Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration

  1. Kyung Won Kim  Is a corresponding author
  2. Ngang Heok Tang
  3. Christopher A Piggott
  4. Matthew G Andrusiak
  5. Seungmee Park
  6. Ming Zhu
  7. Naina Kurup
  8. Salvatore J Cherra III
  9. Zilu Wu
  10. Andrew D Chisholm  Is a corresponding author
  11. Yishi Jin  Is a corresponding author
  1. University of California, San Diego, United States
  2. University of California, San Diego, School of Medicine, United States
5 figures, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Overview and results of expanded axon regrowth screen.

(A) Pie chart showing fraction of genes screened displaying significantly reduced or increased regrowth at 24 hr. Right: representative inverted grayscale images of PLM 24 hr post-axotomy in wild type (black box), and mutants with reduced (red boxes) or increased regrowth (blue boxes). Orange arrowhead, site of axotomy. (B) Distribution of reduced/increased regrowth mutants among nine functional or structural gene classes, shown as percentage of genes in each class. See Figure 1—source data 1 for lists of genes in each class.

https://doi.org/10.7554/eLife.39756.002
Figure 1—source data 1

List of screened genes, reference alleles, and the functional categories.

https://doi.org/10.7554/eLife.39756.006
Figure 1—figure supplement 1
The overview and results of axon regrowth screen combined with our previous study.

(A) Pie chart showing combined genes from both screens (Chen et al., 2011)(this work) displaying significantly reduced or increased regrowth at 24 hr. (B) Distribution of reduced/increased regrowth mutants among nine functional or structural gene classes, shown as percentage of genes in each class.

https://doi.org/10.7554/eLife.39756.003
Figure 1—figure supplement 2
Mutants affecting multiple biological processes required for normal axon regrowth.

(A–E) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting: (A) components of basement membrane (BM) and ADAMTS, (B) components of trafficking and Annexins, (C) microtubule acetyltransferases, (D) microtubule isotypes, and (E) actin filament regulators. Statistics, Student’s t-test with same day controls. (F) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting mfsd-6 mutants. Pan-neuronal expression of MFSD-6 [Pn::mfsd-6] in mfsd-6(tm3356) animals rescues axon regeneration. Statistics, one-way ANOVA followed by Tukey’s multiple comparison tests. Left: MFSD-6 protein structure. Loss-of-function alleles are indicated above (tm3356, deletion; ju833, missense point mutation). Blue rectangles represent transmembrane domains. Data are shown as mean ±SEM. n, number of animals shown within columns. ns, not significant. *p<0.05; **p<0.01; ***p<0.001. In Figure 1—figure supplement 2D, tba-1, tbb-4, and tbb-6 (Chen et al., 2011) are included for comparison.

https://doi.org/10.7554/eLife.39756.004
Figure 1—figure supplement 2—source data 1

Each data point in all graphs.

https://doi.org/10.7554/eLife.39756.005
Figure 2 with 1 supplement
NMNAT/NMAT-2 inhibits PLM axon regrowth via its catalytic domain.

(A) Overview of NAD+ salvage biosynthesis pathway. Top, key enzymes; Bottom, C. elegans orthologs (Shaye and Greenwald, 2011). (B) Top, nmat-2 gene structure and mutant alleles. NMAT-2 contains an NMNAT domain. nmat-2(ju1514) point mutation and nmat-2(ju1512) deletion alleles were generated using CRISPR-Cas9 genome editing. Bottom, sequence alignment of the active site of NMNAT domain of C. elegans NMAT-2 (accession number: NP_492480.1; amino acids 4–14) with human NMNAT1–3 (NP_073624.2, NP_055854, NP_001307441) and C. elegans NMAT-1 (NP_510010.2). Sequences were analyzed using Clustal Omega. (C) Normalized regrowth 24 hr post-axotomy in mutants lacking genes encoding enzymes in the NAD+ biosynthesis pathway. Statistics, Student’s t-test with same day controls. For the statistical test of transgene analysis, one-way ANOVA followed by Tukey’s multiple comparison test. (D) PLM axon regrowth 24 hr post-axotomy in transgenic animals expressing nmat-2(+) driven by tissue-specific promoters for mechanosensory neurons (Pmec-4), epidermis (Pcol-12) or intestine (Pmtl-2) in a nmat-2(ju1512) background. One-way ANOVA followed by Tukey’s multiple comparison test. Data are shown as mean ± SEM. n, number of animals shown within columns. ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.39756.009
Figure 2—figure supplement 1
nmat-1 show no defect in PLM axon regrowth even when its germline is defective.

Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting nmat-1 mutants when treated with 50 µg/ml FUdR. Data are shown as mean ± SEM. n, number of animals shown within columns. ns, not significant.

https://doi.org/10.7554/eLife.39756.010
Figure 3 with 1 supplement
Select ER-PM membrane contact site proteins are required for axon regeneration and are sensitive to injury.

