The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.
The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.
Skin wound healing mechanisms are of basic biological interest and relevant to human health. Wound healing in vertebrates and mammals comprises a complex series of coordinated responses of multiple tissues and signaling1. Many simple genetic model organisms are also capable of healing skin wounds2. It is therefore of interest to analyze genetically tractable models of skin wound repair. We and others have begun to use Caenorhabditis elegans as new model for skin wound healing3,4. The goal of this protocol is to enable a broader set of researchers to use C. elegans as a tool to investigate molecular and cellular mechanisms of epidermal wound healing.
The C. elegans skin comprises the epidermis (also known as hypodermis) and the extracellular cuticle5. The adult epidermis is formed from a small number of multinucleate syncytia, of which the largest is the syncytium known as hyp7. The epidermis is a simple epithelium that secretes the cuticle on its apical surface. The skin can actively defend against skin-penetrating pathogens and repair small wounds4. Wound repair of the C. elegans skin is robust, as almost all wild type animals survive can survive small puncture wounds caused by needles, or local skin damage caused by laser irradiation. C. elegans skin wounding triggers a suite of responses, including an epidermal innate immune response, wound closure, and scar formation4. The adult epidermis is post-mitotic, and wound healing involves local cellular responses as opposed to epidermal proliferation or cell migration. We have shown that skin wounding triggers a large and sustained increase in epidermal Ca2+, requiring the membrane TRPM channel GTL-2 and internal Ca2+ stores3. The epidermal Ca2+ signal is required for formation and closure of F-actin rings at the wound site. Wounding also induces innate immune responses that activate the transcription of AMPs such as nlp-29. The wounding-induced transcription of AMPs is dependent on a TIR-1/PMK-1 p38 MAP kinase cascade acting autonomously in the epidermis4. Defects in either the Ca2+ signaling pathway or in the innate immune response will lead to lower survival post-wounding. The relatively simple structure of the C. elegans epidermis, its genetic tractability and advantages for in vivo imaging make it an excellent system to study multiple aspects of wound repair.
Here we present protocols for the two common methods of wounding: needle wounding and laser wounding. Needle wounding requires no specialized equipment (other than the needle puller), and with experience can be performed on hundreds of worms per day. Needle wounding is performed on animals growing on agar plates. In contrast, laser wounding is performed on anesthetized animals mounted on agar pads under a coverslip, and is suited for live imaging of the cellular responses to damage.
The following protocols describe the detailed procedure for C. elegans skin wounding and for assaying wound responses.
1. Needle Wounding3,4
2. Laser Wounding3
Use femtosecond laser irradiation (800 nm) to perform more precise wounding.
3. Visualization of Epidermal Ca2+ Responses to Wounding
4. Visualization of F-actin Dynamics After Wounding
5. Assay Induction of Epidermal Antimicrobial Peptides
NOTE: Wounding also triggers innate immune responses in the C. elegans skin. Transcription of genes encoding antimicrobial peptides (AMPs) is induced in the epidermis. The nlp-29 (neuropeptide-like protein) AMP is strongly induced by wounding4. To assay how the epidermis controls AMP expression after wounding, use transgenic reporters or real-time PCR to detect individual AMP transcript levels.
6. Assay Survival After Wounding
Laser or needle wounding will trigger rapid and sustained elevation in epidermal Ca levels, as visualized with Ca sensors such as GCaMPs (Figure 1). The Ca elevation occurs within seconds, and remains elevated for tens of minutes. Needle wounding reproducibly results in the formation of F-actin rings at wound sites (Figure 2); these appear within minutes, and gradually close over 1-2 hr after wounding. Actin rings are less often formed after more precise laser wounding. Both needle and laser wounding induce expression of epidermal antimicrobial peptides (AMPs) such as nlp-29, detected with transcriptional reporters (Figure 3). AMP induction is apparent within 2-4 hr after wounding and lasts approximately 24 hr. Needle wounding of the wild type should not significantly affect viability (measured at 24 hr after wounding) or life span.
Figure 1. Laser wounding triggers a Ca2+ response in the C. elegans epidermis. (A) Epidermal GCaMP3 fluorescence (Pcol-19-GCaMP3 (juIs319)) levels after femtosecond laser wounding. Lateral view of epidermis in anterior-body; x, laser wound; N, epidermal nucleus. Spinning disk confocal images, intensity code. UW: unwounded, W: wounded. (B) Quantitation of GCaMP3 ΔF/F0 at 20, 40, and 60 μm from wound site; representative trace. Please click here to view a larger version of this figure.
