Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Summary

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

Abstract

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.

Introduction

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 healing^3,4. 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 mechan....

Protocol

The following protocols describe the detailed procedure for C. elegans skin wounding and for assaying wound responses.

1. Needle Wounding^3,4

1. Grow healthy unstarved worms on standard NGM (nematode growth medium) plates with E. coli OP50 bacteria as food, maintained in a 20 °C incubator.
   NOTE: Methods for NGM agar plates and routine cultivation of C. elegans can be found at www.wormbook.org⁶.
2. Pick 25 L4 stage worms to a freshly seeded plate 1 day before wounding and culture at 20 °C O/N.
3. Before wounding, pull needles from capillaries using a need....

Results

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 l.......

Discussion

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 requ.......

Materials

List of materials used in this article

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 liter. 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