(A) Junctophilin-1 protein structure. From top to bottom: C. elegans JPH-1 (NP_492193.2), its Drosophila ortholog JP (NP_523525.2), and human ortholog JPH1 (NP_065698.1). Junctophilins contain N-terminal MORN (Membrane Occupation and Recognition Nexus) repeats (green) and a C-terminal transmembrane domain (blue). C. elegans deletion allele is indicated above (ok2823). (B) Percentage of axons that exhibit fusion between the regrowing axon and distal fragment 24 hr post-axotomy. Upper image shows a regrowing axon that has not fused with the distal fragment in a wild-type animal. Lower image shows fusion between the regrowing axon and the distal fragment in a jph-1(ok2823) animal. Fisher’s exact test. *p<0.05; **p<0.01. (C) Normalized regrowth 24 hr post-axotomy in mutants lacking selected genes encoding ER-PM MCS proteins. Data are shown as mean ± SEM. n, number of animals shown within columns. Student’s t-test with same day controls. ns, not significant; *p<0.05; **p<0.01. (D) E-Syt protein structure. From top to bottom: C. elegans ESYT-2 and its human orthologs E-Syt2, E-Syt3, and E-Syt1 (NP_065779.1, NP_114119.2, NP_056107.1, respectively). Amino acid length is indicated to the right of each protein. E-Syt proteins contain N-terminal hydrophobic regions (blue), SMP (Synaptotagmin-like Mitochondrial and lipid-binding Protein) domains (yellow), and C-terminal C2 domains (red). C. elegans deletion allele is indicated above (ju1409). (E) Images of the PLM cell body and surrounding neurites. Left, GFP::PISY-1 ER marker; Middle, mKate2::ESYT-2 driven by the mec-4 promoter; Right, Image overlays. Images show single slices taken at 1 μm intervals. (F) Representative inverted grayscale images of GFP::ESYT-2 in the axon of the PLM neuron before and immediately after axotomy (upper and lower panels, respectively). Site of laser axotomy indicated by asterisk; puncta indicated by arrowheads.

https://doi.org/10.7554/eLife.39756.012
Figure 3—figure supplement 1
esyt-2 is widely expressed in the nervous system.

Confocal images of Pesyt-2::GFP transcriptional reporter showing widespread expression in the nervous system of L4 stage animals, including in head ganglia, ventral nerve cord, and tail ganglia. Maximum intensity projection.

https://doi.org/10.7554/eLife.39756.013
PLM axon regeneration involves membrane lipid biosynthesis pathway.

(A) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting neutral lipolysis. (B) Normalized PLM axon regrowth 24 hr post-axotomy in mutants affecting acid lipolysis. (C) Overview of C.elegans Kennedy pathway for de novo biosynthesis of PE and PC, the major phospholipids in the PM. (D) Normalized PLM axon regrowth 24 hr post-axotomy in mutants lacking select genes encoding enzymes in the Kennedy pathway. Data are shown as mean ±SEM. n, number of animals shown within columns. Student’s t-test with same day controls. ns, not significant; *p<0.05.

https://doi.org/10.7554/eLife.39756.015
The Kelch-domain protein IVNS-1 inhibits axon regeneration.

(A) ivns-1 gene structure. Left: Loss-of-function alleles are indicated below (gk252 and ok3171). Right: Alignment of the C. elegans IVNS-1 (NP_510109.1) with its human ortholog IVNS1ABP (NP_006460.1) and mouse ortholog ND1-L (NP_473443.2). Number indicates percentage identity of protein sequences. Sequences were analyzed using Clustal Omega. (B) Normalized PLM axon regrowth 24 hr post-axotomy in mutants of Kelch-domain proteins. Data are shown as mean ±SEM. n, number of animals shown within columns. Student’s t-test with same day controls. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. Right: representative inverted grayscale images of PLM 24 hr post-axotomy. Scale bar, 25 μm. (C) Normalized PLM axon regrowth 6 hr post-axotomy. Data are shown as mean ±SEM. One-way ANOVA followed by Tukey’s multiple comparison test. n, number of animals shown within columns. **p<0.01. (D) Percentage of axons with growth cones (GCs) 6 hr post-axotomy. n, Number of animals shown below columns. Fisher’s exact test. ns, not significant.

https://doi.org/10.7554/eLife.39756.017

Tables

Table 1
Mutants displaying reduced PLM regrowth
https://doi.org/10.7554/eLife.39756.007
Gene nameMutationsNormalized regrowth (24 hr)NP valueMolecular functionClosest human Genea
A. Cell Adhesion and ECM
adt-1cn300.7521**ADAM metalloproteaseADAMTS3
adt-3ok9230.7146**ADAM metalloproteaseADAMTS2
C05D9.7ok29310.6028***UnknownN/A
dpy-10e1280.6728**5FMC ribosome biogenesis complexPELP1
F35G2.1ok16690.6827***Quiescin sulfhydryl oxidaseQSOX1
gly-2tm8390.6916*Mannosyl-glycoprotein N-acetylglucosaminyltransferasesMGAT5
osm-11rt1420.7213**Secreted proteinN/A
zig-1ok7840.6812*BasiginBSG
zig-3ok14760.7318**Kazal type serine peptidase inhibitor domainKAZALD1
B. Channels and transporters
abts-1ok15660.7434***Anion exchange proteinSLC4A7
mfsd-6ju8330.6815**Major facilitatorMFSD6
C. Cytoskeleton and motors
fli-1ky5350.6127***Actin remodeling proteinFLII
mec-12e16050.7315*Tubulin α−3 chainTUBA1C
mec-17ok21090.5514**α-Tubulin N-acetyltransferaseATAT1
tba-9ok18580.7024**α-TubulinTUBA3
vab-10e6980.7811*SpectraplakinDST
D. Protein kinases and phosphatases
plk-1or683ts0.5913**Polo like kinasePLK1
svh-2tm7370.6830***Receptor Tyrosine kinaseMET
E. Neurotransmission, metabolism, and lipid
cept-2ok31350.6827*Choline/ethanolamine phosphotransferaseCEPT1
cpr-1ok13440.6032***Cysteine proteinaseCTSB
dhhc-11gk11050.7532***PalmitoyltransferaseZDHHC11
eat-3tm11070.7416**Mitochondrial dynamin like GTPaseOPA1
npr-20ok25750.4944***G-protein coupled receptorCCKBR/TRHR
ptps-1tm19840.5527***6-pyruvoyl tetrahydrobiopterin synthasePTS
supr-1ju11180.7830**UnknownN/A
F. Trafficking
jph-1ok28230.7714**JunctophilinJPH1
rab-8tm25260.7728**Ras GTPaseRAB8B
rsef-1ok13560.6617*Endosomal Rab family GTPaseRASEF
G. Signaling and interactions
osm-7tm22560.5728**UnknownN/A
par-2or3730.6347***C3HC4-type RING-fingerTRIM
rgl-1ok19210.7430**Ral guanine nucleotide dissociation stimulatorRGL1
wdfy-3ok9120.5217***WD40 and FYVE domainWDFY3
wdr-23tm18170.6630***DDB1 and CUL4 associated factorDCAF11
H. Protein turnover, proteases, cell death
brap-2ok14920.6162***BRCA1-associated protein; zinc ion binding activityBRAP
cdc-48.1tm5440.6919**Transitional ER ATPase homologVCP
ced-9n19500.7316*Cell-death inhibitor Bcl-2 homologBCL2
dnj-23tm71020.6932**DNaJ domain (prokaryotic heat shock protein)DNAJC9
fbxc-50tm51540.7312*F-box proteinN/A
math-33ok29740.6412***Ubiquitin-specific proteaseUSP7
skr-5ok30680.6912*S-phase kinase associated proteinSKP1
tep-1tm37200.5336***ThiolEster containing Protein; endopeptidase inhibitor activityCD109
I. Gene expression and RNA regulation
mec-8e3980.2316***RNA binding protein, mRNA processing factorRBPMS
rict-1mg3600.5826***Subunit of TORC2RICTOR
rtcb-1 bgk4510.5825***tRNA-splicing ligase RtcB homologRTCB
skn-1ok23150.7810*Basic leucine zipper proteinNFE2
smg-3r9300.6828***Nonsense mediated mRNA decay regulatorUPF2
syd-9ju490.4715***Zinc finger E-box binding homeoboxZEB1
tdp-1ok8030.7036***TAR DNA-binding proteinTARDBP/TDP-43
wdr-5.1ok14170.7026***WD repeat-containing proteinWDR5
  1. Genes are classified in nine functional or structural classes. Mutations are genetic or predicted molecular nulls, or partial loss-of-function. Normalized regrowth is relative to matched same-day controls or to pooled controls. Significant levels (*p<0.05; **p<0.01; ***p<0.001) based on Student’s t-test.

