Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.
Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.
At the core of most progressive hearing loss lies the destruction of sensory hair cells, which transduce extrinsic auditory stimuli into nerve impulses detectable by the brain. Cochlear hair cell death causes permanent hearing loss, as adult mammals lack the ability to regenerate these cells following damage. Conversely, non-mammalian vertebrates such as zebrafish can regenerate hair cells lost to acoustic trauma or ototoxic insult. The zebrafish mechanosensory lateral line is a simple and easily manipulated organ system that can be used to study hair cell regeneration1,2,3.
The lateral line consists of small sensory patches called neuromasts, which are connected during development by a string of elongated cells known as interneuromast cells (INMCs). Given their proliferative capacity as apparent stem cells that give rise to new hair cell-containing organs, the behavior of INMCs is of great interest to the community4,5. Much of the research pertaining to INMCs has characterized suppression of their proliferation by nearby Schwann cells that wrap around the lateral line nerve, likely through an inhibitor of Wnt/β-catenin signaling.6,7,8. While the regulation of INMC proliferation and differentiation into new neuromasts has been well-described, the mechanisms governing INMC regrowth after ablation have not been elucidated. This protocol serves as a means of ablating individual INMCs and analyzing their subsequent regenerative behavior.
A variety of methods for destroying hair cells, supporting cells, and entire neuromasts have been published, along with methodologies for monitoring recovery. Chemical ablation works well for hair cells, but doses of toxic chemicals such as CuSO4 must be significantly increased to remove other lateral line cell types9. While electroablation under fluorescence microscopy is an effective and simple means of destroying INMCs to study regeneration, it is difficult to target narrowly and, therefore, is likely to produce significant collateral damage. As a result, this can mask aspects of INMC-specific behaviors10,11. Laser ablation protocols have been employed, but they require the use of specialized equipment not necessarily present in the average laboratory or imaging facility (i.e., high-powered pulsed UV lasers12). The method described here can be performed with any laser-scanning confocal microscope outfitted with the common 405 nm laser, usually used for imaging DAPI and other blue fluorophores. An advantage of this method is that confocal imaging can be performed immediately before and after cell damage without engaging any additional laser control systems or transferring to another microscope equipped with a pulsed laser.
A similar method has been described for ablating neurons in the zebrafish spinal cord13. However, the previous method requires a FRAP module for cell ablation, which is not a requirement for this protocol. Furthermore, given the distinct properties of different cell types (i.e., brightness of transgene fluorescence, depth within the body, and cell shape), it is likely that significant modifications will be needed depending on the cell type used. The protocol detailed here is more likely to be effective for more superficial cells, as supported by a demonstration of sensory hair cell ablation using the same method. Also outlined is a regeneration assay to assess INMC recovery rates after ablation, which was not a feature of the aforementioned study.
The laser-based cell ablation protocol detailed herein captures the regenerative capacity of INMCs through the optimization of laser conditions, pre- and post-ablation imaging, and time-lapse microscopy. These elements come together to comprehensively portray INMC regrowth and gap closure in its entirety. While this protocol is used here to destroy INMCs, it may be applied to other work that requires reliable and effective assessment of cellular ablation. It is also an accessible technology, since it can be conducted on any confocal microscope equipped with a 405 nm laser.
All animal work was approved by the Pace University Institutional Animal Care and Use Committee under protocol 2018-1.
1. Preparation of zebrafish larvae
2. Preparation of low gelling agarose and tricaine solutions
3. Anesthetization of zebrafish larvae
4. Fluorescent screening of anesthetized zebrafish larvae
5. Mounting larvae for laser ablation and imaging
6. Locating prospective targets and pre-ablation imaging
7. Laser ablation of cell bodies
8. Post-ablation imaging and time-lapse microscopy to study regeneration
9. Image analysis
In order to ablate INMCs and record regeneration, screened GFP-expressing zebrafish larvae of the ET20 transgenic line were mounted at 2- or 3-days post-fertilization for ablation as described in step 5.4. Several fish can be mounted simultaneously so that multiple time lapses can be captured in a single experiment. The region of the lateral line located between primIL3 and primIL4 neuromasts was identified, and pre-ablation images were captured as described in steps 6.5–6.7 (Figure 1A,B).
