorized as: (1) normal (these reached and bifurcated at the dorsal midline and then extended along the anteroposterior axis); (2) extension defective (reached the dorsal midline but failed to bifurcate and extend along the anteroposterior axis); (3) branched (contained multiple branches that extended to the dorsal midline); or (4) terminated (axons and branches failed to reach the dorsal nerve cord). Finally, we noted if there were supernumerary growth cones extending directly from DD cell bodies.
UNC-119 immunocytochemistry
To determine where the UNC-119 protein was located, we generated antibodies against UNC-119. The DNA encoding the N-terminal 48 amino acids of UNC-119 was subcloned into the pGEX-3X cloning vector (Pharmacia) to produce GST:Nu119 (pKK20). Bacteria were transformed with pKK20 and induced to express the fusion protein with 1 mM IPTG. Bacteria were grown at 37°C for two hours, harvested, washed and sonicated to separate soluble proteins from the membrane fraction. The GST:Nu119 fusion protein was isolated from the soluble fraction. GST:Nu119 was purified on a Glutathione SepharoseTM column (Pharmacia), lyophilized, and injected into rats to produce antisera against the fusion protein (Pokono Rabbit Farms and Laboratory). The GST:Nu119 antibody was purified from antisera by binding and removal from a CnBr-activated SepharoseTM column (Pharmacia) containing immobilized GST:Nu119. For immunocytochemical labeling wild-type adults were isolated, immobilized on siliconized coverslips and dissected (Richmond and Jorgensen, 1999). Worms were fixed for 1 hour at room temperature in fresh 2% paraformaldehyde, washed and placed briefly in pre-block (10% fetal calf serum in PGT: 1x PBS, 0.25% Triton X-100, 0.1% gelatin). Fixed worms were washed, incubated with antibody overnight at 4°C, washed again, and incubated in secondary antibody (Alexa FluorTM 568 goat anti-rat IgG; Molecular Probes) for 1 hour at room temperature. After washing, worms were mounted and examined using a BioRad Radiance Laser 2000 laser scanning confocal microscope.
unc-119 rescue experiments
A Punc-47::UNC-119genomic:GFP construct was prepared by PCR amplifying plasmid DP#MM016 containing genomic DNA encoding UNC-119 (Maduro and Pilgrim, 1995) using primers containing novel BamHI (5' N-U119: 5'-CGGGGATCCATGAAGGCAGAGCAACAA-3') and KpnI (3', C-U119: 5'-GACTACTCGTATGATGCAGAGGTACCCC-3') sites. The plasmid pJL35, containing Punc-47::synaptobrevin:GFP, was digested with BamHI and KpnI and gel purified to remove the synaptobrevin fragment, which was replaced by the digested PCR product encoding genomic UNC-119. The resulting ligation product, Punc-47::UNC-119genomic:GFP (pKK11) contained the GFP reporter fused in frame to the C terminus of the full-length UNC-119 protein. Similarly, the Punc-47::UNC-119cDNA:GFP (pKK12) reporter was prepared by PCR amplifying a fragment from the plasmid unc-119 cDNA5' using these same primers. Ligations were performed as with pKK11. Punc-47::UNC-119cDNA:GFP (pKK12) contained the GFP reporter fused in frame to the C terminus of a cDNA minigene encoding the full-length UNC-119. All constructs were sequenced to check for errors introduced into our constructs by PCR. Transgenic strains expressing pKK11 or pKK12 (60 ng/µl) and EK L15 (lin-15+ DNA; 60 ng/µl) plasmid DNA were generated by microinjection into unc-119(ed3); lin-15(n765ts) or lin-15(n765ts) mutants (Mello et al., 1991). F1 progeny with a wild-type vulval phenotype were selected and scored for GFP expression using fluorescence microscopy. unc-119(ed3); lin-15(n765ts) oxEx150 [pKK11; EK L15] and lin-15(n765ts) oxEx267[pKK11; EK L15] were generated in this manner. We generated unc-119(ed3); lin-15(n765ts) oxEx151[pKK12; EK L15] and then outcrossed this strain to produce lin-15(n765ts) oxEx151[pKK12; EK L15]. All strains were scored for rescue of GABA motor neuron outgrowth, generation time and locomotion. Interestingly, inclusion of unc-119 introns in the GABA neuron expression construct (pKK11) rescued most of the locomotory phenotype of unc-119(ed3); lin-15(n765ts) oxEx150 mutants. This rescue is likely to be the result of expression of GFP-tagged UNC-119 in several unidentified head neurons in these strains. Expression in these cells was probably caused by enhancers found in the introns of the genomic construct, since such expression did not occur in unc-119(ed3); lin-15(n765ts) oxEx151.