    a Closest human gene based on BLASTP score in Wormbase WS263; Ensembl/HGNC symbol.

  2. brtcb-1(gk451) mutant reported to show increased regrowth in the C. elegans motor neurons (Kosmaczewski et al., 2015).

Table 2
Mutants displaying increased PLM regrowth
https://doi.org/10.7554/eLife.39756.008
Gene nameMutationsNormalized regrowth (24 hr)NP valueMolecular functionClosest human Genea
A. Cell adhesion and ECM
emb-9tk75 b1.3728***Collagen type IV α3 chainCOL4A3
epi-1gm1211.3336***LamininLAMA
mig-17k1741.2437***ADAM metalloproteaseADAMTS5
ZC116.3ok16181.4026***CubilinCUBN
B. Channels and transporters
lgc-12ok35461.3326**Serotonin receptor 3EHTR3E
tmc-1ok18591.3130**Transmembrane channel-like proteinTMC1
C. Cytoskeleton and motors
ivns-1ok31711.3118**Actin-binding; splicingIVNS1ABP
twf-2ok35641.3339**Twinfilin actin binding proteinTWF
nud-1ok5521.3025**Nuclear distribution C, Dynein complex regulatorNUDC
tba-7gk7879391.4515***α-tubulinTUBA
E. Neurotransmission, metabolism, and lipid
nmat-2tm29051.5538***Nicotinamide mononucleotide adenylyltransferaseNMNAT1
qns-1ju15631.1340*NAD + synthetaseNADSYN1
mgl-1tm18111.2428**Glutamate metabotropic receptorGRM3
mgl-3tm17661.3222**Glutamate metabotropic receptorGRM6
npr-25ok20081.2726**Coagulation factor II thrombin receptorF2RL2
ucr-2.3ok30731.4124***Ubiquinol-cytochrome C reductase core proteinUQCRC2
F. Trafficking
nex-1 cgk1481.3827***AnnexinANXA13
nex-2 dok7641.2330**AnnexinANXA7
snb-6tm51951.3038**Vesicle associated membrane proteinVAMP1
G. Signaling and interactions
drag-1tm37731.5226**Repulsive guidance molecule BMP co-receptorRGMB
ect-2ku4271.3131**RhoGEFECT2
lin-2e13091.3127***Membrane associated guanylate kinaseCASK
magi-1zh661.4029***Membrane associated guanylate kinaseMAGI2
prmt-5gk3571.2624**Protein arginine N-methyltransferasePRMT5
rap-1tm8611.3310***Ras small GTPaseRAP1
smz-1ok35761.3913*PDZ domain-containing proteinN/A
trxr-1tm20471.331*Thioredoxin reductaseTXNRD2
H. Protein turnover, proteases, cell death
natb-1ju14051.2914*N(α)-acetyltransferase 20NAA20
rnf-5tm7941.2815*Ring finger proteinRNF5
ulp-5tm30631.2230***SUMO specific peptidaseSENP7
I. Gene expression and RNA regulation
csr-1fj541.2438***ArgonauteAGO1
hda-6tm34361.2942***Histone deacetylaseHDAC6
elpc-3ok24521.3126***Elongator acetyltransferase complex subunitELP3
  1. Genes are classified in nine functional or structural classes. Mutations are genetic or predicted molecular nulls, or partial loss-of-function. Normalized regrowth is relative to matched same-day controls or to pooled controls. Significant levels (*p<0.05; **p<0.01; ***p<0.001) based on Student’s t-test.

    a Closest human gene based on BLASTP score in Wormbase WS263; Ensembl/HGNC symbol.