Three cell bodies were targeted for laser ablation to create a gap in the INMC string of approximately 40 µm. Post-ablation scanning with high gain and subsequent imaging confirmed that no cell bodies remained in the ablated region, leaving a gap between elongated projections of the adjacent INMCs (Figure 1C). Cell death was further demonstrated by examining the T-PMT channel after ablation. Damaged and dying cells were marked by swollen and irregularly shaped nuclei as well as a granular appearance (Figure 2, outlined). In some cases, time-lapse imaging also revealed the recruitment of large amoeboid cells that were likely macrophages (Figure 2, asterisk). Dark areas surrounding the ablated INMCs indicated photobleaching in overlying periderm cells. These cells did not appear to be damaged or destroyed by laser irradiation in these experiments; rather, their fluorescence was temporarily reduced. Time-lapse microscopy reveals that these cells normally recover after ablation and do not change shape or otherwise appear damaged (unpublished results, Volpe et al.).
To support the specificity of INMC destruction, ablations were performed in double-transgenic ET20 and Tg(neuroD:tdTomato) larvae in which the lateral line nerve (which runs just microns below the interneuromast cells) is labeled with red fluorescent protein. Ablations of several cells that created sizeable gaps in the INMC string had little or no effect on the lateral line nerve based on red fluorescence (Figure 3). Flexibility of the technique for ablating superficially localized cells was demonstrated by selective destruction of individual sensory hair cells within neuromasts of the lateral line. Using essentially the same protocol detailed above (sections 7 and 8), single hair cells in transgenic Tg(myo6b:βactin-GFP) larvae were destroyed without apparent damage to adjacent cells (Figure 4). The ablated hair cells did not recover fluorescence after time-lapse microscopy, and their nuclei were noticeably granular and misshapen after laser exposure (Figure 4B). Similar to INMC ablations, apparent macrophages were frequently recruited to the laser exposure site (unpublished results, Volpe et al.).
Following laser ablation and post-ablation image capture, gap size was measured using freely available image analysis software. The measure tool demonstrated gap sizes ranging from just a few microns up to 100 microns, depending upon the width of individual INMCs and how many cells were chosen for ablation (Figure 5). Timelapse microscopy was employed to record INMC behaviors during regeneration. In 50 trials, 15 gaps (30%) were closed by regeneration within a 24 h period. In most cases, INMCs that were able to recover did so within the first several hours of imaging. Recovery was defined as the timepoint at which projections from neighboring cells came into contact, which occurred 16 h after ablation for a gap of approximately 40 µm (Movie 1). Z-stacks were carefully examined to ensure that cell-cell contact occurred within a single z-plane, avoiding possible artifacts due to z-projections. Logistic regression analysis revealed that the probability of gap closure correlated with gap size (p = 0.0453), with smaller gaps being more likely to heal. A gap of 55.5 µm yielded a closure probability of 50% (Figure 5).
In 70% of cases, INMCs were unable to completely close the gap created by ablation. However, even in these cases we were able to monitor the formation of long projections from neighboring INMCs, which resemble in some respects extending neuronal growth cones (Movie 2). Thus, these experiments can also provide insight into the behaviors of INMCs after damage.
Figure 1: Selective ablation of interneuromast cells by laser irradiation. (A) Interneuromast cells between neuromasts primIL3 and primIL4 were selected for ablation. These cells display a characteristic spindle shape with elongated projections overlapping adjacent cell bodies. (B) Pre-ablation imaging identified cell bodies that could be ablated to produce a gap. (C) Post-ablation imaging confirms successful ablation as indicated by the absence of cell bodies in the gap region. Scale bars = 10 µm. Please click here to view a larger version of this figure.
