This work was supported by the German Research Foundation (DFG) Transregio 67 (project 387653785), the DFG SPP 2084 &#181;Bone (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 (&#39;Landtag&#39;).

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 materials and solutions
<ol>
	<li>Maintain the embryos in 1x E3 media. Prepare the 1x E3 media by diluting 170 mL of a 60x E3 stock in 10 L of deionized water and adding 2 mL 0.1% methylene blue.
	<ol>
		<li>Prepare the 60x E3 media stock by mixing 17.2 g of NaCl, 0.76 g of KCl, 2.90 g of CaCl2&#183;2 H2O, 4.90 g of MgSO4&#183;7 H2O in 1 L of deionized autoclaved water.</li>
	</ol>
	</li>
	<li>For the larvae embedding, prepare aliquots of 1% low melting point agarose (LMA) in advance and keep them at room temperature.
	<ol>
		<li>To prepare aliquots, dissolve LMA in 1x E3 media, e.g., 500 mg of LMA in 50 mL of 1x E3 in an Erlenmeyer flask, heat the solution in the microwave until the agarose completely dissolves, and dispense it into 1 mL aliquots in 1.5 mL tubes.</li>
	</ol>
	</li>
</ol>
2. Zebrafish breeding and embryo collection
NOTE: Double transgenic zebrafish larvae reporting the presence of committed osteoblasts [osterix:nGFP = Tg(Ola.Sp7:NLS-GFP)zf132]29 and macrophages [mpeg:mCherry = Tg(mpeg:mCherry)gl23] were used here30 (Figure 1A).
<ol>
	<li>Set up parental zebrafish with the desired single transgenes in standard mating cages the evening before mating. 
	NOTE: Standard mating cages allow the eggs to pass to a lower compartment where the parental zebrafish cannot reach them.
	<ol>
		<li>Keep males and females separated until mating using a divider in the mating cage. Remove the divider in the morning of the next day to initiate mating.</li>
	</ol>
	</li>
	<li>At noon, at the latest, remove parental zebrafish from the breeding tanks and collect the embryos, straining the water from the mating cage through a sieve. 
	NOTE: In case the exact age of the larvae is decisive, harvest embryos in regular intervals of approximately 30 min. Clutch mates should be used in experiments with different experimental groups.</li>
	<li>Place the embryos in a 100 mm diameter dish containing 1x E3 media and keep them in an incubator at 28 &#176;C31.</li>
	<li>Remove unfertilized eggs and dead embryos from the dish using a plastic pipette with the help of a stereomicroscope and change 1x E3 media daily.</li>
	<li>Before the experiment, sort larvae positive for the reporter transgenes using a fluorescence stereomicroscope.</li>
</ol>
3. Larvae embedding in low-melting agarose
<ol>
	<li>Melt LMA aliquots in a thermoblock or water bath at 65 &#176;C. Once the LMA is melted, keep it at 42 &#176;C until embedding (Figure 1B). Add 50 &#181;L of 0.4% MS-222 to the melted 1 mL of LMA aliquot to achieve a 0.02% MS-222 concentration for anesthesia.</li>
	<li>Anaesthetize 4 dpf (days post fertilization) larvae (or older larvae) in 0.02% MS-222 E3 media.</li>
	<li>Transfer the&#160;anesthetized larvae to a glass-bottom microwell dish using a 3 mL plastic Pasteur pipette and remove the excess E3 media with the same pipette (Figure 1B). Avoid drying up of the larvae. 
	NOTE: More than one larva can be embedded in a dish simultaneously; it is recommended to embed 3 to 5 larvae to allow sufficient time for placement.</li>
	<li>Take the melted LMA agarose from the water bath/thermoblock and add a (cooling) drop of LMA to the dish, covering the glass bottom where the larvae were positioned. Pay attention that the LMA is not too hot but still liquid.</li>
	<li>Then, place the larvae into the desired lateral position before the agarose solidifies. Here, ensure a lateral position in which the opercle is as close as possible to the glass bottom of the dish as a preparation for the inverse microscope setup (Figure 1B).</li>
	<li>Let the agarose solidify and then add E3 media (containing a maximum of 0.02% MS-222) to the dish to avoid drying of the agarose (Figure 1B).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67279/67279fig01.jpg" /> 
Figure 1: Schematic representation of the embedding procedure. (A) Double transgenic Tg(osterix:nGFP; mpeg1:mCherry)29,30&#160;zebrafish larvae with green labeled opercular osteoblasts and macrophages labeled in red were used. (B) The workflow of the embedding procedure, as described in step 3 of the protocol. <a href="https://www.jove.com/files/ftp_upload/67279/67279fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. In vivo imaging of the larvae and laser ablation
<ol>
	<li>To laser-ablate osteoblasts in the developing opercle of transgenic zebrafish Tg(osterix:nGFP; mpeg1:mCherry)29,30, use an inverted two-photon laser-scanning microscope with a 25x/0.95 water objective or an equivalent setup.</li>