  2. b emb-9(tk75) mutant reported to be a gain-of-function allele that makes stable EMB-9/Type IV collagen (Kubota et al., 2012).

    cnex-1(gk148) mutant reported to show reduced regrowth in the C. elegans motor neurons (Nix et al., 2014).

  3. d nex-2(bas4) mutant reported to show normal regrowth in the C. elegans motor neurons (Nix et al., 2014).

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Bacterial strainE. coli: OP50Caenorhabditis
Genetics Center
RRID:
WB-STRAIN:OP50
Genetic reagent
(C. elegans)
Strain wild type N2Caenorhabditis
Genetics Center
RRID:WB-STRAIN:N2_
(ancestral)
Genetic reagent
(C. elegans)
CZ10969: Pmec-7-GFP(muIs32) IIConsidered as‘WT’ in many axotomy experiments
Genetic reagent
(C. elegans)
CZ10175: Pmec-4-GFP(zdIs5) IConsidered as ‘WT’ in many axotomy experiments
Genetic reagent
(C. elegans)
CZ25411: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ25415: nmat-2(ju1514) I/hT2 I, III; 
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ17633: nmat-2(tm2905) I/hT2 I, III; 
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ24324: Pmec-7-GFP(muIs32) II; qns-1(ju1563) IV/mIs11 sd IV
Genetic reagent
(C. elegans)
CZ25642: Pmec-4-GFP(zdIs5) I; 
Pmec-7-GFP(muIs32) II
Genetic reagent
(C.elegans)
CZ25534: nmrk-1(ok2571) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ24241: Pmec-4-GFP(zdIs5) I; 
nprt-1(tm6342) IV
Genetic reagent
(C. elegans)
CZ24242: Pmec-4-GFP(zdIs5) I; 
pnc-1(tm3502) IV
Genetic reagent
(C. elegans)
CZ24802: Pmec-4-GFP(zdIs5) I;
pnc-2(tm6438) IV
Genetic reagent
(C. elegans)
CZ25466: Pmec-7-GFP(muIs32) II;
juSi347[nmat-2 gDNA] IV
Genetic reagent
(C. elegans)
CZ25469: nmat-2(tm2905) I;Pmec-7-GFP(muIs32) II;
juSi347[nmat-2 gDNA] IV
Genetic reagent
(C. elegans)
CZ26216: Pmec-7-GFP(muIs32) II;
Ex[Pmec-4-nmat-2(juEx7834)]
Genetic reagent
(C. elegans)
CZ26217: Pmec-7-GFP(muIs32) II;
Ex[Pmec-4-nmat-2(juEx7835)]
Genetic reagent
(C. elegans)
CZ26220: Pmec-7-GFP(muIs32) II;
Ex[Pcol-12-nmat-2(juEx7838)]
Genetic reagent
(C. elegans)
CZ26221: Pmec-7-GFP(muIs32) II;
Ex[Pcol-12-nmat-2(juEx7839)]
Genetic reagent
(C. elegans)
CZ26218: Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2-nmat-2(juEx7836)]
Genetic reagent
(C. elegans)
CZ26219: Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2-nmat-2(juEx7837)]
Genetic reagent
(C. elegans)
CZ26222: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmec-4-nmat-2(juEx7840)]
Genetic reagent
(C. elegans)
CZ26223: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmec-4-nmat-2(juEx7841)]
Genetic reagent
(C. elegans)
CZ26224: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pcol-12-nmat-2(juEx7842)]
Genetic reagent
(C. elegans)
CZ26225: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pcol-12-nmat-2(juEx7843)]
Genetic reagent
(C. elegans)
CZ26310: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2-nmat-2(juEx7836)]
Genetic reagent
(C. elegans)
CZ26311: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2-nmat-2(juEx7837)]
Genetic reagent
(C. elegans)
CZ26332: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2, col-12::nmat-2(juEx7853)]
Genetic reagent
(C. elegans)
CZ26333: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2, col-12::nmat-2(juEx7854)]
Genetic reagent
(C. elegans)
CZ26285: nmat-2(ju1512) I/hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2, col-12, mec-4::nmat-2(juEx7850)]
Genetic reagent
(C. elegans)
CZ26286: nmat-2(ju1512) I / hT2 I, III;
Pmec-7-GFP(muIs32) II;
Ex[Pmtl-2, col-12, mec-4::nmat-2(juEx7851)]
Genetic reagent
(C. elegans)
CZ26391: jph-1(ok2823) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ22032: Pmec-4-GFP(zdIs5) I;
anoh-1(tm4762) III
Genetic reagent
(C. elegans)
CZ22033: Pmec-4-GFP(zdIs5) I; 
anoh-2(tm4796) IV
Genetic reagent
