The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans

Abstract

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.

Keywords: DLK; Robo; Slit; Wnt; WormBook; actin; ephrin; fasciculation; growth cone; microtubule; netrin; semaphorin.

Figure 1

Overall architecture of axon tracts in C. elegans. (A) Adult animal, side view, showing major ganglia, process bundles (dorsal and ventral nerve cords, DNC and VNC), and lateral neurons ALM, AVM, BDU, and SDQ. (B) Head region, ventral view, showing the left and right bundles of the VNC. (C) Midbody region, dorsal view, showing motor commissures, the DNC and dorsal sublateral tracts. Images are of the pan neural marker Prgef-1-GFP(evIs111); Bars, 50 μm (A), 20 μm (B, C).

Figure 2

Schematic drawing of the nervous system and dorso-ventral navigation. (A) Cross-section of the mid body showing positions of longitudinal axon tracts relative to the epidermis and muscle. (B) Axonal trajectories along the dorso-ventral axis and idealized representation of gradients of guidance cues. (C) Schematic of the VNC from a dorsal viewpoint, showing the relative locations of cell bodies, left and right bundles, commissures, and axons projecting into the VNC.

Figure 3

Important proteins controlling axonal navigation. This summarizes major pathways focusing on those more directly implicated in growth cone guidance.

Figure 4

Axon guidance models. (A) In the attraction-repulsion model, extracellular cues act as attractants or repellents. Here, the neuron only responds to the attractant ligand and the receptor mediates outgrowth activity that is directed toward the source of the attractant. In the self-organizing polarization model, a signal triggers feedback loops that asymmetrically localizes receptor signaling. The response to the cues increases or decreases the probability of where the receptor will mediate outgrowth activity. (B) Because the direction of outgrowth stochastically fluctuates at any instant of time, the movement of outgrowth can be considered as a succession of random steps. Depicted here are different objects (any mass) on a line, and 50 random walks of 50 steps from each point according to the probability distribution below. A probability distribution for the location of the object after the walk is given by the endpoints of the lines. The displacement of an object varies according to the degree of fluctuation. Because the outgrowth activity along the surface of an axon fluctuates, the moments of the membrane (a unit of mass) would move according to the same properties. (C) To illustrate how cues control the direction of outgrowth according to each model, an axon is depicted that approaches and crosses over an intermediate target, which expresses two different cues. In the attraction-repulsion model, the axon must switch its response from attraction to repulsion. In the self-organizing polarization model, the cues continue to promote outgrowth activity, but there is a change in the degree to which the direction of outgrowth fluctuates. The random walk models illustrate that the direction of outgrowth does not change, although the displacement does.

Figure 5

Axon regeneration in touch neurons and motor neurons. (A) Cartoons of morphological stages in regrowth after laser injury, based on studies of mechanosensory neurons (Wu et al. 2007). (B) Images from time lapse movie of PLM axotomy and regeneration. PLM soma is to the right. At 2 hr post axotomy the proximal and distal severed ends have retracted a few microns. By 3 hr, 50 min, the proximal end is extending filopodia; by 4 hr, 30 min, a morphologically distinct growth cone has reformed, which begins to extend anteriorly by 6 hr. (C) Images of PLM regrowth in the wild type, regeneration defective mutants (dlk-1, unc-75), and regeneration-enhanced mutants (efa-6). Images are 24 hr post axotomy; Bar, 10 μm; transgene Pmec-4-GFP(zdIs5) or Pmec-7-GFP(muIs32). (D) Confocal images of GABAergic motor neuron regrowth in wild type and in regeneration-defective mutant (unc-75). Marker, Punc-25-GFP(juIs76).

Figure 6

Pathways and genes involved in axon regrowth. Overview of stages and selected regulators in C. elegans axon regeneration. Growth promoting factors are in green, and growth-inhibiting factors in red. See text for details.