	<li>Place the dish with the embedded larvae onto the microscopic stage at the inverted microscope and position the desired region for ablation in the center of the field of view. The opercle can be easily identified with the help of the GFP fluorescence signal.</li>
	<li>Once the opercle is focused and centered in the field of view of the microscope, choose the z-stack settings (often 1-2 &#181;m z-intervals are used) to image the entire breadth of the opercle.&#160;At the same time, choose the area in a specific z-plane to be ablated (usually in the posterior zone of the opercle). The two-photon laser ablation is only exerted in this plane. 
	NOTE: Higher resolution in the z dimension will lead to longer imaging times but will enable volumetric analyses of the acquired data later.&#160;</li>
	<li>Image the opercle area of interest before ablation. For the GFP detection, use a white-light laser at 488 nm and a hybrid detector ranging from 495 nm to 550 nm or equivalent. Image the macrophages using 555 nm (white-light laser) excitation, with a mCherry detection range of 595 to 780 nm on a hybrid detector (or equivalent).
	<ol>
		<li>Use the image format 512 x 512 for a good quality/time ratio (other formats can be used if higher resolution or alternatively higher speed are desired). 
		NOTE: A temperature-controlled incubator installed at the microscope can be used and adjusted, e.g., to 28 &#176;C.</li>
	</ol>
	</li>
	<li>Draw a circular region with a diameter of 25 &#181;m (other shapes and sizes can be selected depending on the experiment) in a selected 2D plane, focusing the opercle using the ROI tool.</li>
	<li>To perform the ablation, apply ablation power (measured without objective) at&#160;450 mW and expose the selected area to the two-photon laser at a scan speed of 400 Hz and a total duration of 10 s. During ablation, the area can be visualized using a hybrid detector in the range of 401-443 nm or equivalent.</li>
	<li>After ablation, confirm the ablation structurally by imaging again the same z-stack with the same settings as used prior to ablation. The ablated area does not contain fluorescent cells anymore.</li>
</ol>
5. Recovery of larvae and imaging to monitor immune cell recruitment
<ol>
	<li>If the imaging of immune cell recruitment needs to be performed with a delay, recover the larvae from the imaging and anesthesia. Remove the larvae carefully from the LMA using forceps or a dissection needle.
	<ol>
		<li>First, remove a layer of agarose covering the larvae. Second, remove the agarose in direct contact with the larvae. Omit this step if immediate imaging of immune cell recruitment is taking place.</li>
	</ol>
	</li>
	<li>Place the larvae back in a dish with pure E3 media and check their recovery. Recovery is evident by swimming of the larvae.</li>
	<li>At 4 or 6 hpl (hours post-lesion), image the opercle area again using the same settings at the same microscope to check for the effects of the ablation on macrophage recruitment. To do so, repeat the embedding (step 3) and imaging as performed before (step 4).</li>
	<li>After imaging, remove the larvae from the LMA. Sacrificing of the larvae can either be performed by incubation in ice water or with an overdose of MS-222 in E3, depending on the legal requirements. Specimens can then be used for follow-up studies or be disposed of.</li>
</ol>
6. Image analysis and statistical analysis
<ol>
	<li>Process the images using an image processing software such as Fiji32. Depending on the z-resolution of the acquired data, analyses can be performed in 2D or 3D.</li>
	<li>Quantify the macrophages manually by either including cells that are clearly separate, such as by discerning the main cell bodies, or alternatively quantifying cells with a nuclear label or by measuring the area in case cells are overlapping (next step).</li>
	<li>For quantification of the area, create a mask encompassing the macrophages. To do that, use the Image &#62; Adjust &#62; Threshold tool and create an ROI (Analyze &#62; Tools &#62; ROI Manager &#62; Add) in Fiji. Exclude small particles in the background for quantification.
	<ol>
		<li>Then, measure the area (Analyze &#62; Measure). Consider whether areas of overlapping cellular fluorescence need to be included in the measurement. 
		NOTE: Here, the circular ablation area was omitted from the area measurements since recruited macrophages cannot be distinguished in this area.</li>
	</ol>
	</li>
	<li>Generate graphs plotting the measurement results using suitable software. To compare the data before and after ablation, perform an appropriate statistical test (here, a paired one-tailed t-test was performed). 
	NOTE: The figures can be prepared using photo editing and graphic design software.</li>
</ol>

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

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The authors have nothing to disclose.

