Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Summary

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Abstract

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see ¹). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel² or sapje³) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes⁴. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy⁵. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally¹. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo⁶, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells⁷.

Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.

Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail^{8, 9}. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish^{10, 11}.

Protocol

1. Labeling Single Cells

Inject one-cell stage embryos with a plasmid encoding GFP or any GFP-fusion protein under the control of a β-Actin promoter. During development, GFP is then expressed in a mosaic fashion. Here, we used the transgenic construct Tg[β-actin:α-actinin-GFP], which places the α-Actinin-GFP fusion protein under the control of the β-actin promoter.

2. Embryo Embedding

1. Collect zebrafish embryos and maintain under normal conditions until 28 hpf (staging according to¹²).
2. Dechorionate embryos under the stereomi....

Results

Laser-mediated injury was performed on immobilized 1 day-old embryos. As shown in Figure 1, a few laser pulses can generate a small wound, easily recognizable by the damaged, coiled, Actin-rich myofibrils that are normally stretched between the somite boundaries. A higher number of laser pulses will however result in a massively damaged somite block, where most myofibrils are destroyed. Nevertheless, we can literally observe that "time heals all wounds", with small injuries healing faster than larger one.......

Discussion

Laser-mediated injury is a powerful method for inflicting wounds of a desired size by ablating cells in order to study regeneration under controlled conditions in the zebrafish embryo. Notably, cells can be targeted precisely (Figure 2) and both the injury area as well as timing can be controlled. Subsequently, the injury site and regeneration processes are easily monitored, recorded (Figure 3) and analyzed (Figure 1). However, although the laser is well focused in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Heating block
Pair #5 forceps	Dumont
Glass slides	Menzel		76 x 26 mm
Coverslips	Roth		50 x 24 mm #1
Petroleum jelly
Stereomicroscope	Leica		MZFLIII
Micropoint laser	Andor Technology
Fluorescence microscope	Zeiss		Axioplan II
Metamorph software	Molecular devices
Reagents Low-melting point agarose ( #50081, Lonza) Tricaine stock solution: 400 mg Tricaine (#A-5040, Sigma-Aldrich ) / 100 ml dH2O pH 9.0 E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4)