(C. elegans)
CZ26325: Pmec-4-GFP(zdIs5) I;
anoh-1(tm4762) III;
anoh-2(tm4796) IV
Genetic reagent
(C. elegans)
CZ26069: Pmec-4-GFP(zdIs5) I;
obr-1(xh16) III
Genetic reagent
(C. elegans)
CZ24555: Pmec-4-GFP(zdIs5) I; 
obr-2 (xh17) V
Genetic reagent
(C. elegans)
CZ24556: Pmec-4-GFP(zdIs5) I;
obr-3(tm1087) X
Genetic reagent
(C. elegans)
CZ24557: obr-4(tm1567) I;
Pmec-4-GFP(zdIs5) I
Genetic reagent
(C. elegans)
CZ25696: obr-4(tm1567) I;
Pmec-4-GFP(zdIs5) I;
obr-1(xh16) III; obr-2(xh17) V; 
obr-3(tm1087) X
Genetic reagent
(C. elegans)
CZ26375: Pmec-4-GFP(zdIs5) I; 
esyt-2(ju1409) III
Genetic reagent
(C. elegans)
CZ26570: juIs540 [Pmec-4-mKate2-ESYT-2]; 
juEx7807[Pmec-4-GFP-PISY-1]
Genetic reagent
(C. elegans)
CZ24897: juEx7604 [Pmec-4-GFP-ESYT-2]
Genetic reagent
(C. elegans)
CZ22087: Pmec-7-GFP(muIs32) II;
atgl-1(tm3116) III / hT2 I, III
Genetic reagent
(C. elegans)
CZ22536: Pmec-7-GFP(muIs32) II;
hosl-1(gk278589) X
Genetic reagent
(C. elegans)
CZ22006: Pmec-7-GFP(muIs32) II;
abhd-5.1(ok3722) V
Genetic reagent
(C. elegans)
CZ21968: Pmec-7-GFP(muIs32) II;
abhd-5.2(ok3245) V
Genetic reagent
(C. elegans)
CZ22007: lid-1(gk575511) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ22163: Pmec-7-GFP(muIs32) II;
abhd-5.2(ok3245) V abhd-5.1(ju1282) V
Genetic reagent
(C. elegans)
CZ22166: lid-1(gk575511) I;
Pmec-7-GFP(muIs32) II;
abhd-5.2(ok3245) abhd-5.1(ju1282) V
Genetic reagent
(C. elegans)
CZ22686: Pmec-7-GFP(muIs32) II;
lipl-1(tm1954) V
Genetic reagent
(C. elegans)
CZ22688: Pmec-7-GFP(muIs32) II;
lipl-3(tm4498) V
Genetic reagent
(C. elegans)
CZ22535: Pmec-7-GFP(muIs32) II;
lipl-4(tm4417) V
Genetic reagent
(C. elegans)
CZ24364: Pmec-4-GFP(zdIs5) I;
lgg-1(tm3489) II/ mIn1 II
Genetic reagent
(C. elegans)
CZ23325: Pmec-7-GFP(muIs32) II;
lgg-2(tm6474) IV
Genetic reagent
(C. elegans)
CZ23322: Pmec-7-GFP(muIs32) II;
hlh-30(tm1978) IV
Genetic reagent
(C. elegans)
CZ14408: Pmec-7-GFP(muIs32) II;
mxl-3(ok1947) X
Genetic reagent
(C. elegans)
CZ22541: nhr-49(nr2041) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ22510: Pmec-7-GFP(muIs32) II;
nhr-80(tm1011) III
Genetic reagent
(C. elegans)
CZ25587: Pmec-4-GFP(zdIs5) I;
cka-1(tm1241) IV
Genetic reagent
(C. elegans)
CZ25549: Pmec-4-GFP(zdIs5) I; 
cka-2(tm841) X
Genetic reagent
(C. elegans)
CZ25403: Pmec-4-GFP(zdIs5) I; 
cka-1(tm1241) IV; cka-2(tm841) X.
Genetic reagent
(C. elegans)
CZ25370: Pmec-4-GFP(zdIs5) I;
pcyt-1(et9) X
Genetic reagent
(C. elegans)
CZ25790: Pmec-4-GFP(zdIs5) I;
pcyt-2.1(gk440213) I
Genetic reagent
(C. elegans)
CZ25368: Pmec-4-GFP(zdIs5) I; 
pcyt-2.2(ok2179) X
Genetic reagent
(C. elegans)
CZ25992: Pmec-4-GFP(zdIs5) I; pcyt-2.1(gk440213) I;
pcyt-2.2(ok2179) X
Genetic reagent
(C. elegans)
CZ26521: Pmec-4-GFP(zdIs5) I; pcyt-2.1(gk440213) I;
pcyt-1(et9) X 
Genetic reagent
(C. elegans)
CZ25369: Pmec-4-GFP(zdIs5) I; 
cept-1(et10) X
Genetic reagent
(C. elegans)
CZ26423: Pmec-4-GFP(zdIs5) I; 
cept-2(ok3135) V
Genetic reagent
(C. elegans)
CZ19835: Pmec-4-GFP(zdIs5) I; 
kel-8(tm5214) V
Genetic reagent
(C. elegans)
CZ23911: kel-20(tm3676) I;
mec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ18224: Pmec-4-GFP(zdIs5) I;
ivns-1(ok3171) X
Genetic reagent
(C. elegans)
CZ18225: Pmec-4-GFP(zdIs5) I;
ivns-1(gk252) X
Genetic reagent
(C. elegans)
CZ25508: Pmec-4-GFP(zdIs5) I;
ivns-1(ok3171) X; 
Ex[ivns-1_gDNA(juEx7673)]
Genetic reagent
(C. elegans)
CZ25509: Pmec-4-GFP(zdIs5) I;
ivns-1(ok3171) X;
Ex[ivns-1_gDNA(juEx7674)]
Genetic reagent
(C. elegans)
CZ25510: Pmec-4-GFP(zdIs5) I;
ivns-1(gk252) X;
Ex[ivns-1_gDNA(juEx7673)]
Genetic reagent
(C. elegans)
CZ25511: Pmec-4-GFP(zdIs5) I;
ivns-1(gk252) X;
Ex[ivns-1_gDNA(juEx7674)]
Genetic reagent
(C. elegans)
CZ24755: juEx7584[Pesyt-2-GFP]
Genetic reagent
(C. elegans)
CZ21465: Pmec-4-GFP(zdIs5) I;
