Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Sep 18;104(38):15132-7.
doi: 10.1073/pnas.0707001104. Epub 2007 Sep 11.

Caenorhabditis Elegans Neuronal Regeneration Is Influenced by Life Stage, Ephrin Signaling, and Synaptic Branching

Affiliations
Free PMC article

Caenorhabditis Elegans Neuronal Regeneration Is Influenced by Life Stage, Ephrin Signaling, and Synaptic Branching

Zilu Wu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

We previously reported functional regeneration of Caenorhabditis elegans motor neurons after femtosecond laser axotomy. We report here that multiple neuronal types can regrow after laser axotomy using a variety of lasers. The precise pattern of regrowth varies with cell type, stage of animal, and position of axotomy. Mechanosensory axons cut in late larval or adult stages displayed extensive regrowth, yet failed to reach their target area because of guidance errors in the anteroposterior axis. By contrast, mechanosensory axons cut in early larval stages regrew at the same rate but with fewer anteroposterior guidance errors, and were more likely to reach their target area. In adult animals lacking the VAB-1 Eph receptor tyrosine kinase, mechanosensory axon regrowth was more accurate than in the wild type, suggesting that guidance errors of regrowing touch neuron axons are the result of Eph signaling. Kinase-dependent and kinase-independent Eph signaling influenced outgrowth and guidance of regrowing touch neurons, respectively. Mechanosensory neurons regrew when severed proximal to their collateral synaptic branch but did not regrow when severed distal to the branch point. However, the distal axon could regrow if the branch is removed surgically at the same time as distal axotomy, or at a later time. The touch neuron synaptic branch point may act as a sorting area to regulate growth. These findings reveal that multiple influences affect regenerative growth in C. elegans neurons.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Regrowth of D motor neurons after axotomy. (A) D neuron morphology and synaptic polarity; triangles indicate neuromuscular junctions; X marks indicate sites of axotomy in mid lateral commissure and in ventral cord. Confocal of D neuron morphology in L4 stage, juIs76 marker. (Scale bar: 20 μm.) (B) When D neuron commissures were cut at the mid lateral position (arrow), they retracted as far as the lateral edges of the muscle quadrants. Proximal stumps of cut D commissures became swollen and began to sprout within 6–12 h of cutting. Most DD neurons cut in the L4 stage regrew to the dorsal cord within 8–10 h (e.g., 21/23 DD5s). In all figures, red asterisks indicate a regrown process, and df denotes the distal fragment of the cut axon. Midbody D neurons become unable to regrow dorsally as the reproductive system develops. Neurons DD4 + DD5 (marker juIs145) were cut in L4 stage. At 30 h, DD4 has not grown, whereas DD5 has reached the dorsal cord. (C) DD4 and DD5 (marker ynIs37) cut in L3 stage; both regrow to dorsal cord by 10 h. Note splitting and lateral extension of both regrowing processes as they meet dorsal muscle (*), similar to the behavior of VD growth cones in development (31); one of the DD4 branches has been pruned by 24 h. (D) Growth of new DD5 commissure to dorsal cord after cutting in ventral process (arrow, 0 h), L4 stage juIs145.
Fig. 2.
Fig. 2.
Decline in guidance of touch neuron regrowth during larval development. (A) Anatomy of anterior touch neurons ALM and AVM. X, site of axotomy in ALM, proximal to the synaptic branch in the nerve ring (nr). (B) Aberrant regrowth of ALML after axotomy in mid L4 stage. A growth cone is visible at 24 h; by 48 h, the regrown process has reversed posteriorly; the distal fragment (df) is beaded yet clearly visible at 48 h. (C) Regrowth of ALML after axotomy in L1 stage. At 12 h, the proximal stump of ALMR has formed a growth cone but has not yet begun to extend; the distal fragment is faint and beaded. By 21 h, ALMR has extended anteriorly and ventrally, sending two branches to the ventral cord (marked by AVM process); the ALMR distal fragment is no longer visible. By 29 h, ALMR has reached the nerve ring; one of the ventral cord branches has retracted. (Scale bars: 10 μm.) (D) ALML regrowth into the nerve ring (nr) 24 h after axotomy in L1. The regenerated ALM (*) lacks the anterior process distal to the synaptic branch. (E) Growth rates of ALM between 10 and 24 h and 24 and 48 h (gray bars) after axotomy in different stages; mean ± SEM; n > 5 for each; only adult growth rates are significantly slower (t test). *, P < 0.05.
Fig. 3.
Fig. 3.
Eph signaling influences guidance of regrowing touch neurons. (A) PLM processes cut in L4 stage proximal to the branch point by using MHz laser, marker zdIs5. Note prominent growth cones at 9–25 h and posterior turning between 25 and 56 h. (B) PLM axons turn less in vab-1(e2027) background; b, PLM branch. (Scale bar: 10 μm.) (C) Growth ratio (total distance grown by axon divided by net distance from cut site to tip of axon) in wild type, vab-1 null and kinase-dead (e118) mutants, and efn-1(ju1). The total regrowth of PLMs at 24 h was not significantly different (wt, 100 ± 18 μm; vab-1, 64 ± 8 μm; P = 0.09 by t test). (D) maximum dorsoventral distance (DV, dashed line in 56 h panel) of regrown processes from distal fragment, in wild type, vab-1(e2027), vab-1(e118) animals cut as L4s and scored 48 h later. (E) Regrowth of anterior and posterior processes after anterior axotomy in MYR::VAB-1 (quIs5) and MYR::VAB-1(G912E) (quIs4) L4 animals, and WT controls; all strains contain the zdIs5 marker. Anterior regrowth is significantly reduced only in quIs5 expressing animals. *, P < 0.05; **, P < 0.01.
Fig. 4.
Fig. 4.
The touch neuron synaptic branch inhibits regrowth of the distal axon. All animals were cut in mid L4 stage, zdIs5 marker. (A) The PLM distal process does not regrow after cutting; in some animals, the synaptic branch grows in response (*). (B) Retraction of PLM synaptic branch stump after cutting (arrow). (C) The distal process regrows when the branch is also cut. (D) Distal axon regrowth stimulated when the branch is cut 12 h after the distal axon. Anterior is to the left and dorsal up in all panels. (Scale bars: A–C, 5 μm; D, 10 μm.) (E) Process regrowth in 24 h after kHz or MHz axotomy of proximal (P), distal (D) axon, or distal + branch (D+B), or branch point (BP); also plotted is branch growth after cutting distal axon [D(B)]; n = 6–15 for each group; comparisons use t test. ***, P < 0.001; ns, not significant. Axon growth after the delayed branch cut (D) is measured between 24 and 48 h after the first cut.

Similar articles

See all similar articles

Cited by 94 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback