Axon Regeneration Pathways Identified
by Systematic Genetic Screening in C. elegans
Lizhen Chen,1Zhiping Wang,1Anindya Ghosh-Roy,1Thomas Hubert,1Dong Yan,1,2Sean O’Rourke,3Bruce Bowerman,3
Zilu Wu,1,2Yishi Jin,1,2,4,* and Andrew D. Chisholm1,*
1Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
2Howard Hughes Medical Institute
3Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
4Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
*Correspondence: firstname.lastname@example.org (Y.J.), email@example.com (A.D.C.)
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. Weuncoverseveral functionalclus-
ters of genes that promote or repress regrowth,
including genes classically known to affect axon
guidance, membrane excitability, neurotransmis-
sion, and synaptic vesicle endocytosis. The con-
served Arf Guanine nucleotide Exchange Factor
(GEF), EFA-6, acts as an intrinsic inhibitor of re-
growth. By combining genetics and in vivo imaging,
tion in EFA-6 partially bypasses the requirement for
icantly expands our understanding of the genetic
basis of axonal injury responses and repair.
Damage to the adult mammalian CNS, in stroke or in spinal cord
injury, remains devastatingly untreatable. Despite significant
recent advances in our understanding of selected pathways,
strategies for treating CNS injury remain limited. Axon injury in
mature neurons triggers injury responses and repair pathways
(Abe and Cavalli, 2008). These pathways activate regrowth
programs whose effectiveness depends on both the intrinsic
growth competence of the neuron (Sun and He, 2010) and the
local extracellular environment (Busch and Silver, 2007). Much
attention has focused on the regrowth-inhibiting properties of
CNS myelin components such as Nogo (Schwab, 2010).
However, the roles of specific myelin components in vivo remain
a matter of debate (Cafferty et al., 2010; Lee et al., 2010).
Compared to the effects of extrinsic cues, less is known about
intrinsic mechanisms affecting regrowth competence. Experi-
mental paradigms such as the conditioning lesion show that
neuronal sensitivity to extrinsic influences in regeneration is
under the control of intrinsic pathways (Enes et al., 2010; Hannila
and Filbin, 2008; Ylera et al., 2009). Intrinsic triggers of regrowth
include positive injury signaling pathways such as the MAP
kinases Erk and JNK, which are activated by injury and retro-
gradely transported from sites of damage (Perlson et al., 2005).
Differences in regenerative ability at different stages also reflect
alterations in intrinsic growth capacity (Moore et al., 2009).
Analysis of regeneration-competent neurons in the vertebrate
PNS and in model organisms has given insight into pathways
that promote axon regrowth after injury (Ambron et al., 1996;
Chen et al., 2007). Several studies have used genomic or pro-
teomic approaches to identify regeneration-associated genes
(Michaelevski et al., 2010). As yet, a limited number of such
genes have been tested for function in vivo. An important goal
is to exploit new models for large-scale screening and gene
discovery that will open up additional therapeutic avenues.
The nematode C. elegans is an emerging model for genetic
and chemical screens for factors affecting axon regeneration
after injury (Ghosh-Roy and Chisholm, 2010; Samara et al.,
2010; Wang and Jin, 2011). Axons labeled with GFP transgenes
can be severed precisely with ultrafast laser irradiation (Yanik
et al., 2004). Although laser axotomy of single axons differs in
the precise mechanism of damage from mechanical severing
or crush injuries of vertebrate nerves, at least some regrowth
mechanisms are conserved. In C. elegans, as in vertebrate
neurons, the second messengers Ca2+and cAMP are rate
limiting for axonal regrowth (Ghosh-Roy et al., 2010; Neumann
et al., 2002; Qiu et al., 2002). Pharmacological screening in
C. elegans revealed a conserved role for protein kinase C in
regenerative growth (Samara et al., 2010). Finally, the Dual
Leucine Zipper Kinase/DLK-1 cascade was first demonstrated
in C. elegans as essential for axonal regrowth (Hammarlund
et al., 2009; Yan et al., 2009) and is required for axon regenera-
tion in Drosophila (Xiong et al., 2010) and likely in mouse (Itoh
et al., 2009). These results suggest that axon regrowth after
laser surgery involves pathways required in other models of
Here, we exploit the rich genetic resources of C. elegans
to perform a large-scale mutation-based screen for genes
with roles in adult axon regrowth. We identify many genes
required for axonal regrowth, most of which are not required
Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc. 1043
for developmental axon outgrowth and have not previously been
implicated in axon regeneration. By analyzing regeneration at
multiple time points and in double mutants, we order the activity
of newly characterized genes relative to each other and to the
DLK-1 cascade. Manipulation of the conserved pathways
identified here could significantly expand current strategies to
augment the regenerative abilities of damaged neurons.
Functional Screen for Axon Regrowth Genes
To identify conserved genetic pathways affecting axon regrowth
we selected >650 C. elegans genes based on their orthology to
human genes and potential neuronal function or known bio-
chemical role (Figure 1A; see Experimental Procedures). We
focused on genes not essential for overall health or growth
rate; for >90% of the genes, we examined genetic null mutants
(see Table S1 available online). To assay axon regrowth in vivo,
we used mechanosensory PLM neurons, which consistently
regrow after laser axotomy (Wu et al., 2007). Over 95% of
mutants displayed normal PLM axon development; mutants
with aberrant development are summarized in Table 3. In the
primary screen, we severed the PLM axon using femtosecond
laser surgery in 10–20 animals per genotype. Under our condi-
tions >95% of PLM neurons survive surgery (Wu et al., 2007).
After 2–4 hr, the proximal axon stump swells and forms a
growth cone-like structure that extends over the next 24–48 hr.
Wild-type PLM axons regrow in an error-prone manner and
can reestablish synaptic connections in certain genetic back-
Figure 1. Overview and Results of Axon
(A) Flow chart of screen strategy.
(B) Pie chart showing fraction of genes screened
displaying significantly reduced or increased re-
growth at 24 hr.
(C) Distribution of increased/decreased regrowth
(p < 0.01 and p < 0.05) mutants among nine
functional or structural gene classes, shown as
percentage of genes in each class. Color coding
as in (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 hr and at 24 hr are signifi-
cantly 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 6 hr and reduced regrowth at 24 hr suggesting
mitochondrial function becomes important during
later regrowth; see also Figure S1.
All bar charts in this and other figures display
mean ± SEM.
grounds (Ghosh-Roy et al., 2010). Mu-
tants showing altered regrowth at 24 hr
(Figures 1B and 1C and Tables 1 and 2)
were retested in a secondary screen
(?200 genes). As we sever axons in the
mid-L4 stage when animals are growing, reduced regrowth
could also reflect developmental delay or arrest in response to
our axotomy procedure. We measured the growth of intact
neurons in selected strains and found no significant effects on
organismal growth rate (Figure S1A).
Altered regrowth 24 hr postaxotomy could reflect defects in
growth cone formation or in later processes of axon extension.
We analyzed 60 mutants with altered regrowth at 24 hr for their
effects at 6 hr postaxotomy, when wild-type axons have just
begun to extend (Figure S1B). Most mutants with reduced re-
growth at 24 hr displayed proportional effects at 6 hr (Figure 1D),
suggesting these genes act throughout regrowth. However,
some mutants displaying increased regrowth at 24 hr (e.g.,
slt-1, sax-3; see Figures 3E and 3F) did not significantly affect
regrowth at 6 hr, suggesting these genes affect later axon exten-
sion. Conversely, a few mutants (efa-6; see Figure 4C) displayed
larger increases in regrowth at 6 hr than at 24 hr, suggesting
a preferential effect on early stages of regrowth.
initial regrowth were involved in growth cone formation. By 6 hr
postaxotomy, between 40% and 60% of wild-type PLM axon
stumps form growth cones; on average, axons with growth
cones at 6 hr extend further than those without growth cones
(Figure S1C), suggesting growth cones reflect growth rather
than stalling. Among 50 mutants tested at 6 hr the fraction of
growth cones positively correlated with regrowth (R2= 0.11,
p = 0.01, Figure S1D), suggesting many genes required for re-
growth affect growth cone formation. At 24 hr, the fraction of
growth cones did not correlate with regrowth (data not shown),
Genetics of Axon Regeneration
1044 Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc.
possibly reflecting a more stochastic presence of growth cones
in axon extension. However, mutants displaying increased re-
growth at 6 hr (e.g., efa-6) did not display a higher fraction of
growth cones than wild-type (Figure S1D), suggesting the wild-
type level of growth cone formation is a phenotypic ceiling. We
conclude that growth cones correlate with early regrowth but
not with overall regrowth at later time points.
Clusters of Genes Required for Axonal Regrowth
but Not for Development
We found genes affecting PLM regrowth among all structural
and functional classes tested (Figures 1B and 1C and Tables
1–4). When analyzed as nine gene classes (Figure 1C), genes
promoting regrowth (i.e., those displaying reduced growth in
loss-of-function mutants) were more frequent in the ‘‘cytoskel-
eton and motors’’ and ‘‘neurotransmission’’ classes. Genes in-
hibiting regrowth (i.e., increased regrowth in loss-of-function
mutants) were concentrated in the ‘‘cell adhesion/extracellular
matrix’’ class (Figure 1C). Here, for reasons of space limitations,
transporters, neurotransmitters, and gene expression.
Neuronal excitability can promote regrowth (Brushart et al.,
2002), but can also act as an intrinsic negative signal via
L-type voltage gated calcium channels (Enes et al., 2010). In
C. elegans, neuronal excitability is generally influenced by the
opposing action of voltage-gated calcium and potassium chan-
nels (Goodman et al., 1998); the voltage-gated Ca2+channel
EGL-19 is required for regrowth of PLM neurons (Ghosh-Roy
et al., 2010). We tested 53 additional channels and associated
proteins (Figure S2A) and found a cluster of genes affecting
both Ca2+and Na+ionic balance to be critical for regrowth,
including the Ca2+channel regulator UNC-80 (Jospin et al.,
2007), the Na+pump NKB-1 (Doi and Iwasaki, 2008), the stoma-
tins UNC-1 and UNC-24 (Sedensky et al., 2004), and the Deg/
ENaC Na+channel UNC-8 (Tavernarakis et al., 1997). Among
these genes, UNC-24 and UNC-1 interact with UNC-8 and with
the mechanosensory channel complex, suggesting electrical
activity regulated by mechanosensory channels could promote
regrowth (Bounoutas and Chalfie, 2007). Conversely, loss of
function in the BK type K+channel SLO-1 (Wang et al., 2001)
or in the conserved K+channel regulatory protein MPS-1 (Cai
et al., 2005) resulted in enhanced regrowth. As loss of function
in K+channels should tend to increase membrane excitability,
these findings suggest excitability promotes PLM regrowth.
PLM regrowth was strongly reduced in mutants affecting
chemical neurotransmitters, including acetylcholine (cha-1/
ChAT and unc-17/vesicular ACh transporter), GABA (unc-25/
GAD), and biogenic amines (tph-1/Tryptophan hydroxylase)
(Figure S2B). Mutants affecting ACh synthesis or packaging
(cha-1, unc-17) or AChR biosynthesis (ric-3) displayed reduced
regrowth, suggestinga neurotransmitterroleof AChisimportant.
Chalfie, 1995), and we find that deg-3 mutants display strongly
reduced regrowth (Table 3). Although deg-3(u662) mutants also
display aberrant PLM development, PLM morphology was
normal in other cholinergic mutants tested (cha-1, etc), suggest-
ing the requirement for ACh in regrowth is separable from any
role in development.
Mechanosensory neurons are neither GABAergic nor receive
GABAergic input, suggesting an indirect role of GABA in
regrowth. Notably, regrowth did not require genes involved in
GABA vesicular packaging (unc-46, unc-47) or the postsynaptic
promoting roles in vertebrate neuronal development (Akerman
and Cline, 2007) and a trophic role in regenerating vertebrate
neurons (Shim and Ming, 2010; Toyoda et al., 2003). Specula-
tively, regenerating neurons may become more dependent on
trophic factors whose roles in development are masked by
The DLK-1 MAPK cascade is essential for axon regrowth after
injury (Hammarlund et al., 2009; Yan et al., 2009). We screened
over 80 additional protein kinases, representing approximately
one-fourth of all conserved C. elegans kinases (Manning, 2005),
as well as selected protein phosphatases (Figure S3). In addition
to the members of the DLK-1 MAPK cascade, several cytosolic
kinases were important for regrowth, including the stress-acti-
vated KGB/MEK-1 pathway, the p21-activated kinase MAX-2
and the Atg1 kinase UNC-51 kinase. Of these, only MAX-2
and UNC-51 have been previously linked to axonogenesis in
C. elegans (Lucanic et al., 2006; Ogura et al., 1994); UNC-51,
but not MAX-2 is required for PLM developmental outgrowth
(Table 3). We also find that PKC-1/protein kinase C can promote
PLM regrowth, consistent with a recent report (Samara et al.,
2010). Additionally, among 12 protein phosphatases tested, we
identified the LAR-like receptor tyrosine phosphatase PTP-3
(Ackley et al., 2005) and the PP2A regulatory subunit PPTR-1
as critical for regrowth (Table 1; Figure S3C). LAR has been
implicated in axon regrowth in vertebrates (Xie et al., 2001). To
our knowledge PP2A has not been linked to axon regrowth. In
C. elegans PPTR-1 negatively regulates Akt signaling (Padma-
(Gallo et al., 2010). Loss of function in akt-1 or akt-2 did not
significantly affect regrowth (Figure S3A). AKT-1 and AKT-2
could play redundant roles; alternatively PPTR-1 may promote
regrowth via RNP stabilization.
Axonal injury induces pervasive changes in gene expression
(Yang et al., 2006) and our previous studies implicated bZip
proteins in regrowth (Ghosh-Roy et al., 2010; Yan et al., 2009).
We tested 130 additional genes implicated in RNA metabolism,
transcription, and translation, as well as specific transcription
factors. The Argonaute-like protein ALG-1 (Grishok et al., 2001)
was critical for regrowth, implying a regrowth-promoting role
for microRNAs. Several proteins affecting chromatin remodeling
were required, including the SWI/SNF complex component
XNP-1/ATR-X. Conversely, loss of function in the histone deace-
tylase HDA-3/HDAC3 improved regrowth (Table 2); as loss of
HDA-3 function is neuroprotective in a C. elegans model of
polyglutamine toxicity (Bates et al., 2006), HDA-3 may act
generally to repress neuroprotective genes. Of 63 transcription
factors tested, the neurogenin bHLH family member NGN-1
(Nakano et al., 2010) showed a strong requirement (Table 1).
As PLM neuron differentiation was normal in ngn-1 mutants,
NGN-1/neurogenin may specifically promote regrowth. The
range of gene expression regulators identified here underscores
the complexity of the changes in gene expression following
Genetics of Axon Regeneration
Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc. 1045
Table 1. Selected Mutants Displaying Reduced PLM Regrowth
Regrowth (24 hr)p Value
A. Cell Adhesion and ECM
sdn-1zh20 0.25 ± 0.06 <0.001Normal Syndecan SDC2
sax-7/lad-1 ok12440.30 ± 0.050.001Normal L1 IgCAM NRCAM
rig-3 ok21560.43 ± 0.040.001 NormalGPI-linked IgCAMNCAM1
rig-4 ok11600.52 ± 0.060.001 NormalSidekick IgCAM SDK1
B. Channels and Transporters
unc-1e719 0.67 ± 0.080.037Normal Stomatin STOM
unc-32e189(lf)0.41 ± 0.05 0.020 NormalV0 ATPase
Na+/K+ ATPase b subunit
nkb-1ok1089 0.45 ± 0.05 0.039 12% US
C. Cytoskeleton and Transport
ebp-1 tm13570.29 ± 0.02 0.001 Normal MT End-binding proteinMAPRE1
unc-14ju56 0.13 ± 0.02***NormalRUN domainRUSC2
unc-104 e1265 0.37 ± 0.120.009 NormalKIF1A kinesin KIF1B
kap-1ok676 0.46 ± 0.060.002 NormalKinesin-associatedKIFAP3
jip-1km18 0.71 ± 0.080.018Normal JNK interacting proteinMAPK8IP2
D. Protein Kinases and Phosphatases
max-2 ok19040.35 ± 0.090.054 Normal p21 activated kinasePAK3
ptp-3 ok2440.63 ± 0.1 ***NormalLAR receptor tyrosine
pptr-1tm2954 0.51 ± 0.090.031NormalPP2A regulatory subunitPPP2R5E
E. Neurotransmission, Lipid Signaling, Metabolism
dgk-1 nu62, ok14820.49 ± 0.070.001 NormalDiacylglycerol kinase q
sphk-1 ok10970.65 ± 0.050.039 NormalSphingosine kinaseSPHK1
nduf-2.2 ok4370.52 ± 0.08**NormalNADH Ubiquinone reductaseNDUFS2
isp-1 qm150 0.46 ± 0.090.017Normal Fe-S proteinUQCRFS1
F45E4.5ok560, ok2285 0.48 ± 0.09 0.008NormalPiccolo/Aczonin PCLO
ric-3md1580.34 ± 0.05 **NormalAChR maturationRIC3
unc-25e156 0.61 ± 0.080.047 NormalGlutamic acid
cha-1 p1152 0.61 ± 0.08 <0.001NormalCholine acetyltransferase CHAT
unc-17e245, e113 0.41 ± 0.09 <0.001 40% BranchlessACh vesicular transporterSLC18A3
tph-1mg280 0.50 ± 0.070.024Normal Tryptophan hydroxylase TPH2
F. Cell Signaling and Protein Interaction Domains
cwn-2 ok895 0.48 ± 0.05 0.001NormalWnt ligand WNT5B
tag-60ok2292 0.30 ± 0.06***NormalPDZ domainSLC9A3R2
tag-68 gk1850.48 ± 0.07***Normal I-SmadSMAD6
dab-1gk2910.51 ± 0.08 0.001 NormalDisabledDAB2
G. Membrane Trafficking
unc-26e1196 0.31 ± 0.050.002 NormalSynaptojanin SYNJ1
unc-57e406 0.21 ± 0.040.001 NormalEndophilinSH3GL3
sec-22ok3053 0.63 ± 0.04 0.01728% OSTI-VAMPSEC22B
unc-41e2680.42 ± 0.08<0.001NormalStonin STON2
H. Protein Degradation, Proteases, Cell Death
uev-3ju6390.51 ± 0.14<0.001NormalUbiquitin E2 ligase variant UBE2N
I. RNA Metabolism, Chromatin, Transcription Factors, Translation
alg-1gk214 0.72 ± 0.150.012NormalArgonaute-like EIF2C4
unc-75e9500.52 ± 0.060.001 Normal RRM RNA-bindingTNRC4
ngn-1 ok22000.41 ± 0.05 ** 10% OSNeurogenin bHLHNEUROD1
Genetics of Axon Regeneration
1046 Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc.
Axon Regrowth Requires Genes Functioning in Synaptic
Axon regrowth was strongly reduced in a cluster of mutants
previously thought to be dedicated to synaptic vesicle (SV) re-
cycling (Figure 2A), including unc-26/Synaptojanin, unc-57/
Endophilin, and unc-41/Stonin. These are ‘‘core module’’
proteins or ‘‘secondary effectors’’ in SV endocytosis (Dittman
and Ryan, 2009). In contrast, genes involved in SV exocytosis,
such as unc-13/mUnc13, unc-18/mUnc18, or unc-10/Rim,
were not required for regrowth (Figure 2A). Both unc-26 and
unc-57 mutants displayed significantly reduced regrowth at
6 hr; unc-57 mutants displayed reduced regrowth from 6 to
24 hr, but not from 24 to 48 hr (Figure 2B). Expression of
UNC-57 driven by its own promoter, or pan-neural expression
of UNC-26 rescued axon regrowth defects, supporting the
view that the SV endocytosis genes are required cell-autono-
mously for axon regrowth (Figure 2C). To address whether
UNC-57 acts continuously in regrowth, we expressed it under
the control of a heat shock promoter and induced UNC-57
expression by heat shock at times before and after axotomy.
Heat shock-induced expression of UNC-57 either 7 hr before
or 6 hr after axotomy could rescue the defects of unc-57
mutants (Figure 2D), suggesting a continuous requirement in
regenerative growth. As we sever the PLM axon at sites distant
from synapses, and SV exocytosis genes appear to be
dispensable for axon regrowth, the requirement for SV endocy-
tosis genes in regrowth may be independent of known roles in
Permissive and Inhibitory Roles of Extracellular Factors
Axon regeneration is influenced in many ways by the extracel-
lular environment. We tested approximately 60 genes encoding
extracellular matrix components, putative cell adhesion pro-
teins, and cell surface receptors (Figure 3A). Several such genes
were required for regrowth (Table 1), including the cell surface
proteoglycan SDN-1/Syndecan (Rhiner et al., 2005), the
L1CAM ortholog SAX-7/LAD-1 (Chen et al., 2001), the novel
GPIlinked IgCAM RIG-3, and the IgCAM RIG-4/Sidekick
(Schwarz et al., 2009). In vertebrate axons L1 is upregulated
after injury and required for regrowth (Becker et al., 2004);
however, syndecans or sidekick family members have not
previously been implicated in axon regeneration. Conversely,
loss of function in several putative basement membrane compo-
nents, such as spon-1/F-spondin (Woo et al., 2008) or pxn-2/
Peroxidasin (Gotenstein et al., 2010) resulted in enhanced
regrowth (Table 2). In vertebrates the ‘‘glial scar’’ is an ECM
barrier to CNS regeneration (Busch and Silver, 2007); although
C. elegans does not encode orthologs of glial scar components
such as chondroitin sulfate proteoglycans, these observations
raise the possibility that the basement membrane forms an
analogous barrier to PLM regrowth.
Wnt signals regulate the polarity of PLM neurite outgrowth in
development (Hilliard and Bargmann, 2006). We find PLM re-
growth involves distinct Wnt signals. For example the Wnt
CWN-2 is not required for PLM development yet is required for
regrowth (Table 1). CWN-2 is expressed anterior to PLM, sug-
gesting it could be permissive or attractive in PLM regrowth,
Table 1. Continued
Regrowth (24 hr)p Value
npp-12ok3350.64 ± 0.130.013NormalGp210nuclear pore complexNUP210
spr-1ok21440.55 ± 0.080.015NDCoRESTRCOR3
xpo-3ok12710.50 ± 0.080.01460% OS Exportin-tXPOT
egl-27ok1510.44 ± 0.080.025NormalNucleosome remodelingMTA1
Genes are classified in nine functional or structural classes. Mutations are genetic or predicted molecular nulls, unless partial loss of function (lf) indi-
cated. Normalized regrowth is relative to matched same-day controls or to pooled controls. Significance levels (*, p < 0.05; **, p < 0.01; ***; p < 0.001)
based on Mann-Whitney test in primary screen; numerical p values are from repeats, after correction for multiple comparisons.
aPLM development is scored in at least 20 worms per genotype; defects in other touch neurons are not indicated here; OS, overshooting axon.
bClosest human gene based on BLASTP score in Wormbase WS213; Ensembl/HGNC gene symbol.
Table 2. Selected Mutants Displaying Increased PLM Regrowth
pxn-2ju3581.97 ± 0.190.009NormalPeroxidasin ECM enzymePXDN
spon-1ju430ts1.77 ± 0.41(0.082)NormalF-spondin ECMSPON1
vab-19e1036cs1.63 ± 0.19(0.0003)NormalAnkyrin repeat proteinKANK1
sax-3ky1231.43 ± 0.100.009(mixed defects)Robo receptor for SLT-1ROBO2
efa-6ok3533, tm31241.42 ± 0.110.01440% OSArf6 GEFPSD4
mps-1ok13761.41 ± 0.100.043NormalK+ channel accessory subunitKCNE2
slt-1eh151.39 ± 0.100.013NormalSlit ligandSLIT1
hda-3ok19911.58 ± 0.14(0.1051)NormalHistone deacetylaseHDAC1
Conventions as for Table 1. The increased regrowth of pxn-2 mutants (Gotenstein et al., 2010) is included for comparison.
Genetics of Axon Regeneration
Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc. 1047
similar to its roles in other neurons (Kennerdell et al., 2009; Song
et al., 2010).
Among tested axon guidance pathways, Slit-Robo signaling
had an inhibitory effect on regeneration. Both slt-1/Slit and
sax-3/Robo null mutants displayed increased PLM regrowth,
and slt-1 sax-3 double mutants showed no further enhancement
in axon regeneration than either single mutant (Figures 3B and
3C). Further, overexpression of SAX-3 in touch neurons inhibited
PLM regrowth, indicating SAX-3/Robo can act cell autono-
mously to restrain regrowth (Figure 3B). Constitutive expression
of SLT-1 from body wall muscles also reduced PLM regrowth in
a SAX-3-dependent manner (Figure 3B). In development, SAX-3
activity has a minor role in promoting PLM outgrowth (Li et al.,
2008). To address whether SAX-3 acts at the time of regrowth
or earlier we performed temperature shift experiments on
sax-3(ky200ts) (Zallen et al., 1998) and found that animals
shifted to the restrictive temperature immediately postaxotomy
exhibited increased regrowth equivalent to sax-3 null mutants
(Figure 3D), indicating that SAX-3 acts at the time of regrowth.
Last, we addressed when in regrowth SLT-1 and SAX-3 signals
acted. slt-1 and sax-3 mutants displayed normal regrowth from
0 to 6 hr then increased regrowth during the 6–24 hr period
(Figures 3E and 3F), suggesting SLT-1 and SAX-3 signals inhibit
extension of the regrowing axon. In ventrally guided AVM axons,
SLT-1 signals play repulsive roles in development and regrowth
(Gabel et al., 2008; Hao et al., 2001). In contrast, in PLM neurons
SAX-3/Robo appears to switch from a growth-promoting role
during development to an inhibitory role in regrowth.
The Conserved Signaling Protein EFA-6 Is an Intrinsic
Inhibitor of Regeneration
Among the few genes with inhibitory effects on regrowth we
focused on EFA-6, the C. elegans member of the EFA6
(Exchange Factor for Arf6) family. EFA6 proteins contain a vari-
able N-terminal region, a Sec7 homology domain with GEF
activity specific to ARF6 GTPases (Franco et al., 1999), a pleck-
strin homology (PH) domain, and a coiled-coil domain (Fig-
ure S4A). C. elegans efa-6 mutants displayed mild PLM axon
overshooting in development (Figures S4B and S4C) and
enhanced regrowth of PLM (Figures 4A and 4B). Cell-type
specific transgenic expression of EFA-6 from pan-neural or
promoter, rescued efa-6 developmental defects (Figure S4C)
and inhibited PLM regrowth after axotomy both in efa-6(lf)
(Figure 4E) and efa-6(+) backgrounds (data not shown; see
also Figure 5), indicating EFA-6 acts cell autonomously and
Table 3. Roles of Axon Outgrowth and Branching Genes in PLM Regrowth
Regrowthp ValuePLM DevelopmentMolecular Function
A. Known Axon Outgrowth Mutants
unc-53e4040.12 ± 0.05 ***100% US NAVNAV2
unc-115e22250.28 ± 0.04**5% OS, 14% branchlessActin-binding LIMABLIM1
unc-73e936(lf), rh400.31 ± 0.1 ***100% US GEFTRIO
unc-69e587, ju690.43 ± 0.09*60% US (e587); Normal (ju69)Kinesin interactorFEX2
unc-51e369, ky347(lf)0.47 ± 0.10*30% US (ky347) Atg1 kinaseULK2
unc-76e911 0.67 ± 0.12NS 95% US, 80% branchlessKinesin interactorSCOC
unc-16e1090.68 ± 0.09 NS7% OS, 10% branchless JIP1 scaffoldSPAG9
unc-44e3620.77 ± 0.22*5% OS, 5% USAnkyrinANK1
unc-33 e204 0.79 ± 0.12NS2% OS, 10% branchless CRMP/TOADDPYSL3
B. Other Mutants with Reduced PLM Axon Outgrowth or Branching Defects
deg-3u6620.42 ± 0.07 ***50% short axons nAChR subunitCHRNA7
pig-1 gm144 0.55 ± 0.08* 40% USleucine zipper kinaseMELK
wsp-1gm324 0.83 ± 0.08 NS90% branchless WASPWASL
ddr-1 ok8740.74 ± 0.12 NS25% branchless Discoidin domain receptorDDR2
hst-2 ok5950.56 ± 0.10*11% branchlessHeparan sulfotransferase HS2ST1
klc-1ok2609 0.82 ± 0.07NS 10% extra branch Kinesin light chainhKLC1B
klp-10 ok704 0.85 ± 0.06NS15% extra branchKinesin-like KIF15
nfi-1 qa5240.79 ± 0.13 NS50% wavy processNuclear factor INF1A
oig-1ok1687 0.70 ± 0.10 NS26% extra branchOne Ig DomainPTPRF?
rack-1tm2262 0.74 ± 0.10 NS 10% extra branch Receptor of C-kinaseGNB2L1
ver-3gk227 1.28 ± 0.17 NS 10% extra branchVEGFR FLT1
F09A5.1ok2286 0.86 ± 0.08* 18% extra branch Spinster SPINL
vang-1ok11420.78 ± 0.10 NS40% proximal branch Van Gogh/Strabismus VANGL1
Y38H8A.4 ok27930.73 ± 0.07* 20% branchlessSerine/Threonine kinaseTSSK2
Conventions as for Tables 1 and 2; NS, not significant.
Genetics of Axon Regeneration
1048 Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc.
or sax-3, efa-6 mutants displayed enhanced regrowth during the
0–6 hr period (Figures 1D and 4C), implying EFA-6 acts early in
regrowth. Furthermore, heat shock induced EFA-6 overexpres-
sion 1 hr before axotomy inhibited PLM regrowth, whereas
induction earlier or later had little effect (Figure 4D).
To investigate the mechanism underlying EFA-6 function, we
next examined arf-6(lf) mutants. arf-6(lf) mutants displayed
modestly increased regrowth and did not further enhance
transgenes potently inhibited regrowth in arf-6(lf) backgrounds
(Figure 4E), suggesting EFA-6 acts on regrowth independent of
ARF-6. To dissect which functional domains of EFA-6 were
important in axon regrowth we expressed mutant EFA-6 lacking
either the Sec7 domain, the PH domain, or the C-terminal coiled
coil domain (Table S2). Each of these ‘‘gain-of-function’’ trans-
genes rescued efa-6 developmental overgrowth (Figure S4B)
and inhibited regrowth, as did constructs in which the conserved
catalytic residue of the Sec7 domain was mutated (E447K). In
contrast, expression of an EFA-6 variant lacking the N terminus
did not block PLM regrowth (Figure 4E). As overexpression of
EFA-6 might affect nonphysiological pathways, we made
single-copy insertion transgenes expressing full length EFA-6
or the E447K mutant and found that both rescued efa-6 devel-
opmental and regrowth phenotypes (Figure 4F), suggesting a
GEF-independent role for EFA-6 in inhibiting regrowth.
Our recent studies on C. elegans embryos indicate that EFA-6
(O’Rourke et al., 2010). Axon regeneration also involves precise
regulation of axonal MT dynamics (Ertu ¨rk et al., 2007). In our
screen we found that the MT plus-end binding protein EBP-1
Table 4. Genes Required for Normal Regrowth of PLM
A. Highly Significant Effect B. Significant Effect
Seq NameGene NameSeq Name Gene Name
W01B6.1cwn-2 F18F11.3 cdh-8
C09E10.2dgk-1 F56D1.6 cex-1
F47A4.2 dpy-22 Y73F8A.19 cpna-4
C07H6.7 lin-39H09G03.2 frm-8
Y38F1A.10 max-2F47D12.1 gar-2
T26A5.3 nduf-2.2 Y81G3A.3gcn-2
ZK1290.4 nfi-1 F38A6.3hif-1
T23H12.1 npp-12C34F6.4 hst-2
W03G1.6pig-1 F46F2.2 kin-20
W07A12.7 rhy-1R10E9.1 msi-1
T14A8.1ric-3 F44G3.9 nhr-111
C18F3.2 sax-7K06B9.5 pax-2
F57C7.3 sdn-1 T07E3.6pdf-1
Y52D3.1 strd-1Y42H9AR.3 rabs-5
F31E8.3 tab-1 C18H9.7rpy-1
F37D6.6 tag-68T03D8.3 sbt-1
F39B2.2 uev-1F31E8.2 snt-1
Table 4. Continued
A. Highly Significant EffectB. Significant Effect
Seq NameGene NameSeq NameGene Name
ZK637.8a unc-32 R13A1.4unc-8
(A) Seventy genes with significantly reduced regrowth in primary screen
(p < 0.01), listed by sequence (seq) name and CGC name. (B) Sixty-one
genes displaying reduced regrowth with p < 0.05.
Genetics of Axon Regeneration
Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc. 1049
(Srayko et al., 2005) was required for regrowth (Figure 5A). The
reduced regrowth of ebp-1 mutants could not be bypassed by
efa-6(lf) and was not further decreased by EFA-6 overexpression
(Figure 5A). Notably, the morphology of the axon stumps in
ebp-1 mutants resembled those in EFA-6 overexpressors (Fig-
ure 5B), suggesting the increased regrowth in efa-6 mutants
might reflect altered axonal MT dynamics.
To test whether EFA-6 affected axonal MT dynamics, we ex-
pressed the MT plus-end binding protein EBP-2 fused to GFP
(see Experimental Procedures). End binding protein GFP fusions
are established markers of growing ends of MTs in vertebrate
neurons (Stepanova et al., 2003) and in C. elegans embryos
(Srayko et al., 2005). In wild-type axons within 3 hr of axotomy,
before overt regrowth, axonal MTs (defined as motile EBP-
2::GFP puncta) became highly dynamic close to the severed
end of the axon (arrows, Figure 5D). In contrast, in efa-6(lf)
mutants axonal MTs were more abundant and regrew for longer
times and distances than in the wild-type (Figures 5C and 5D).
Conversely, in EFA-6 overexpressing axons the number of
Normalized regrowth (24 h)
synaptic vesicle recycling
Normalized regrowth (24 h)
regrowth rate (µm/h)
unc-57 unc-57; Ex[Phsp-UNC-57]
Normalized regrowth (24 h)
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) Time course 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
(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
dynamic axonal MTs was significantly reduced
(Figures 5C and 5D). Axonal MT dynamics
were normal in arf-6 mutants (data not shown),
suggesting enhanced regrowth in efa-6 mutants
is mainly due to the microtubule destabilizing
role of EFA-6.
To directly address whether the reduced re-
bilization of the MT cytoskeleton, we tested
whether the MT stabilizing drug taxol could
overcome regrowth inhibition. Delivery of taxol
by microinjection into the body cavity did not
affect regrowth in the wild-type, yet significantly
rescued regrowth of EFA-6-overexpressing
axons (Figure 5E). Conversely, incubation in
colchicine blocked axonal regrowth (data not
shown). These findings show that MT polymeri-
zation is critical for C. elegans axon regrowth
and support a specific role for EFA-6 promoting
catastrophe of axonal MTs.
Interactions among Regrowth Pathways
Our screen identified many genes with positive
and negative influences on PLM axonal re-
growth. To address how these pathways interact, we analyzed
regrowth in double and triple mutants. Genetic backgrounds
that elevate cAMP signaling (kin-2) display enhanced PLM
axon regeneration but do not overcome the block in regrowth
in dlk-1 mutants (Ghosh-Roy et al., 2010) (Figure 6A). Examina-
tion of double mutants between dlk-1 and other enhanced-
regrowth mutants revealed similar dependence on dlk-1 (Fig-
ure 6A). In contrast, elevated Ca2+or cAMP signaling in
egl-19(gf)/VGCC or pde-4(lf)/Phosphodiesterase mutants en-
suggesting Ca2+and cAMP act in parallel to UNC-57 and
upstream of DLK-1. However, lack of SLT-1 did not promote
regrowth in unc-51/Atg1 or unc-57/Endophilin mutants (Fig-
ure 6C). These findings suggest Slit/Robo signals play a
modulatory role, dependent on intrinsic pathways such as
DLK-1, UNC-51, and UNC-57.
As loss of function in dlk-1 and other regrowth-promoting
genes results in similar phenotypes, we used a gain-of-function
effect caused by overexpression of DLK-1 [dlk-1(++)] to address
Genetics of Axon Regeneration
1050 Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc.
their order of activity. Overexpression of DLK-1 is sufficient to
enhance PLM axon regeneration (Yan et al., 2009). DLK-1 over-
expression completely suppressed the regrowth defects of
unc-51/Atg1and unc-57/Endophilin mutants (Figure6E), consis-
tent with DLK-1 acting downstream or in parallel to UNC-51/
ATG1 and the SV endocytosis genes. Loss of function in RPM-
1, a negative regulator of DLK-1, did not suppress unc-57/
Endophilin regrowth defects (data not shown), consistent with
previous findings that PLM regrows normally in rpm-1 mutants
(Yan et al., 2009).
Time window (h)
Regrowth rate (µm/h)
Normalized total regrowth
06 12 24
Total regrowth (µm)
23 23 292925 253434
Figure 3. Slit/Robo Signals Inhibit PLM Axon Extension
(A) Normalized 24 hr PLM regrowth in mutants affecting axon guidance, cell adhesion, and extracellular matrix.
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 hr; red arrows indicate lesion sites, yellow dotted lines indicate original path of
PLM. Scale, 10 mm.
(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 with unshifted ky200 animals (black).
(E and F) slt-1 and sax-3 mutants display faster axon extension in the 6–24 hr time period.
Statistics, t test; n values in columns; ***p < 0.001; ##, p < 0.01; *p < 0.05; ns, not significant.
Genetics of Axon Regeneration
Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc. 1051
Among all double mutants tested, only efa-6(lf) suppressed
regeneration defects of dlk-1 mutants (Figures 5F and 6E). In
efa-6 dlk-1 double mutants the proximal stumps of severed
axons extended significantly further than in dlk-1(lf) although
they did not form growth cones (Figure 5G). efa-6 mutations
also partially suppressed the regrowth defects of unc-26/Synap-
tojanin and unc-51/Atg1 mutants (Figure 6E), consistent with
EFA-6 acting downstream or in parallel to DLK-1, UNC-26, and
UNC-51 in axon regrowth.
Genes with inhibitory roles, such as slt-1 and efa-6, affect
different stages of regrowth and therefore likely act in distinct
pathways. To test whether elimination of multiple inhibitory path-
ways could further enhance regrowth relative to single mutants,
we analyzed slt-1 efa-6 double mutants. We found that regrowth
at the 24 hr time point was not further enhanced in slt-1 efa-6
double mutants compared with the highest single mutant (Fig-
enhanced in efa-6 slt-1 double mutants compared with single
mutants. Thus, the combined loss of two inhibitory pathways
can result in further increases in regrowth at later time points.
Our results establish the feasibility of systematic genetic
screening for axon regeneration phenotypes using genetically
amenable model organisms. Our findings underscore the molec-
ular complexity of axon regeneration and provide a genetic
framework for a more comprehensive understanding of axonal
repair and regrowth mechanisms.
Genetic Complexity of Axon Regrowth
As a forward genetic phenotype-based screen in axon regener-
ation remains technically challenging, we have focused on
systematic large-scale testing of conserved candidate genes.