Figure 2: Post-ablation imaging demonstrates cell death in response to laser exposure in GFP-labeled interneuromast cells. Transmitted light photomultiplier imaging (T-PMT) indicates the presence of a necrotic cell with a granular appearance (encircled) as well as the recruitment of irregularly shaped cells that are likely macrophages (*). Merging of GFP and T-PMT channels confirms that cell death and macrophage activity occurred at the site of ablation. Scale bars = 10 µm. Please click here to view a larger version of this figure.
Figure 3: Specificity of interneuromast ablation. (A) Pre-ablation GFP-labeled interneuromast cells (green) and tdTomato-labeled lateral line nerve (red) in a double-transgenic Tg(ET20;NeuroD:tdTomato) larva. Cell bodies in close proximity to the lateral line nerve were targeted for ablation. (B) Post-ablation imaging demonstrates ablation of targeted cell bodies with an intact lateral line nerve. Scale bars = 10 μm. Please click here to view a larger version of this figure.
Figure 4: Ablation of sensory hair cells using confocal microscopy. (A) Pre-ablation imaging identified a GFP labeled sensory hair cell (*) targeted for ablation in double-transgenic Tg(ET20; myo6b:βactin-GFP) zebrafish. Transmitted light photomultiplier tube (T-PMT) imaging discloses normal hair cell morphology, with a round cross-section. (B) Post-ablation imaging confirms successful ablation of the targeted hair cell. A T-PMT image indicates irregularity in hair cell shape and increased granularity after laser exposure, suggesting cell death rather than photobleaching. Scale bars = 10 μm. Please click here to view a larger version of this figure.
Figure 5: Logistic regression models the probability of gap closure as a function of gap width (in µm). A score of 0 represents complete gap closure, whereas a score of 1 represents incomplete gap closure. Results indicate that the effect of gap width on the ability of interneuromast cells to close respective gaps is statistically significant (p = 0.0453, n = 24 total; n = 12 closed gaps, n = 12 incompletely closed gaps). Please click here to view a larger version of this figure.
Movie 1: Time-lapse microscopy demonstrating gap closure (~40 µm) after 16 h. Images were captured every 15 min, and a maximum-intensity z-projection was made for all timepoints. Please click here to download this video.
Movie 2: Time-lapse microscopy of an INMC gap that did not close. The elongated and branching projections of the remaining INMCs are shown. Please click here to download this video.
The protocol describes a versatile method for laser cell ablation that can be performed on any confocal microscope equipped with a near-ultraviolet laser (405 nm wavelength). This protocol addresses limitations of previously employed methods such as electroablation (which causes more widespread damage11) and pulsed UV-laser ablation (which requires additional specialized equipment12). Pre- and post-ablation confocal imaging provide rapid feedback regarding success of the experiment. Subsequent time-lapse microscopy offers a simple assay for regeneration in a cell type critical for sensory system development.
It is of vital importance to prescreen for transgenic zebrafish that express GFP strongly in neuromasts and INMCs. Larvae with bright fluorescence and approximately even spacing of the primI-derived neuromasts are ideal candidates for laser ablation. Even in these fish, there are occasionally dimmer INMCs that may at first escape detection. These cells can remain behind after laser ablation, preventing the formation of a true gap between INMCs. The digital gain should be adjusted to reveal these dimmer cells during pre-ablation imaging such that they can also be targeted with the 405 nm laser (step 8.2).
Similarly, laser targeting sometimes will not entirely destroy a cell; rather, it will bleach the fluorescence, resulting in a failure to create a real gap. These cells will generally increase in fluorescence during timelapse microscopy. Gain adjustment in the post-ablation imaging step along with T-PMT imaging (Figure 2) help ensure that all cells targeted have been effectively removed. It frequently requires two or three individual cell ablations to produce a gap in the INMC string. Cell death can be detected by blebbing or a granular appearance in the targeted cells; although, this may require a brief waiting period after laser targeting (and in some cases, apparent apoptotic or necrotic figures are observed shortly thereafter).
