In a relatively short time, C. elegans has emerged as a highly tractable model to study axon regeneration. Current data suggest that many genes function similarly in worm and mammalian regenerating axons (see Introduction above). C. elegans offers the axon regeneration field a simple, highly invariant nervous system, genetic tractability, and the ability to study individual neurons in vivo. Although many novel signaling pathways that function in regeneration have been identified, many questions remain.
What Activates Regeneration Pathways?
MAPK pathways (48
) and probably also Notch signaling (21
) are activated by injury. The cellular mechanism that links injury to activation is currently unknown. A variety of injury signaling mechanisms have been proposed, including calcium entry, electrical signals, and changes in trafficking of a regeneration factor. Of these, only calcium is known to function in C. elegans
). One possibility is that calcium signaling is the single cue that activates all of the acute responses to injury. For example, modulation of calcium in vivo and at physiological conditions can activate Notch (69
). Alternatively, other injury signals may exist. For example, disruption of the microtubule cytoskeleton in nerve injury may be an injury signal. In support of this idea, microtubule depolymerization can activate dlk-1
). However, it remains to be determined whether this activation mechanism occurs in regenerating axons.
Answers to these questions and others may come via genetic screens. So far, two screens (an unbiased RNAi screen in unc-70
/β-spectrin mutants and systematic screening of existing mutant alleles using laser axotomy) identified many genes involved in regeneration (15
). Additional genetic screens are likely to provide more details on the function of known pathways and also to identify additional factors that mediate regeneration.
What Are the Effectors of Regeneration Pathways?
In the end, regeneration signaling must converge on growth mechanisms that enable the injured neuron to generate a new growth cone, maneuver the growth cone to its target, and reconnect. These growth mechanisms are for the most part not understood in C. elegans. However, the recent description of microtubule dynamics in regenerating axons and the discovery of a novel regulator of these dynamics (see section on efa-6 and Microtubule Dynamics) suggest that it will be possible to analyze regeneration at the cell-biological level. Further application of cell-biological techniques, such as electron microscopy and super-resolution imaging, in combination with genetic analysis, should result in a better understanding of the growth mechanisms that mediate regeneration.
A second approach to cell biology that also has potential translational applications is the use of C. elegans
as a platform to screen for drugs that are effective in improving regeneration after injury. A high-throughput screen using microfluidics identified a chemical enhancer of regeneration (75
). Further, microinjecting compounds directly into the pseudocoelom of animals immediately after axotomy can enhance regeneration (15
). This technique may be used in the future to assess limited numbers of compounds for effects on regeneration. Such compounds may identify particular cell-biological processes that are important for regeneration.
How Good is Regeneration?
Most regeneration studies in C. elegans
have used the morphology of the regenerating neuron as a measure of regenerative success. To date, two studies in the GABA motor neurons have shown that regeneration is accompanied by functional recovery at the level of whole-animal behavior (see section on Fusion and Functional Regeneration). However, such experiments are limited in their ability to accurately assess the function of individual regenerated neurons compared with their uninjured counterparts. C. elegans
is a tractable model for a more detailed study of neuronal function using electrophysiological and optogenetic techniques (23
). The future application of these techniques to the study of regeneration will yield information about how effective functional recovery is and may identify new pathways that are required for functional, rather than merely morphological, regeneration.
Why Inhibit Regeneration?
Axon regeneration can restore function, so why inhibit it? In the vertebrate CNS, pathways that inhibit regeneration also function in uninjured nervous systems and help maintain a stable, functional system by inhibiting aberrant growth and plasticity (2
). For example, CSPGs inhibit regeneration after injury in the CNS (11
). In uninjured animals, enzymatic degradation of CSPGs results in ectopic growth and sprouting (5
). These data suggest that inhibition of the injury response in the CNS is part of a broader program to limit plasticity. Similarly, it is possible that the C. elegans
pathways that inhibit regeneration also function to stabilize the mature, uninjured nervous system.
Neurons in old C. elegans
animals show a loss of stability and accumulate ectopic branches (85
). Loss of jnk-1
causes an increase in ectopic neuronal branching in old animals, suggesting that jnk-1
contributes to nervous system stability (85
). Consistent with the idea that stability pathways can also inhibit regeneration, jnk-1
mutants also display increased regeneration after nerve injury (36
). In other ways, however, stability and regeneration seem to be separate processes. The dlk-1
pathway is required for regeneration but not for spontaneous branching in old animals. Loss of mlk-1
increases the incidence of age-dependent branching but decreases regeneration (36
). This suggests that in C. elegans
, regeneration and spontaneous branching are promoted by different mechanisms. It remains to be seen whether other inhibitors of regeneration, such as Notch (21
) and efa-6
), affect spontaneous branching and the overall stability of the nervous system.
Why Can Worms Regenerate at All?
Neurons in C. elegans can regenerate in response to injury. Is this ability due to a specialized regeneration mechanism that has evolved specifically to respond to nerve injury? Or is the response to injury part of a more general mechanism, such as homeostasis or stress response? Or is it even a pathological and undirected response to trauma?
During their life span in the wild, worms are subjected to desiccation, mechanical trauma, and predators. Thus, the ability to regenerate neurons quickly after they have been severed and to regain movement may pose a significant selective advantage. Alternatively, neuronal regeneration may be a particular manifestation of some broader biological process. A more complete understanding of the mechanisms that mediate regeneration might eventually help answer this question.
C. elegans has advanced the field of axon regeneration by providing a genetic system that is compatible with both high-throughput screening and single-neuron analysis. Additional forward genetics, a wider use of high-throughput techniques, functional regeneration assays, and drug validation will make C. elegans an even more robust model and further advance the study of axon regeneration.