Axon regeneration pathways identified by systematic genetic screening in C. elegans

Abstract

The mechanisms underlying the ability of axons to regrow after injury remain poorly explored at the molecular genetic level. We used a laser injury model in Caenorhabditis elegans mechanosensory neurons to screen 654 conserved genes for regulators of axonal regrowth. We uncover several functional clusters of genes that promote or repress regrowth, including genes classically known to affect axon guidance, membrane excitability, neurotransmission, and synaptic vesicle endocytosis. The conserved Arf Guanine nucleotide Exchange Factor (GEF), EFA-6, acts as an intrinsic inhibitor of regrowth. By combining genetics and in vivo imaging, we show that EFA-6 inhibits regrowth via microtubule dynamics, independent of its Arf GEF activity. Among newly identified regrowth inhibitors, only loss of function in EFA-6 partially bypasses the requirement for DLK-1 kinase. Identification of these pathways significantly expands our understanding of the genetic basis of axonal injury responses and repair.

Figure 1. Overview and results of axon regrowth screen

(A) Flow chart of screen strategy. (B) Pie chart showing fraction of genes screened displaying significantly reduced or increased regrowth at 24 h. (C) Distribution of increased/decreased regrowth (P < 0.01 and P < 0.05) mutants among nine functional or structural gene classes, shown as % of genes in each class. Color code as (B) except that genes with P < 0.05 (orange, light blue) are omitted. See Table S1 for lists of genes in each class. (D) Total regrowth at 6 h and at 24 h are significantly correlated among 50 genes tested (Pearson r = 0.7, P < 0.0001). Each dot represents a single gene/mutant. Red line, linear regression; slope = 0.72, R² = 0.52, P < 0.001. Two mitochondrial mutants (isp-1, nduf-2.2) display normal regrowth at 6h and reduced regrowth at 24 h suggesting mitochondrial function becomes important during later regrowth; see also Figure S1.

Figure 2. Regrowth requires a subset of synaptic vesicle recycling genes

(A) Normalized regrowth in mutants lacking selected synaptic vesicle and trafficking genes (mean ± SEM). (B) Timecourse of regrowth in unc-57/Endophilin mutants (mean ± SEM); growth rates plotted for each time period. (C) Transgenic rescue of the unc-26 and unc-57 regrowth phenotypes. (D) Heat shock induced expression of UNC-57 can rescue the unc-57 regrowth phenotypes when animals are heat shocked before or after axotomy. Statistics, t test. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

Figure 3. Slit/Robo signals inhibit PLM axon extension

(A) Normalized 24 h PLM regrowth in mutants affecting axon guidance, cell adhesion, and extracellular matrix. (B) slt-1 and sax-3 mutants display increased PLM regrowth at 24 h; overexpression of SLT-1 in body wall muscles (kyEx441) or of SAX-3 in touch neurons (juEx2219) caused reduced regrowth. The inhibitory effect of SLT-1 overexpression is dependent on sax-3. Regrowth normalized to WT (zdIs5) = 1 ± 0.04 (mean ± SEM). (C) Representative images of PLM axon regrowth in slt-1 and sax-3 mutants at 24 h; red arrows indicate lesion sites, yellow dotted lines indicate original path of PLM. Scale, 10 μm. (D) Reduced SAX-3 activity after axotomy enhances regrowth. When shifted from 20°C to 25°C immediately after axotomy (red), sax-3(ky200ts) mutants displayed increased regrowth compared to unshifted ky200 animals (black). (E, F) slt-1 and sax-3 mutants display faster axon extension in the 6–24 h time period. Statistics, t test; n values in columns; ***, P < 0.001; ##, P < 0.01; *, P < 0.05; ns, not significant.