epi-1(gm121) IV
Genetic reagent
(C. elegans)
CZ21463: Pmec-7-GFP(muIs32) II;
emb-9(tk75) III
Genetic reagent
(C. elegans)
CZ21198: Pmec-7-GFP(muIs32) II;
adt-1(cn30) X
Genetic reagent
(C. elegans)
CZ20937: Pmec-4-GFP(zdIs5) I;
adt-2(wk156) X
Genetic reagent
(C. elegans)
CZ21004: Pmec-4-GFP(zdIs5) I;
adt-3 (T19D2.1) (ok923) X
Genetic reagent
(C. elegans)
CZ26611: Pmec-7-GFP(muIs32) II;
gon-1(e1254) IV / +
Genetic reagent
(C. elegans)
CZ23908: rab-8(tm2526) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ23909: rab-11.2(tm2081) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ20682: Pmec-7-GFP(muIs32) II;
nex-1(gk148) III
Genetic reagent
(C. elegans)
CZ20683: Pmec-7-GFP(muIs32) II;
nex-2(ok764) III
Genetic reagent
(C. elegans)
CZ20684: Pmec-7-GFP(muIs32) II;
nex-3(gk385) III
Genetic reagent
(C. elegans)
CZ20685: Pmec-7-GFP(muIs32) II;
nex-4(gk102) V
Genetic reagent
(C. elegans)
CZ14006: Pmec-7-GFP(muIs32) II;
mec-17(ok2109) IV
Genetic reagent
(C. elegans)
CZ14008: Pmec-7-GFP(muIs32) II;
atat-2(ok2415) X
Genetic reagent
(C. elegans)
CZ14848: mec-17(ok2109) IV; atat-2(ok2415) X;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ17720: Pmec-7-GFP(muIs32) II;
mec-12(tm5083) III
Genetic reagent
(C. elegans)
CZ9247: tba-1(ok1135) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ26688: Pmec-4-GFP(zdIs5) I;
tba-7(gk787939) III
Genetic reagent
(C. elegans)
CZ26833: Pmec-4-GFP(zdIs5) I;
tba-7(u1015) III
Genetic reagent
(C. elegans)
CZ26635: Pmec-4-GFP(zdIs5) I;
mec-7(ok2152) X
Genetic reagent
(C. elegans)
CZ10615: Pmec-4-GFP(zdIs5) I;
tbb-2(gk129) III
Genetic reagent
(C. elegans)
CZ11083: Pmec-4-GFP(zdIs5) I;
tbb-4(ok1461) X
Genetic reagent
(C. elegans)
CZ10810: Pmec-4-GFP(zdIs5) I;
tbb-6(tm2004) V
Genetic reagent
(C. elegans)
CZ21461: Pmec-4-GFP(zdIs5) I;
fli-1(ky535) III
Genetic reagent
(C. elegans)
CZ21199: Pmec-7-GFP(muIs32) II;
gsnl-1(ok2979) V
Genetic reagent
(C. elegans)
CZ10888: viln-1(ok2413) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ13606: Pmec-4-GFP(zdIs5) I;
twf-2(ok3564) X
Genetic reagent
(C. elegans)
CZ20063: Pmec-4-GFP(zdIs5) I;
mfsd-6(ju833) III
Genetic reagent
(C. elegans)
CZ19827: Pmec-4-GFP(zdIs5) I;
mfsd-6(tm3356) III
Genetic reagent
(C. elegans)
CZ24417: Pmec-4-GFP(zdIs5) I;
mfsd-6(tm3356) III;
Prgef-1-mfsd-6(juEx6079)
Genetic reagent
(C. elegans)
CZ21030: Pmec-4-GFP(zdIs5) I;
C05D9.7(ok2931) X
Genetic reagent
(C. elegans)
CZ25317: Pmec-4-GFP(zdIs5) I;dpy-10(e128) II
Genetic reagent
(C. elegans)
CZ23667: Pmec-4-GFP(zdIs5) I;F35G2.1(ok1669) IV
Genetic reagent
(C. elegans)
CZ23772: Pmec-4-GFP(zdIs5) I;gly-2(tm839) I
Genetic reagent
(C. elegans)
CZ17890: Pmec-4-GFP(zdIs5) I;osm-11(rt142) X
Genetic reagent
(C. elegans)
CZ17021: Pmec-4-GFP(zdIs5) I;zig-1(ok784) II
Genetic reagent
(C. elegans)
CZ17023: Pmec-4-GFP(zdIs5) I;zig-3(gk33) X
Genetic reagent
(C. elegans)
CZ17024: Pmec-4-GFP(zdIs5) I;zig-3(ok1476) X
Genetic reagent
(C. elegans)
CZ22031: abts-1(ok1566) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ21461: Pmec-4-GFP(zdIs5) I; 
fli-1(ky535) III
Genetic reagent
(C. elegans)
CZ17435: Pmec-7-GFP(muIs32) II;mec-12(e1605) III
Genetic reagent
(C. elegans)
CZ17637: Pmec-4-GFP(zdIs5) I;tba-9(ok1858) X
Genetic reagent
(C. elegans)
CZ20033: vab-10(e698) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ17099: Pmec-4-GFP(zdIs5) I;
plk-1(or683ts) III
Genetic reagent
(C. elegans)
CZ17285: Pmec-4-GFP(zdIs5) I;
svh-2(tm737) X
Genetic reagent
(C. elegans)
CZ19343: Pmec-4-GFP(zdIs5) I;
cpr-1(ok1344) V
Genetic reagent
(C. elegans)
CZ19200:dhhc-11(gk1105) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ22823: Pmec-4-GFP(zdIs5) I;eat-3(tm1107) II
Genetic reagent
(C. elegans)
CZ16134: Pmec-4-GFP(zdIs5) I;npr-20(ok2575) II
Genetic reagent
(C. elegans)