To determine the structure of other neurons in unc-119(ed3) mutants, we analyzed the CAN neuron in the lateral cord and the DB neurons in the ventral cord using Pacr-5::GAP-43:GFP (pJL1), and the A- and B-type motor neurons using Paex-3::SPECTRIN:GFP (pMH50). We crossed the extrachromosomal arrays oxEx81[pJL1 (30 ng/µl); EK L15 (60 ng/µl)] and oxEx287[pMH50 (30 ng/µl); EK L15 (60 ng/µl)] into unc-119(ed3) III; lin-15(n765ts) and characterized the outgrowth pattern of these neurons using confocal microscopy. To determine if other subsets of neurons were rescued by GFP-tagged UNC-119 expression in the GABA neurons we crossed transgenic animals expressing oxEx151[pKK12; EK L15] with worms expressing oxEx81[pJL1; EK L15] and isolated cross progeny. Thus, the unc-119 (ed3); lin-15(n765ts); oxEx81[pJL1; EK L15]; oxEx151 [pKK12; EK L15] (EG2440) and lin-15(n765ts); oxEx81[pJL1; EK L15]; oxEx151[pKK12; EK L15] (EG2441) strains were generated. We characterized the axon morphology of the GABA motor neurons using Punc-47::UNC-119:GFP (oxEx151) and the CAN lateral cord neuron using Pacr-5::GAP-43:GFP (oxEx81) with confocal microscopy.
We failed to detect the circumferential extensions of DB motor neurons in unc-119(ed3) mutants expressing Pacr-5::GFP (Fig. 3B-D). We characterized the morphology of the cholinergic motor axons in unc-119(ed3) mutants expressing a pan-neuronal marker Paex-3::SPECTRIN:GFP. In both wild-type and mutant worms expressing this marker the DB motor axons extended to the dorsal nerve cord. In unc-119(ed3) mutants the DB axons exhibited minor branching defects in 75% of the worms scored (n=8 worms, data not shown). These data indicate that the defects in DB axon extension observed using the Pacr-5::GFP marker were caused by expression of the Pacr-5::GFP marker in the unc-119(ed3) strain and are not caused by mutations in the unc-119 gene.
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Rescue of unc-119(ed3) axon morphology using heat-shock promoters
Two constructs expressing UNC-119 in a regulated manner (Phsp16-48::UNC-119 and Phsp16-2::UNC-119) were generated by subcloning the unc-119 cDNA into constructs containing the separate heat-shock promoters (pJL26: Phsp16-48, pPD49.78: Phsp16-2). The hsp16-48 promoter is strongly expressed in the developing embryo (Stringham et al., 1992), and the hsp16-2 promoter drives expression in neural and hypodermal cells (Mello and Fire, 1995). Transgenic unc-119(ed3) worms carrying either the Phsp16-48 construct (oxEx321) or the Phsp16-2 construct (oxEx322) and a marker were generated by co-injection. Injection mixes included a heat-shock construct at 2 ng/µl, herring sperm DNA at 40 ng/µl and marker DNA at 60 ng/µl. The marker, pPD97/98 is expressed in coelomocytes (Miyabayashi et al., 1999).
Heat-shock experiments were performed as follows. Animals in all groups were maintained at 20°C, heat shocked at 33°C for 1 hour, and returned to 20°C to develop. We maintained non-heat-shocked siblings as controls. Transgenic embryos were collected from gravid adults en masse and heat shocked (Lewis and Fleming, 1995). Newly hatched larvae were collected within 2 hours of heat shock (late embryonic heat-shock group) and 10 hours after embryonic heat shock (early embryonic heat-shock group). For the larval heat-shock experiments, embryos were collected and hatched in M9. Staged L1 larvae were heat shocked 24 hours later when all larvae had arrested development (corresponding to a stage equivalent to 3-5 hours after hatching). These animals were placed on plates with food and allowed to develop a few hours (L1 group). Non-heat-shocked sibling larvae were also allowed to develop for an additional 30-36 hours after hatching before being heat shocked as L3s (L3 group). For all groups (including non-heat-shocked controls) the morphologies of the DD axons were scored in worms expressing the co-injection marker at the L1, L2, L4, and adult stage of development. Data were combined from both heat-shock promoters and analyzed using the unpaired Students t-test. Nearly complete rescue could be obtained by increasing the concentration of the heat-shock construct. However, we observed that at these concentrations leaky expression resulted in morphological rescue of non-heat-shocked controls. We did not include these data in our results.
