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First published online February 22, 2008
doi: 10.1242/10.1242/dev.013995

Development 135, 1129-1136 (2008)
Published by The Company of Biologists 2008
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Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

Christopher V. Gabel1, Faustine Antoine2, Chiou-Fen Chuang3, Aravinthan D. T. Samuel1,* and Chieh Chang2,*

1 Department of Physics and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA.
2 Department of Biology and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 1B1, Canada.
3 Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.


Figure 1
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  		Fig. 1. Axon regeneration is exhibited by specific neuronal types. (A) Schematic of femtosecond laser system. (B-F) Axon trajectories of ASH, AWC, DA/DB, HSN and AVM neurons. In each case, an image is shown before, immediately after and 24 hours after surgery. D indicates the distal end and P the proximal end of severed axons. Red arrows point to the laser target. ASH and AWC axons were snipped at their posterior ventral projections; ASH axons did not noticeably grow out after 24 hours (B; n=12); AWC axons did not grow out at all (C; n=12). Representative examples of successful axon regeneration in the cholinergic DA/DB motoneurons (D), serotonergic HSN motoneurons (E) and AVM mechanosensory neurons (F).

 

Figure 2
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  		Fig. 2. Proximity of the axotomy point affects the manner of axon regeneration in DA/DB neurons. DA/DB motoneurons were cut at defined distances from the cell body. The total contour length of new outgrowth from the neuron was measured 24 hours after surgery and binned into two categories: continuing growth from the proximal end of the severed axon or new growth initiated at the cell body. Error bars represent ±1 s.e.m.

 

Figure 3
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  		Fig. 3. Spatiotemporal dynamics of regenerative axon growth. Representative patterns in axon regeneration are shown with this mechanosensory neuron, imaged at 30-minute intervals for 15 hours following femtosecond laser axotomy using fluorescence microscopy. The dorsal and anterior directions are upwards and leftwards, respectively.

 

Figure 4
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  		Fig. 4. Excess axons remain 24 hours after laser axotomy. (A) A representative example of an injured neuron with only a primary axon 24 hours after surgery. (B) An injured neuron with an ectopic axon from the cell body 24 hours after surgery. (C) Excessive axon sprouting from an injured neuron 24 hours after surgery. Arrows indicate primary axons and arrowheads denote ectopic axons. Scale bar: 20 µm.

 

Figure 5
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  		Fig. 5. Developmental stage affects axon regeneration. (A,B) Representative images showing the morphology of successful and unsuccessful AVM axon regeneration 24 hours after injury. The dorsal and anterior directions are upwards and leftwards, respectively. In cases of successful regeneration to the ventral nerve cord (A), we label the primary axon terminus (blue circle) at the point of innervation to the ventral nerve cord, and label all other axon termini (red circles) as the ends of any other axon branches or outgrowths. In cases of unsuccessful regeneration to the ventral nerve cord (B), we label the primary axon terminus (blue circle) as the end of the longest axon shaft, and label the remaining axon termini (red circles) as the ends of any axon branches from the main shaft (e.g. the uppermost branch in B) or the ends of shorter axon shafts. (C) Scatter plots showing the positions of regenerated axon termini in all L3, L4 and young adult animals. In the case of young adult animals, surgery was performed shortly after the last larval molt. In the manner shown in A,B, all primary and secondary regenerating axon termini are indicated by large blue and small red circles, respectively. In order to consolidate data from different worms, all scatter plots are scaled by each worm's circumference. In each scatter plot, the top line indicates the dorsal nerve cord, the bottom line indicates the ventral nerve cord and the broken line indicates the longitudinal axis halfway between the dorsal and ventral nerve cords. The wild-type morphology of the AVM axon before surgery is drawn in green. The distance between the top and bottom lines corresponds to half of total worm circumference, and the horizontal axis shows a proportion of body length equivalent to about 3.5 circumferences. (D) Bar chart showing the percentage of successful ventral guidance of the regenerating AVM axons in various stage animals based on the scatter plots shown in Fig. 5C. Asterisk indicates a case in which an early developmental stage differs significantly from the young adult (P<0.05). (E) Bar charts of average axon length based on the scatter plots shown in C, scaled by worm circumference (in which 1 corresponds to half of total worm circumference) and unscaled (in µm). Axon length corresponds to the contour length between the cell body and axon termini. Asterisks indicate cases in which an early developmental stage is significantly different from the young adult (P<0.05). Error bars represent ±1 s.e.m.

 

Figure 6
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  		Fig. 6. Cellular and molecular determinants of initial axon development in AVM neuron. (A) Schematic of the axon guidance systems used by the AVM neuron during development. Dorsal muscles express the repellent SLT-1/Slit (red). Ventral axons express the attractant UNC-6/netrin (blue). (B) Schematic of signaling molecules that operate downstream of the guidance receptors in AVM axon development.

 

Figure 7
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  		Fig. 7. Molecular requirements for regenerative axon growth and guidance. (A) Scatter plots (as in Fig. 5C) showing the positions of regenerated axon termini in all wild-type, unc-6, unc-40, unc-5, slt-1, sax-3, unc-6 slt-1, unc-129 slt-1, unc-34, ced-10 and mig-10 animals. All surgeries were performed on young adult worms, shortly after they exited the last larval molt. (B-D) Bar charts (as in Fig. 5D,E) showing the percentage of successful ventral guidance and average axon length of the regenerating AVM axons in wild-type and mutant worms based on the scatter plots shown in A. (B,C) Asterisks indicate cases in which mutant differs from wild-type or specific comparisons are significantly different (P<0.05). (D) Asterisks indicate cases in which mutant differs from wild-type or rescued worms (P<0.05). Error bars represent ±1 s.e.m.

 

Figure 8
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  		Fig. 8. Representative patterns of regenerated AVM axon in wild-type and mutant worms 24 hours after axotomy in the young adult stage. In each image, the dorsal and anterior directions are upwards and leftwards, respectively. Scale bar: 20 µm. (A) The wild-type regenerated axon reaches the ventral nerve cord. (B) The AVM axon of a slt-1 mutant reaches the dorsal midline. Anteriorly (C) and posteriorly (D) projecting AVM axons in unc-6/netrin mutants. (E) An AVM axon in an unc-6 slt-1 double mutant is considerably longer than in average wild-type worms. Defective axon outgrowth in ced-10/rac1 (F) and unc-34/Ena (G) mutants. (H) Aberrant axon rewiring in a mig-10/Lpd mutant.

 

Figure 9
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  		Fig. 9. Expression patterns of slt-1/slit and unc-129/TGF-β in adults. (A) SLT-1 maintains its expression in dorsal body wall muscles into adulthood. Only the mid-body region is shown. (B) UNC-129/TGF-β is expressed at high levels in dorsal body wall muscles into adulthood. Anterior to mid-body region is shown. Asterisks indicate the positions of the vulvae and the arrowhead indicates the nerve ring in the head. In each image, dorsal is upwards and anterior is leftwards. Scale bar: 50 µm.

 




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