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Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae
In This Article
Summary
This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.
Abstract
Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using different methods such as fin amputation, scale plucking, skull trepanation, and microscopic approaches. Using a confocal laser scanning setup equipped with a two-photon laser, a laser ablation method was developed as a lesion paradigm to ablate bone-forming cells (osteoblasts) in the developing opercle of zebrafish larvae. The method described here allows the ablation of cells in a precise manner, as the area, shape, and depth can be finely adjusted. In addition, this method allows imaging of the area before and just after the ablation, so that short-term effects of the injury can be analyzed. In this experimental setup, the immune response after ablation of osteoblasts in the injured area was studied. An increase in the recruitment of macrophages was observed after ablation, indicating the relevance of their presence during bone regeneration.
Introduction
Zebrafish regenerate diverse organs such as the retina, brain, heart, and pancreas1. In addition, zebrafish regenerate skeletal elements, which is why they have been used to study the regeneration of fins, scales, and calvariae (skull caps)2. Different experimental paradigms have been used to study tissue and bone regeneration, such as fin resection (amputation), fin fracture, skull trepanation3,4, cryoinjury5,6, or genetic ablation7,8,....
Protocol
The study protocol received approval from the Landesdirektion Sachsen, permit numbers 25-5131/564/2, DD25-5131/450/4, 25-5131/496/56, DD25.1-5131/354/87. The zebrafish strains used were maintained according to national law and under standardized conditions as previously described27,28. The details of all the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of mater.......
Representative Results
Laser ablation was performed as indicated in the protocol above. The GFP signal of the osteoblasts in the ablated area disappeared instantaneously after ablation. To study the response of the osteoblast ablation in terms of the immune response, the presence of macrophages in 6 dpf larvae before and at 6 hpl was imaged. Before ablation, very few macrophages were observed in the opercle area10 (Figure 2). At 6 hpl, a strong accumulation of macrophages in the ablated ope.......
Discussion
Laser ablation has been applied in various disciplines of biological research. In particular, it has been useful as a method to study tissue regeneration10,11,12. For example, the recruitment and phenotype changes of innate immune cells were recently analyzed over time using UV laser or two-photon laser ablation assays, along with the recovery of osteoblasts at the laser ablation site10. Live imaging of i.......
Acknowledgements
This work was supported by the German Research Foundation (DFG) Transregio 67 (project 387653785), the DFG SPP 2084 µBone (project KN 1102/2-1) to FK. This work was supported by the Light Microscopy Facility (DFG project 413875620), a Core Facility of the CMCB Technology Platform at TU Dresden. The work at the TU Dresden was co-financed with tax revenues based on the budget agreed by the Saxon Parliament ('Landtag').
....Materials
| Name | Company | Catalog Number | Comments |
| Calcium chloride dihydrate, CaCl2·H2O | Carl Roth | 5239.1 | |
| Cell Culture Dish, PS, 100/20 mm | Greiner Bio-one | 664160 | |
| Dumont #55 Foceps | FST | 11295-51 | Tip shape straight, 11 cm, 0.05 x 0.02 mm |
| Falcon 6-well plate | Corning | 353502 | |
| Glass-bottom microwell dish | MatTek | P35G-1.5-14-C | 35 mm Dish, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated |
| Insight X3 multiphoton laser | Spectra-Physics | ||
| Leica Application Suite | LAS X, Leica Microsystems | ||
| Low melting agarose | Biozyme | 840101 | Biozym Plaque Agarose |
| Magnesium sulfate heptahydrate, MgSO4·7H2O | Sigma-Aldrich | M5921 | |
| Mating cages | many varieties, e.g. Tecniplast | ||
| Methyleneblue | Carl Roth | A514.1 | |
| MS-222 | SIGMA Aldrich | A5040 | |
| Potassium Chloride, KCl | PanReac AppliChem | 131494 | |
| Sodium chloride, NaCl | Carl Roth | 3957.1 | |
| SP8 FALCON | Leica Microsystems | Equipped with a Insight X3 multiphoton laser and Leica Application Suite software | |
| Stainless Steel Dissect Needle | Bochem | 12010 | 140 mm |
| Stereo Microscope System SZX16 | Olympus | Equipped with a LED illumination base SZX2-ILLTQ | |
| Thermostatic Cabinets TS - WTW | xylem | TS 608/2-i | For incubation (embryo) |
| Transfer pipette, 3.5 mL | SARSTEDT | 861171 | 155 x 15 mm, LD-PE, transparent |
| Zeiss SteREO Discovery.V12 version 4.7.1.0. | Zeiss | Equipped with Axiocam MRm camera and AxioVision sofware |
References
- Marques, I. J., Lupi, E., Mercader, N. Model systems for regeneration: Zebrafish. Dev. 146 (18), dev167692 (2019).
- Dietrich, K., Fiedler, I. A. K., Kurzyukova, A. Skeletal biology and disease modeling in zebrafish. J Bone Min....
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