Synapse analysis
Presynaptic varicosities in wild-type and unc-119(ed3) worms were characterized using the synapse markers Punc-25::synaptobrevin:GFP (juIs1) (Hallam and Jin, 1998; Nonet, 1999), Pstr-3::synaptobrevin:GFP (kyIs105; a gift from C. Bargmann and G. Crump), and Pmec-7::synaptobrevin:GFP (jsIs37) (Nonet, 1999). The following strains were evaluated using confocal microscopy: unc-119(ed3) III; juIs1 lin-15(n765ts) X (EG1978); unc-119(ed3) III; kyIs105 V; lin-15(n765ts) X (EG1460) and unc-119(ed3) III; jsIs37; lin-15(n765ts) X (EG1885). We characterized DD synapse localization in L1 larvae isolated within 1 hour of hatching and VD synapse localization in L3 larvae using fluorescence confocal microscopy. GABA motor neuron synapses were evaluated in the region of the anterior reflex of the gonad, and PLM synapses posterior of the vulva were evaluated. Data was analyzed for significance using unpaired Students t-tests. We characterized the structure of GABAA receptor clusters in oxIs22; lin-15(n765ts)(EG1653) and unc-119(ed3); oxIs22; lin-15(n765ts) (EG1790) using an integrated Punc-49::UNC-49B:GFP marker (oxIs22) (Bamber et al., 1999).
Electron microscopy
Adult nematodes were prepared for transmission electron microscopy as described (Richmond et al., 1999). Specimens were immersed in ice cold fixative (0.7% glutaraldehyde/0.7% osmium tetroxide in 10 mM Hepes buffer) for 1 hour. Animals were then washed thoroughly in buffer and anterior and posterior extremities were excised in buffer. Postfixation was in 2% osmium tetroxide in 10 mM Hepes buffer for 3 hours. Specimens were then washed in water, stained en bloc in 1% uranyl acetate, dehydrated through an ethanol series, passed through propylene oxide and embedded in epoxy resin. Ribbons of ultrathin sections (35 nm) were collected and examined on an Hitachi H-7100 TEM equipped with a Gatan slow-scan digital camera. Morphometric analysis was performed using the public domain software package NIH Image. For analysis, an active zone was defined as the set of serial sections containing a discernable presynaptic density, as well as two adjacent sections anterior and posterior to the sections containing the density. Docked vesicles were defined as those vesicles appearing within a single vesicle radius (
30 µm) of the presynaptic plasma membrane. Significance values were calculated using Students t-tests or Wilcoxin rank-sum tests.
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RESULTS |
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UNC-119 protein is located in axons
Previous experiments using the unc-119 promoter to drive the expression of reporter constructs demonstrated that the unc-119 gene is expressed primarily in neurons (Maduro and Pilgrim, 1995). However, we were unable to detect any subcellular localization using our GFP-tagged UNC-119 constructs. To determine the subcellular location of UNC-119 we generated antibodies recognizing the N terminus of the protein. The nerve cords and axons of wild-type worms were labeled with purified antibodies (Fig. 2). Immunoreactivity was absent in unc-119(ed3) animals, and anti-UNC-119 antibodies exclusively labeled the GABA neurons expressing GFP-tagged UNC-119 protein in transgenic unc-119(ed3) (data not shown). Thus, the antibody is specific for UNC-119. UNC-119 immunoreactivity was not restricted to a specific subcellular location although the labeling was enriched in axonal processes and weakly expressed in the cytoplasm of neuronal cell bodies.