A limitation of this procedure is that it may require multiple experimental trials before laser conditions are optimized and INMC regeneration is successful. The laser power and total dwell time of the laser may each require slight adjustment for different samples. It has been found that excessive laser application can impair the ability of the lateral line to repair itself. This includes both 1) overexposure of individual cells to laser irradiation, presumably causing additional damage to surrounding tissues, as well as 2) the ablation of too many cells in the string, leading to a larger gap. Users must achieve a balance between sufficiently irradiating cells to ensure their destruction and not overexpose the cells, which can slow or prevent regeneration. With the appropriate settings, it has been found that the INMCs can be removed without visible damage to the posterior lateral line nerve, indicating that collateral damage is minimized with this protocol unlike electroablation11. In no cases were new neuromasts forming in the gap observed, presumably because the lateral line nerve remains intact and underlying glial cells remain and inhibit the proliferation of INMCs6,7,8,11.
It is demonstrated that this method of confocal laser ablation may be applied to other cell types as well, particularly in those superficially located within the animal including sensory hair cells (Figure 4). A similar protocol may be used to ablate skin cells to examine wound repair or neurons for axonal regeneration studies, as previously described13. It is anticipated that this technique will become a useful addition to the experimental repertoire of laboratories that possess a confocal microscope but no other specialized equipment for laser ablation.
The authors have nothing to disclose.
This work was funded by NIH R15 grant 1R15DC015352-01A1 and Pace University internal funding sources. Fish lines were courtesy of Vladimir Korzh14, Katie Kindt15,16, and the laboratory of A. James Hudspeth. We would like to thank A. James Hudspeth and members of his group for feedback on these experiments, and colleagues at Pace University for their support. Logistic regression analysis in RStudio was aided in particular by our colleague Matthew Aiello-Lammens.
12-well PTFE Printed Slides | Electron Microscopy Sciences | 63425-05 | |
15 mM Tricaine stock solution | Sigma | E10521 | 15 mM Tricaine in reverse osmosis water |
1X E3 + 600 µM Tricaine | Dilute 15 mM Tricaine stock 25X in 1X E3 media | ||
1X E3 media | Dilute 60X E3 media to 1X in reverse osmosis water (16.7 ml/L) | ||
60 X E3 media | All components purchased from Sigma-Aldrich | 34.4 g Nacl, 1.52 g Kcl, 5.8 g CaCl2.2H2O, 9.8 g MgSO4.7H20 in 1 liter reverse-osmosis water | |
Bx60 Compound microscope with mercury arc lamp fluorescence | Olympus | ||
LSM 700 confocal microscope equipped with 405 nm, 488 nm, and 555 or 561 nm lasers | Carl Zeiss Microscopy, LLC | A 5 mW 405 nm laser was used for ablation; Ablations and imaging were performed through a 63x Plan-Apochromat objective with an NA of 1.40 | |
FIJI/ImageJ Image processing software | Multiple contributors | Downloadable at https://fiji.sc/ | |
Dissecting needle modified into hair knife | Fisher Scientific | 19010 | An eyelash was glued onto the end of a wood-handled dissecting needle |
Microsoft Excel software | Microsoft Corp. | Google sheets is a no-cost alternative to Microsoft Excel | |
Glass bottom 35 mm dishes with no. 1.5 coverslip, 20 mm window | Mattek Corporation | P35G-1.5-20-C | 35 mm petri dish, 20 mm glass window |
Immersol 518F Immersion Oil | Fisher Scientific | 12-624-66A | |
Low Gelling Agarose | Sigma Life Science | A9414-256 | |
Corning Netwell Insert with 74 um Polyester Mesh, 24 mm Insert | Millipore Sigma | CLS3479-48EA | |
Rstudio software | Rstudio PBC | Downloadable at https://rstudio.com/ | |
SMX-168-BL Stereo microscope | Motic | ||
Transfer Pipette | Fisher Scientific | 13-711-7M | |
ZEN software | Carl Zeiss Microscopy, LLC |