CZ23845: Pmec-4-GFP(zdIs5) I;
ptps-1(tm1984) I
Genetic reagent
(C. elegans)
CZ17995: supr-1(ju1118) I;Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ12031: Pmec-4-GFP(zdIs5) I;rsef-1(ok1356) X
Genetic reagent
(C. elegans)
CZ17629: Pmec-4-GFP(zdIs5) I;
osm-7(tm2256) III
Genetic reagent
(C. elegans)
CZ17098: Pmec-4-GFP(zdIs5) I;
par-2(or373ts) III
Genetic reagent
(C. elegans)
CZ18676: Pmec-4-GFP(zdIs5) I;
rgl-1(ok1921) X
Genetic reagent
(C. elegans)
CZ20056: Pmec-7-GFP(muIs32) II;
wdfy-3(ok912) IV
Genetic reagent
(C. elegans)
CZ19721: Pmec-4-GFP(zdIs5) I
wdr-23(tm1817) I
Genetic reagent
(C. elegans)
CZ22063: Pmec-4-GFP(zdIs5) I;
brap-2(tm5132) II
Genetic reagent
(C. elegans)
CZ21217: Pmec-4-GFP(zdIs5) I;
brap-2(ok1492) II
Genetic reagent
(C. elegans)
CZ19337: Pmec-4-GFP(zdIs5) I;
cdc-48.1(tm544) II
Genetic reagent
(C. elegans)
CZ19725: Pmec-4-GFP(zdIs5) I;
ced-9(n1950sd) III
Genetic reagent
(C. elegans)
CZ21651: Pmec-4-GFP(zdIs5) I;
dnj-23(tm7102) II
Genetic reagent
(C. elegans)
CZ21356: Pmec-4-GFP(zdIs5) I;
fbxc-50(tm5154) II
Genetic reagent
(C. elegans)
CZ16950: Pmec-7-GFP(muIs32) II;
math-33(ok2974) V
Genetic reagent
(C. elegans)
CZ16951: Pmec-7-GFP(muIs32) II;
skr-5(ok3068) V
Genetic reagent
(C. elegans)
CZ21010: tep-1(tm3720) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ22796: mec-8(e398) I;Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ14510: Pmec-4-GFP(zdIs5) I;rict-1(mg360) II
Genetic reagent
(C. elegans)
CZ22570: rtcb-1(gk451) I /
[bli-4(e937) let-?(q782) qIs48](hT2) I, III;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ21655: Pmec-4-GFP(zdIs5) I;
skn-1(ok2315) IV/nT1(qIs51) IV; V
Genetic reagent
(C. elegans)
CZ23377: Pmec-4-GFP(zdIs5) I;
smg-3(r930) IV
Genetic reagent
(C. elegans)
CZ13997: Pmec-7-GFP(muIs32) II;
syd-9(ju49) X
Genetic reagent
(C. elegans)
CZ21723: Pmec-4-GFP(zdIs5) I;
tdp-1(ok803) II
Genetic reagent
(C. elegans)
CZ23133: Pmec-4-GFP(zdIs5) I;
wdr-5.1(ok1417) III
Genetic reagent
(C. elegans)
CZ21194: Pmec-7-GFP(muIs32) II;
mig-17(k174) V
Genetic reagent
(C. elegans)
CZ22792: Pmec-7-GFP(muIs32) II;
ZC116.3(ok1618) V
Genetic reagent
(C. elegans)
CZ19193: Pmec-7-GFP(muIs32) II;lgc-12(ok3546) III
Genetic reagent
(C. elegans)
CZ18217: Pmec-4-GFP(zdIs5) I;tmc-1(ok1859) X
Genetic reagent
(C. elegans)
CZ17639: Pmec-4-GFP(zdIs5) I;nud-1(ok552) III
Genetic reagent
(C. elegans)
CZ17841: Pmec-4-GFP(zdIs5) I;mgl-1(tm1811) X
Genetic reagent
(C. elegans)
CZ17843: Pmec-4-GFP(zdIs5) I;mgl-3(tm1766) IV
Genetic reagent
(C. elegans)
CZ17848: Pmec-4-GFP(zdIs5) I;npr-25(ok2008) V
Genetic reagent
(C. elegans)
CZ22890: Pmec-4-GFP(zdIs5) I;ucr-2.3(ok3073) III
Genetic reagent
(C. elegans)
CZ15607: drag-1(tm3773) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ17393: Pmec-4-GFP(zdIs5) I;
snb-6(tm5195) II
Genetic reagent
(C. elegans)
CZ17018: Pmec-4-GFP(zdIs5) I;
drag-1(tm3773) I
Genetic reagent
(C. elegans)
CZ18617: Pmec-4-GFP(zdIs5) I;
ect-2(ku427) II
Genetic reagent
(C. elegans)
CZ18818: Pmec-4-GFP(zdIs5) I;
lin-2(e1309) X
Genetic reagent
(C. elegans)
CZ18817: Pmec-4-GFP(zdIs5) I;
magi-1(zh66) IV
Genetic reagent
(C. elegans)
CZ20673: Pmec-4-GFP(zdIs5) I;
prmt-5(gk357) III
Genetic reagent
(C. elegans)
CZ18816: Pmec-4-GFP(zdIs5I;
rap-1(tm861) IV
Genetic reagent
(C. elegans)
CZ18460: Pmec-4-GFP(zdIs5) I;
smz-1(ok3576) IV
Genetic reagent
(C. elegans)
CZ22544: Pmec-4-GFP(zdIs5) I;
trxr-1(sv47) IV
Genetic reagent
(C. elegans)
CZ24963: Pmec-4-GFP(zdIs5) I;
natb-1(ju1405) V/nT1 IV; V
Genetic reagent
(C. elegans)
CZ16946: Pmec-7-GFP(muIs32) II;
rnf-5(tm794) III
Genetic reagent
(C. elegans)
CZ23068: ulp-5/tofu-3(tm3063) I;
Pmec-7-GFP(muIs32) II
Genetic reagent
(C. elegans)
CZ23091: Pmec-7-GFP(muIs32) II;
csr-1(fj54) IV/nT1 IV; V
Genetic reagent