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Migrating unc-119 growth cones are morphologically normal
GABA motor neuron axons were branched in unc-119(ed3) adults. The simplest explanation for this phenotype is that the axons branched inappropriately during outgrowth. In this case, axon branching would be visible during and shortly after the completion of outgrowth. Alternatively, the branching defects could arise after the axon scaffold had been established. To determine when ectopic axon branching occurred in unc-119(ed3) mutants we examined the structure of the DD motor neurons shortly after growth cone migration was completed and again at 48 hours after hatching (Fig. 4). At both of these time points we could distinguish between DD and VD neurons based on cell body location and GFP expression. The DD motor neurons differentiate during embryogenesis and their outgrowth is finished before hatching (Sulston et al., 1983). Surprisingly, we discovered that the majority of DD commissures (58%) were morphologically normal at hatching, that is, they reached the dorsal nerve cord and extended along the dorsal midline. Only 7% of the axons were branched (Fig. 4A,C). However, after 48 hours, branching of the DD axons increased significantly (Fig. 4B,D). Only 25% of axons were normal after 48 hours (n=8 animals, 40 axons scored), while the number of branched axons in mutants had increased to 55%. Thus, DD axon morphology was initially normal but over time DD axons became branched.
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UNC-119 is required for maintenance of the axon scaffold
In unc-119 (ed3) mutants we observed that motor axon branching occurs after outgrowth is completed. This observation suggested that the presence of a functional UNC-119 protein is required to suppress axon branching. When is UNC-119 function required? There are three possibilities: (1) UNC-119 could be required during outgrowth only; axons that developed in the absence of UNC-119 may grow out correctly but they are unstable, (2) UNC-119 could be required immediately after outgrowth during differentiation to stabilize the differentiated state, or (3) UNC-119 could be required throughout the life of the animal to maintain neuronal morphology. To distinguish among these possibilities we expressed the UNC-119 protein before, during and after DD outgrowth in unc-119 (ed3) mutants using two different heat-shock promoters. We present cumulative data from both heat-shock promoters (see Materials and Methods). When UNC-119 was expressed before DD axons had extended to the dorsal nerve cord (early embryonic heat shock; Fig. 7A) we observed that 24 hours after heat shock the number of morphologically normal axons was increased relative to siblings that did not have heat-shock induced expression of UNC-119. Rescue of axon morphology declined over time so that when these animals molted into adults there was no significant difference between treated and untreated siblings. When embryos were heat shocked after the completion of DD outgrowth (late embryonic heat shock, Fig. 7B), we observed that axon morphology was improved compared to untreated animals. Again, there was a decline in the number of wild-type axons in these animals over time as observed within the early embryonic heat-shock group. Expression of a GFP-tagged UNC-119 protein demonstrated that the fusion protein remained present for approximately 24 hours before fluorescence disappeared, presumably the result of degradation (data not shown). The disappearance of UNC-119::GFP fluorescence coincided with the decline of nervous system morphology. Expression of UNC-119 during the L1 and L3 larval stages demonstrated that expression of UNC-119 even after differentiation was complete could halt the further deterioration of the nervous system (Fig. 7C,D). In short, loss of the UNC-119 protein led to a progressive degeneration of axon morphology during larval stages, and conversely, expression of UNC-119 during larval development was sufficient to maintain nervous system morphology. Surprisingly, the expression of UNC-119 after the morphology of the axons had declined could reverse the defects. Specifically, heat shock during the L1 stage resulted in significantly improved morphology (P=0.02 compared to L4; Fig. 7C). These results suggest that even mature neurons whose morphology has declined have the potential to repair defects such as extra branches and thereby reestablish a normal morphology. Interestingly, these DD neurons were reversing their developmental defects during the time when the VD neurons were developing and the DD neurons were rewiring synapses; perhaps this stage represents a period of neuronal plasticity. These data are all consistent with a requirement for UNC-119 throughout the life of the animal.
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DISCUSSION |
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Furthermore, UNC-119 protein is continuously required to maintain the structure of the nervous system, even after development of the nervous system is complete. Transient expression of the wild-type protein in mutants during development rescued axon structure in early larval stages, but the morphology of the neurons became abnormal when the UNC-119 protein degraded. Expression during late larval stages prevented the further degeneration of the axon scaffold. Interestingly, expression of the protein in the L1 stage reversed defects of the motor neuron axons, thus demonstrating that these neurons still possessed the potential to develop normally even after their normal period of outgrowth had passed.
How does UNC-119 stabilize the structure of the nervous system? We propose four potential mechanisms for UNC-119 function: activity-dependent stabilization, target-dependent differentiation, regulation of the cytoskeleton or of membrane trafficking, or maintenance of cell polarity.