Figure 2. Needle wounding triggers actin polymerization around wound site. (A) Epidermal needle wounds trigger formation of actin rings at the wound margin, visualized with Pcol-19-GFP-moesin (juIs352). Scale: 10 μm. UW: unwounded, W: wounded. (B) Quantitation of actin ring diameter from line scans, 4 line scans per animal (dotted red lines in panel (A), WT). Please click here to view a larger version of this figure.
Figure 3. Epidermal wounding activates innate immune response. Representative images (WT unwounded, WT wounded + 3 hr) of Pnlp-29-GFP(frIs7) induction after needle wounding. Scale: 200 μm. Please click here to view a larger version of this figure.
Reporter | Transgene | Fluorescence | Allele |
Calcium | Pcol-19-GCaMP3;Pcol-19-tdTomato | GFP/tdTomato | juIs319 X |
Actin | Pcol-19-GFP::moesin | GFP | juIs352 I |
nlp-29 | Pnlp-29-GFP/Pcol-12-DsRed | GFP/DsRed | frIs7 IV |
Table 1. Transgenes used in wound response assays. This table lists chromosomally integrated transgenes available at the Caenorhabditis Genetics Center (http://www.cbs.umn.edu/research/resources/cgc).
Gene name | Sequence |
nlp-29 | F: cttctcgcctgcttcatggc |
R: gtccgtatccaccatatcctcc | |
nlp-30 | F: TTCTTCTCGCCTGCTTCATGG |
R: CATAACCTCTACCATATCCACCG | |
cnc-1 | F: gccattgtcgccatttcctc |
R: cctccatacattggatatcctc | |
cnc-5 | F: CTTCTTCTAGCAATGCTCGCTC |
R: GTATCCTCCACCATACCCTCC | |
ama-1 | F: ACTCAGATGACACTCAACAC |
R: GAATACAGTCAACGACGGAG | |
snb-1 | F: tccagcagacacaagctcagg |
R: gagacaacttctgatcacgctc |
Table 2. Primers used for RT-PCR of AMP transcripts. Working concentration is 0.2 µM. snb-1 serves as internal control.
The methods presented here for needle and laser wounding provide complementary approaches for assessing the ability of the epidermal epithelium to repair damage. Laser wounding is relatively localized and (depending on laser configuration) can be confined to the epidermis, whereas needle wounding disrupts the epidermis, cuticle, and likely internal basement membranes. Needle wounding may more accurately resemble wounds inflicted by pathogens or mechanical damage in the natural environment. As a rule, needle wounding requires more practice to achieve precise wounds that are compatible with animal survival. The cellular responses to needle and laser wounding display many similarities. However it should be noted that formation of actin rings is not reproducibly observed after femtosecond laser wounding.
A limitation of needle puncture is that it is not a precise wound: the needle also damages internal tissues whose contributions to worm survival have not been investigated. A possible modification of this protocol would be to combine microfluidic-based microinjection system11 and fluorescent reporters to perform more precise physical wounding.
The needle and laser wounding methods detailed here are simple yet fairly labor intensive, and at best medium-throughput, making it challenging to use genomic or biochemical approaches to characterization of the wound response. Cuticle penetrating pathogens such as fungi or protozoa12,13 can be delivered to larger populations of animals, and may trigger responses that overlap with the response to wounding, yet introduce additional complexities of pathogen specific responses. Further work will be required to explore whether other abiotic methods such as ballistic bombardment14 or arrays of micromechanical piercing structures15 can be adapted for large-scale wounding.
The authors have nothing to disclose.
Needle wounding methods for C. elegans were first developed by Nathalie Pujol and Jonathan Ewbank; we thank Nathalie Pujol for comments on the manuscript. Work in our laboratory on C. elegans epidermal wound repair is supported by a grant from the NIH to A.D.C. (R01 GM054657).
Name | Company | Catalog Number | Comments |
Agarose | Denville Scientific | CA3510-8 | |
Plastic Petri plate | Tritech Research | T3308 | 60 mm |
Levamisole | Sigma | L9756 | 12 mM |
Borosilicate Glass Capillary | World Precision Instruments | 1B100F-4 | |
Needle puller | Sutter Instrument | P-2000 | |
Worm pick (Platinum wire) | Tritech Research | PT-9010 | |
M9 solution | Home-made | 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Sterilize by autoclaving | |
Trizol | Invitrogen | 15596026 | Use 500 ml for 50 worms |
iQ SYBR Green | Bio-Rad | 1708882 | |
SuperScript III | Invitrogen | 18080-051 | |
PCR machine | Bio-Rad | C1000/CFX96 | |
96-well PCR tubes | Bio-Rad | MLL9601 | |
Image analysis software | Molecular Devices | Metamorph |