(C. elegans)
CZ17638: Pmec-4-GFP(zdIs5) I;
elpc-3(ok2452) V
Genetic reagent
(C. elegans)
CZ12938: Pmec-4-GFP(zdIs5) I;
hda-6(tm3436) IV
Recombinant DNA reagentPlasmid: pCZGY3260:nmat-2 genomic DNAThis workN/Anmat-2 genomic DNA (~1500 bp upstream;~670 bp downstream); modified pCFJ201
plasmid for modified MosSCI on ChIV
Recombinant DNA reagentPlasmid: pCZ993:Pmec-4-nmat-2 gDNA-let-858 3'UTRThis workN/Anmat-2 expression driven by mec-4
promoter in the
mechanosensory neurons
Recombinant DNA reagentPlasmid: pCZ994: Pmtl-2-nmat-2 gDNA-let-858 3'UTRThis workN/Anmat-2 expression driven by mtl-2 promoter in the intestine
Recombinant DNA reagentPlasmid: pCZ995: Pcol-12-nmat-2 gDNA-let-858 3'UTRThis workN/Anmat-2 expression driven by col-12
promoter in the epiderdims
Recombinant DNA reagentPlasmid: pCZGY3329:
Pmec-4-GFP-ESYT-2
This workN/A
Recombinant DNA reagentPlasmid: pCZGY3344: Pmec-4-mKate2-ESYT-2This workN/A
Recombinant DNA reagentPlasmid: pCZGY3342:
Pmec-4-mKate2-PISY-1
This workN/A
Recombinant DNA reagentPlasmid: pCZGY3302:
ivns-1 genomic DNA
This workN/Aivns-1 genomic DNA (2 kb upstream; 800 bp downstream)
Recombinant
DNA reagent
Plasmid: pCZGY3347: Prgef-1-mfsd-6This workN/A
Recombinant
DNA reagent
Plasmid: pCZGY3346:Pesyt-2-GFPThis workN/AGFP expression driven by esyt-2 promoter
Sequence-
based reagent
crRNA: nmat-2:/AltR1/rCrGrArGrU rCrGrC rUrCrU rUrCrU rUrGrC rCrGrUrGrUrU rUrUrA rGrArG rCrUrA rUrGrC rU/AltR2/IDTN/AcrRNA to makenmat-2(ju1512) and nmat-2(ju1514)
Sequence-
based reagent
crRNA: nmat-2:/AltR1/rCrGrUrGrU rUrGrA rArCrU rArArC rUrCrC rArCrU rGrUrU rUrUrA rGrArG rCrUrArUrGrC rU/AltR2/IDTN/AcrRNA to makenmat-2(ju1512)
Sequence-
based reagent
crRNA: nmat-1:/AltR1/rArA
rCrUrU rUrUrU rCrGrGrUrCrC rCrCrA rUrArG rGrUrU rUrUrA rGrArG rCrUrA rUrGrC rU/AltR2/
IDTN/AcrRNA to mak
e nmat-1(ju1565)
Sequence-
based reagent
crRNA: nmat-1:/AltR1/rArU
rGrUrA rCrUrU rGrArU rUrArC
rGrGrA rArUrC rGrUrU
rUrUrA rGrArG rCrUrA
rUrGrC rU/AltR2/
IDTN/AcrRNA to make nmat-1(ju1565)
Sequence-
based reagent
crRNA: qns-1:/AltR1/rGrGrUrGrU rUrArU rUrCrA
rCrGrU rGrUrU rArCrA
rGrUrU rUrUrA rGrArG
rCrUrA rUrGrC rU/AltR2/
IDTN/AcrRNA to make qns-1(ju1563)
Sequence-based reagentcrRNA: qns-1:/AltR1/rGrA
rUrArA rCrUrG rArArA rUrCrU
rGrGrA rUrArG rGrUrU rUrUrA
rGrArG rCrUrA rUrGrC rU/AltR2/
IDTN/AcrRNA to make qns-1(ju1563)
Sequence-based reagentcrRNA: esyt-2:/AltR1/rGrG rUrUrU rCrArG rUrArA rUrUrGrUrGrG rGrCrU rGrUrUrUrUrA rGrArG rCrUrA rUrGrC rU/AltR2/IDTN/AcrRNA to make esyt-2(ju1409)
Sequence-based reagentcrRNA: esyt-2:/AltR1/rGrU rGrCrA rCrUrU rArCrG rGrGrU rUrGrU rArGrGrGrUrU rUrUrA rGrArGrCrUrA rUrGrC rU/AltR2/IDTN/AcrRNA to makeesyt-2(ju1409)
Peptide,
recombinant
protein
Protein: Cas9-NLS
purified protein
QB3 MacroLab, UC BerkleyN/A
Peptide,
recombinant
protein
Phusion High-Fidelity DNA polymerasesThermo ScientificCat#F530L
Peptide,
recombinant
protein
DreamTaq DNA polymerasesThermo ScientificCat#EP0705
Commercial assay or kit
Chemical compound, drug5-fluoro-2-deoxy uridineSigma-AldrichCat#50-91-9
Software, algorithmImageJNIH imageRRID:SCR_003070
Software, algorithmZENZeisshttps://www.zeiss.com/microscopy/us/downloads/zen.html
Software, algorithmZeiss LSM Data ServerZeisshttps://www.zeiss.com/microscopy/us/downloads/lsm-5-series.html
Software, algorithmGraphPad Prism 5GraphPad
Software, Inc.
RRID:SCR_002798

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  1. Kyung Won Kim
  2. Ngang Heok Tang
  3. Christopher A Piggott
  4. Matthew G Andrusiak
  5. Seungmee Park
  6. Ming Zhu
  7. Naina Kurup
  8. Salvatore J Cherra III
  9. Zilu Wu
  10. Andrew D Chisholm
  11. Yishi Jin
(2018)
Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration
eLife 7:e39756.
https://doi.org/10.7554/eLife.39756