First, neuronal activity is required for the maintenance of nervous system structure in many organisms (Goodman and Shatz, 1993; Katz and Shatz, 1996; Crair, 1999). Increases or decreases in neuronal activity are correlated with excessive axon branching. For example, depolarization of neurons in culture (McCaig, 1990; Perez et al., 1996; Adams et al., 1997; Ramakers et al., 1998) or in living Drosophila (Budnik et al., 1990) causes axonal sprouting. Neuronal damage, induced by epileptic seizures or axotomy, results in an increase in synaptic activity and axonal branching of traumatized cells (Lankford et al., 1998; Angelov et al., 1999; McNamara, 1999; Stoll and Muller, 1999). Decreases in neuronal activity can also cause abnormal axon branching. In C. elegans, mutations in a cGMP-gated channel or calcium channels cause defects in chemosensory axon morphology (Coburn and Bargmann, 1996; Komatsu et al., 1996; Peckol et al., 1999). Thus, the branching defects observed in unc-119(ed3) mutants might be due to a lack of synaptic input into the motor neurons. However, analysis of synaptic function mutants does not support this model. Although weak branching defects in the sensory neurons are observed in the neurotransmission mutant unc-13 (Peckol et al., 1999), data from our lab indicates that GABA motor neurons are not branched in unc-13 mutants (Richmond et al., 1999).
Second, the UNC-119 protein might suppress axon branching in response to a differentiation signal. As they approach and contact the target cell, growth cones begin differentiating into functional nerve terminals (Burden, 1998; Sanes and Lichtman, 1999). Retrograde signals associated with the synaptic target can initiate the differentiation of the neuron (Dai and Peng, 1996; Fitzsimonds and Poo, 1998; Hall et al., 2000). In C. elegans, one source of this differentiation signal may be the body wall muscle. Interestingly, target muscle contact is not required for axon formation (Plunkett et al., 1996). However, muscle contact is necessary for normal synaptogenesis. In fact, the presence of ectopic muscles induces branching of the GABA motor neurons (Plunkett et al., 1996). Thus it is plausible that UNC-119 responds to a target-derived differentiation signal by suppressing further axon outgrowth. In the unc-119 mutant, failure to receive or transmit this information would cause the neuron to actively branch in search of synaptic targets.
Third, the UNC-119 protein might be required to stabilize the axon, by regulating the cytoskeleton or membrane trafficking in the neuron. For example, it has recently been demonstrated that collateral branch formation involves the local fragmentation of microtubule arrays (Yu et al., 1994; Davenport et al., 1999; Dent et al., 1999). In these regions, fragmented microtubules repolymerize and extend laterally to form interstitial branches. During development, microtubule severing proteins destabilize the cytoskeleton (Quarmby and Lohret, 1999; Quarmby, 2000). One such protein, katanin, is expressed in neurons (Ahmad et al., 1999). It is possible that the UNC-119 protein either inhibits microtubule severing proteins or promotes microtubule stabilizing proteins and thereby preserves axon morphology. Alternatively, UNC-119 might be required for membrane trafficking, perhaps by regulating the targeting of vesicles to the correct compartment of the neuron.
Fourth, UNC-119 may be required to maintain cell polarity. Neurons are highly polarized cells (Craig and Banker, 1994; Higgins et al., 1997). Cell polarity appears to be normal initially in unc-119 mutants: motor neurons extend a single growth cone that follows the correct trajectory. However, polarity declines thereafter. Secondary growth cones emerge from the shaft and cell body of the neuron. Moreover, distinctions between axonal and dendritic regions break down. Synapses were observed in the dendritic processes of unc-119 neurons in addition to their normal locations. A differentiated axon tip may exert apical dominance on the axon shaft and cell body, analogous to the tip meristem of a plant. Loss of this polarizing signal may lead to the sprouting of multiple axon shafts and the formation of synapses in dendrites.
In summary, many molecules have been identified that are required during axon outgrowth to regulate growth cone behavior and pathfinding. Our studies demonstrate that UNC-119 belongs to a different class of molecules that maintain the differentiated state of the neuron after axon outgrowth is completed. Understanding how these molecules function may eventually reveal the causes of abnormal axon branching in diseased nervous tissue or in response to neuronal trauma.
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ACKNOWLEDGMENTS |
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