Our selection of candidates is by necessity biased, and we
plan to expand the screen to reduce this bias. Nonetheless,
Normalized regrowth (24 h)
growth rate (µm/h)
Time period (h)
-1h +6h -7hno hs
efa-6 ; Phsp-EFA-6
efa-6 (lf); Prgef-EFA-6
efa-6(lf) ; Pmec-4-
Normalized regrowth (24 h)
Normalized regrowth (24 h)
131414 15 2321
efa-6; SCI (FL)
efa-6; SCI (EK)
Normalized regrowth (24 h)
Figure 4. EFA-6 Inhibits the Early Phase of Axon Regrowth
(A) PLM axon regrowth at 24 hr is increased in efa-6 mutants, normalized to controls (n values in bars).
(B) Images of wild-type and efa-6 axons at 24 hr. Red arrows, site of axotomy. Scale, 10 mm.
(C) Axon growth is increased in efa-6 from 0–14 hr postaxotomy 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 hours).
(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; domain deletions or point mutations indicated below; Sec7 domain (blue box), PH domain (yellow oval), coiled coil domain
(red box); n R 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 R 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.
Genetics of Axon Regeneration
1052 Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc.
our analysis supports the view that regenerative axon regrowth
requires many genetic pathways in addition to those defined in
developmentalaxonoutgrowth, polarity,orguidance. Inaddition
navigation in many contexts, regenerative regrowth involves
pathways that sense damage and trigger the resumption of
developmental programs that may be repressed in mature
neurons. By focusing on genes nonessential for development
or PLM outgrowth we have identified candidates with relatively
specific effects on regrowth.
Overall, approximately 10% of genes tested in our screen dis-
played significant effects on axonal regrowth (<60% of normal
regrowth). In addition to such genes with ‘‘strong’’ requirements,
we found a similar number of genes with smaller yet significant
effects, displaying regrowth 80%–60% of the wild-type (Table
4B). At least some of these genes may define pathways with
modulatory or partly redundant roles. Most such genes have
only been examined at the 24 hr time point; future studies could
address whether such genes have greater effects at different
time points or in different genetic backgrounds.
A Function for the Synaptic Vesicle Endocytic Pathway
Among genes required for regenerative regrowth, we identified
several unexpected functional clusters, including genes impli-
cated in synaptic vesicle (SV) endocytosis and in neurotransmis-
transmission (unc-13, unc-18) did not affect regrowth. Endocytic
trafficking could play several roles in axon regrowth: repair of
damage to the plasma membrane, vesicular transport of retro-
grade injury signals, and membrane addition in axon extension
(Tuck and Cavalli, 2010). Endocytosis can inhibit axon growth
by internalization of Nogo (Joset et al., 2010). Although SV endo-
cytosis genes are required at multiple times in regrowth, the
requirement for UNC-57/Endophilin could be bypassed by
elevated DLK-1 activity. We therefore favor the interpretation
that the SV endocytosis genes may be required for vesicles that
function in injury signaling. For example, the Drosophila DLK
family member Wallenda associates with retrogradely trans-
ported vesicles, and transport is important for the response to
injury (Xiong et al., 2010). The finding that SV endocytosis is crit-
ical for regrowth can be placed in a broader context of evidence
that synaptic growth is neuroprotective (Massaro et al., 2009).
EFA-6 Is an Intrinsic Inhibitor of Regrowth
Precise regulation of microtubule (MT) dynamics is emerging as
a critical factor in axonal regenerative growth (Ertu ¨rk et al.,
2007; Hellal et al., 2011; Sengottuvel et al., 2011; Stone et al.,
tified. Our analysis reveals EFA-6 as a negative regulator of axon
regrowth that affects axonal MT dynamics. Although named for
its presumed GEF activity for Arf6 small GTPases, the Sec7
GEF domain of EFA-6 is not essential for its effects on regrowth.
Total regrowth (µm/6 h)
Total dynamic MTs
WT + M9
WT + taxol
efa-6(gf) + M9
efa-6(gf) + taxol
+ 24 h
+ 6 h
Normalized regrowth (24 h)
Figure 5. EFA-6 Negatively Regulates Axonal
Microtubule Dynamics Downstream or in Parallel
(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 re-
growth 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 hr postaxotomy; cf. the lack
of growth cones in axon stumps of dlk-1 mutants (F).
(C and 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 hr post-
axotomy in the 40 mm region proximal to the site of
axotomy, visualized with Pmec-4-EBP-2::GFP (juEx2843);
scale, 10 mm.
(E) Microinjection of taxol increases regrowth in efa-6(gf)
animals compared with buffer-injected controls. The
effect of taxol on wild-type (muIs32) is not significant.
(F) Regrowth 6 hr postaxotomy is increased in dlk-1 efa-6
double mutants compared with dlk-1 single mutants.
(G) Images of dlk-1 single and double-mutant axons at6 hr
postaxotomy. The region in the boxed area is enlarged at
right. Red arrowheads indicate end of distal fragment
closest to axotomy site.
Scales, 10 mm. All charts show mean ± SEM; statistics,
t test or Mann-Whitney test; ***p < 0.001; **p < 0.01;
*p < 0.05; ns, not significant.
Genetics of Axon Regeneration
Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc. 1053
Instead, growth-inhibitory effects of EFA-6 are mediated by its N
terminus, a region that lacks well-defined domains (Cox et al.,
2004), but which shares motifs with other EFA6 family members
pendently of its Arf GEF activity, arf-6 mutants themselves dis-
played modestly increased regrowth. EFA-6 could inhibit re-
growth by two mechanisms, one involving ARF-6 activation and
the other involving its N terminus.
By characterizing EBP-2::GFP dynamics, we find that efa-6
mutant axons display increased numbers of dynamic MTs after
axotomy, consistent with studies in the early embryo (O’Rourke
et al., 2010). The suppression of the EFA-6 regrowth inhibition
effect by taxol supports the model that the reduced regrowth
of EFA-6overexpressing axons isaconsequence of destabilized
MTs. EFA-6 could directly or indirectly destabilize growing MTs,
functions have yet to be studied in detail (Sakagami, 2008). It will
be important to determine whether mammalian EFA6 family
members also affect axonal regrowth.
Genetically Defined Stages and Pathways
in Axon Regrowth
A key outcome of our screen has been the identification of
pathways with inhibitory influences on axon regrowth, indicating
that PLM axon regrowth in the wild-type is restrained by intrinsic
and extrinsic inhibitory influences. Several mutants display
similarly increased regrowth suggesting PLM axons cannot
extend faster than 6–8 mm/hr. Nevertheless total regrowth can
be further increased, as in slt-1 efa-6 double mutants, by pro-
longing the period over which axons extend. As in vertebrate
spinal cord regeneration, where combinatorial therapies can
enhance regrowth (Kadoya et al., 2009), reduction in multiple
inhibitory pathways may be needed to optimize regrowth in
C. elegans. A remaining question is whether these inhibitory
pathways account for the inability of certain C. elegans neurons
to regrow in the wild-type (Gabel et al., 2008; Wu et al., 2007).