Figure 4. EFA-6 inhibits the early phase of axon regrowth

(A) PLM axon regrowth at 24 h is increased in efa-6 mutants, normalized to controls (n values in bars). (B) Images of wild type and efa-6 axons at 24 h. Red arrows, site of axotomy. Scale, 10 μm. (C) Axon growth is increased in efa-6 from 0–14 h post axotomy but not later. (D) Inducible overexpression of EFA-6 can inhibit regrowth only at the time of axotomy (time of heat shock relative to axotomy in h). (E) The effect of efa-6(tm3124) on axon regrowth can be reversed by overexpression of EFA-6 using the mec-4 (touch neuron) or rgef-1 (pan-neural) promoters, but not the myo-3 (muscle) promoter. The EFA-6 N-terminus, but not the Sec7 GEF domain, is necessary and sufficient to inhibit regrowth. Domain deletions or point mutations indicated below; n ≥ 9 for each condition. (F) Rescue of efa-6(lf) by single-copy insertions (SCI) of full-length EFA-6 (juSi51) or EFA-6(E447K) (juSi53). n ≥ 11 for each condition. All charts show mean ± SEM; statistics, t test; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. See also Figure S4.

Figure 5. EFA-6 negatively regulates axonal microtubule dynamics downstream or in parallel to DLK-1

(A) Normalized regrowth of efa-6, ebp-1, and double mutants. efa-6(lf) does not bypass the requirement for ebp-1, nor does EFA-6 overexpression enhance the regrowth reduction of ebp-1 mutants. (B) Expanded but immotile growth cone like structures formed in severed axon stumps in ebp-1(lf) mutants and EFA-6 overexpressors at 24 h post axotomy; cf. the lack of growth cones in axon stumps of dlk-1 mutants (panel F). (C,D) Analysis of MT dynamics in regrowing axons; number of dynamic MTs (EBP-2::GFP nucleation events) detected in kymographs is indicated in bars (C). efa-6(tm3124) mutants display increased numbers of dynamic MTs. Overexpression of the EFA-6 N-terminus (efa-6(gf), juEx3533) decreases the number of dynamic MTs. (D) Kymographs of MT dynamics in PLM axons 3 h post axotomy in the 40 μm region proximal to the site of axotomy, visualized with Pmec-4-EBP-2::GFP (juEx2843); scale, 10 μm. (E) Microinjection of taxol increases regrowth in efa-6(gf) animals compared to buffer-injected controls. The effect of taxol on wild type (muIs32) is not significant. (F) Regrowth 6 h post axotomy is increased in dlk-1 efa-6 double mutants compared to dlk-1 single mutants. (G) Images of dlk-1 single and double mutant axons at 6 h post axotomy. The region in the boxed area is enlarged at right. Red arrowheads indicate end of distal fragment closest to axotomy site. Scales, 10 μm. All charts show mean ± SEM; statistics, t test or Mann-Whitney test; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

Figure 6. Interactions among growth-promoting and growth-inhibiting pathways

(A) dlk-1(lf) is epistatic to mutants displaying enhanced regrowth. In all panels regrowth is normalized to wild type at 24 h; mean ± SEM. (B) Loss of function in pde-4 or gain of function in egl-19 partly suppress unc-57. (C) unc-51, unc-57, and dlk-1 are epistatic to slt-1 in regrowth. (D) DLK-1 overexpression (Prgef-1-DLK-1, juEx2789) can fully suppress the reduced regrowth of unc-51 and unc-57 mutants. (E) Loss of function in efa-6 partly suppresses the reduced regrowth of unc-26, unc-51, and dlk-1, but does not suppress ebp-1 (ebp-1 efa-6 data from Fig. 5 are included for comparison). (F) efa-6 slt-1 double mutants display enhanced regrowth compared to single mutants at 48 h post axotomy. All statistics, t test; n ≥ 10 for each condition except panel D (n ≥ 5); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7. A genetic pathway for PLM axon regrowth

Summary of relationships between regrowth-promoting (green) and regrowth-inhibiting (red) genes in PLM regrowth, incorporating data reported here and previous results (Ghosh-Roy et al., 2010; Yan et al., 2009).