Overall, our analysis suggests the following model for PLM
axon regrowth (Figure 7). Axonal injury triggers a calcium tran-
sient that activates cAMP and PKA signaling upstream of
DLK-1 (Ghosh-Roy et al., 2010). In parallel, SV endocytosis
may be activated to form signaling vesicles. Such vesicles could
transport DLK-1 itself, or other injury signals. DLK-1 kinase is
activated and triggers local translation (Yan et al., 2009). Each
of these pathways is critical either for competence of injured
axons to regrow or for the initial stages of regeneration in which
the proximal stump reestablishes a growth cone. Axonal MTs
become highly dynamic after axotomy, but their growth is
restrained by factors such as EFA-6. As the newly reformed
growth cone extends, it navigates a microenvironment com-
posed of permissive and inhibitory environmental signals. Inhib-
itory signals include basement membrane components and
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 hr; 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.
included for comparison).
(F) efa-6 slt-1 double mutants display enhanced regrowth compared with single mutants at 48 hr postaxotomy.
All statistics, t test; n R 10 for each condition except panel D (n R 5); *p < 0.05; **p < 0.01; ***p < 0.001.
Genetics of Axon Regeneration
1054 Neuron 71, 1043–1057, September 22, 2011 ª2011 Elsevier Inc.
Slit and Robo signals. As our studies have focused on axons
capable of regeneration, it will be important to test whether
pathways defined here are limiting in axons that do not sponta-
C. elegans Genetics, Transgenes, and Candidate Gene Set
We maintained C. elegans on NGM agar plates as described (Brenner, 1974).
Animals were grown at 20?C unless stated otherwise. For deletion mutations
obtained from the C. elegans gene knockout consortium (ok, gk mutations)
or the Japan National Bioresource Project (tm mutations), we backcrossed
the mutant at least two times to N2 wild-type. For selected ‘‘hit’’ genes, we re-
tested the mutant after a second round of outcrossing and found consistent
effects on regrowth. Deletions were genotyped by PCR; primer sequences
are available on request. Transgenes were generated by standard procedures
(see Supplemental Experimental Procedures).
Wechose aset of 654genes based onthe following criteria: (1)recognizable
C. elegans, human similarity, as assessed by ‘‘best BLAST score’’ in Worm-
base; (2) viable mutant strain; (3) known structural or functional category
(e.g., kinase, channel); (4) expression in neurons (Wormbase). Some genes
were prioritized based on RNAi screens for synaptic function (Sieburth et al.,
2005) or axonal guidance (Schmitz et al., 2007). A few genes were selected
based on expression in touch neurons (Zhang et al., 2002).
Femtosecond Laser Axotomy and Imaging
We performed laser axotomy essentially as described (Wu et al., 2007). To
immobilize worms for EBP-2::GFP imaging without anesthetics, we used
12.5% agarose pads and a suspension of 0.1 mm diameter polystyrene beads
(Polysciences) under the coverslip (C. Fang-Yen, personal communication).
For live imaging of EBP-2::GFP, we collected 200 frames of 114 msec expo-
sure each every 230 msec using the spinning disk confocal and generated
kymographs using Metamorph (Molecular Devices) from a 40 mm ROI on the
PLM axon proximal to the cut site.
To apply taxol to regrowing axons in vivo, we grew animals on NGM agar
plates containing 5 mM taxol (Sigma) for 24 hr prior to axotomy. One hour
before axotomy, we injected 2–5 nl of 50 mM taxol in M9 buffer into the body
cavity using standard injection protocols, and then recovered the animals on
taxol-containing plates for 30 min. We axotomized PLM using our standard
protocol except with 50 mM taxol in solutions. Control animals were injected
with M9 buffer and cultured without taxol. Animals injected with buffer or taxol
were healthy and grew at normal rates.
The distribution of total regrowth length of axons in wild-type and controls
passed standard tests of normality. In preliminary analysis, we used the
Student’s t test or the Mann-Whitney test. Among 650 such two-way compar-
isons, 33 are expected to be significant at the 0.05 level by chance. Most
genes discussed here displayed effects significant at the 0.01 level (red bars
in bar charts of regrowth); we also discuss some genes that gave repeatable
results at the 0.05 level (orange bars). To compare regrowth between experi-
ments with different control means, we normalized each experimental data
point by dividing it by its control mean. To correct for multiple comparisons,
we used two approaches. First, most genes displaying significant differences
in the primary screen were either repeated or retested with a second allele, in
many cases by a different experimenter. We then calculated adjusted P values
for the set of repeat experiments using the Benjamini-Hochberg correction
for false discovery rate (Benjamini and Hochberg, 1995). All other statistical
analyses used Prism (GraphPad).
Supplemental Information includes Supplemental Experimental Procedures,
four figures, three tables and can be found with this article online at doi:10.
We thank Alison Hughes, Niousha Saghafi, Amanda Rajapaksa, Sunny Sun,
Peg Scott, Caroline Yu, Laura Toy, and other members of our labs for strain
construction. We thank Johann Gagnon-Bartsch and Terry Speed for advice
on statistical analysis; Cori Bargmann, Gian Garriga, and Erik Jorgensen for
reagents; Chris Fang-Yen for the bead immobilization protocol; and Emily
Troemel for comments on the manuscript. We thank the C. elegans Gene
Knockout Consortium and the Japanese National Bioresource Project for
deletion mutations, and the Caenorhabditis Genetics Center for strains. L.C.,
A.D.C., and Y.J. designed the screen. Z. Wu performed axotomy, imaging,
and technical development. L.C. constructed strains and analyzed efa-6;
Z. Wang analyzed slt-1/sax-3 signaling. A.G.-R. designed and performed MT
imaging and analysis. L.C., Z. Wang, T.H., A.G.-R. and D.Y. contributed to
the screen and analyzed the results. S.O’R. and B.B. provided reagents
and unpublished data for efa-6. L.C., Z. Wang, Y.J., and A.D.C. wrote the
manuscript. Z. Wang is a Fellow of the Jane Coffin Childs Memorial Fund.
Z. Wu is an Associate and Y.J. is an Investigator of the Howard Hughes
Medical Institute. Supported by grants from the NIH to B.B. (R01 GM049859
and GM058017), Y.J. (R01 NS035546), and A.D.C. (R01 NS057317).
Accepted: July 5, 2011
Published: September 21, 2011
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