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...

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 ablation7,8,9. Recently, laser ablation approaches have been widely used in zebrafish to study the healing and regeneration response after injury10,11,12,13,14.
Laser-mediated ablation has been used in different areas of biological research, such as mechanobiology, developmental biology, regeneration research, and tumor surgery10,11,12,15,16,17,18,19. There are various methodologies of laser ablation, such as using YAG (yttrium aluminum garnet), UV (ultraviolet), or 2p (two-photon) lasers20. Laser ablation allows the removal of single cells or larger portions of tissues in a very accurate manner, enabling the study of different processes, such as the response of the immune system to tumor ablation21 or during zebrafish opercle regeneration. In the latter example, ablation was confirmed by the disappearance of the nuclear and cytoplasmic fluorophore signal and necrosis staining10,22.
It is well known that the innate immune response and its regulated kinetics are essential for appropriate tissue regeneration. Neutrophils and macrophages are the first cells to be recruited to the injury site, where they perform different roles, such as cytokine and growth factor release, cellular debris removal, and extracellular matrix remodeling23. This recruitment and subsequent macrophage functionalization have also been observed in amputated zebrafish fins24 and the developing opercle, which was subjected to laser &#39;nanodissection&#39; leading to osteoblast ablation10. In the latter experiment, recovery of the osteoblast number, normal opercle development, and recruitment of innate immune cells (neutrophils, macrophages, and osteoclast-like cells) to the injured area was observed after UV laser and two-photon laser-mediated ablation10. Experiments in zebrafish in which the immune system was pharmacologically suppressed by using glucocorticoids showed an impairment of regeneration upon misregulated immunity, supporting the functional role of the immune system in tissue repair3,10,25,26.
Here, a two-photon laser-mediated ablation methodology to study the biology of bone regeneration in developing zebrafish bones is described. In particular, the effect of the osteoblast ablation approach in the opercle is shown in terms of the immune response, which is investigated by monitoring the recruitment of macrophages to the ablation site.

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			<td>Calcium chloride dihydrate, CaCl2&#183;H2O</td>
			<td>Carl Roth</td>
			<td>5239.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish, PS, 100/20 mm</td>
			<td>Greiner Bio-one</td>
			<td>664160</td>
			<td></td>
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			<td>Dumont #55 Foceps</td>
			<td>FST</td>
			<td>11295-51</td>
			<td>Tip shape straight, 11 cm, 0.05 x 0.02 mm</td>
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			<td>Falcon 6-well plate</td>
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			<td>353502</td>
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			<td>Glass-bottom microwell dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm Dish, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated</td>
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			<td>Insight X3 multiphoton laser</td>
			<td>Spectra-Physics</td>
			<td></td>
			<td></td>
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			<td>Leica Application Suite</td>
			<td>LAS X, Leica Microsystems</td>
			<td></td>
			<td></td>
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			<td>Low melting agarose&#160;</td>
			<td>Biozyme</td>
			<td>840101</td>
			<td>Biozym Plaque Agarose</td>
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			<td>Magnesium sulfate heptahydrate, MgSO4&#183;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M5921</td>
			<td></td>
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			<td>Mating cages</td>
			<td></td>
			<td></td>
			<td>many varieties, e.g. Tecniplast</td>
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		<tr>
			<td>Methyleneblue</td>
			<td>Carl Roth</td>
			<td>A514.1</td>
			<td></td>
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			<td>MS-222</td>
			<td>SIGMA Aldrich</td>
			<td>A5040</td>
			<td></td>
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			<td>Potassium Chloride, KCl</td>
			<td>PanReac AppliChem</td>
			<td>131494</td>
			<td></td>
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			<td>Sodium chloride, NaCl</td>
			<td>Carl Roth</td>
			<td>3957.1</td>
			<td></td>
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			<td>SP8 FALCON&#160;</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Equipped with a Insight X3 multiphoton laser and Leica Application Suite software</td>
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		<tr>
			<td>Stainless Steel Dissect Needle</td>
			<td>Bochem</td>
			<td>12010</td>
			<td>140 mm</td>
		</tr>
		<tr>
			<td>Stereo Microscope System SZX16</td>
			<td>Olympus</td>
			<td></td>
			<td>Equipped with a LED illumination base SZX2-ILLTQ</td>
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			<td>Thermostatic Cabinets TS - WTW</td>
			<td>xylem</td>
			<td>TS 608/2-i</td>
			<td>For incubation (embryo)</td>
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			<td>Transfer pipette, 3.5 mL</td>
			<td>SARSTEDT</td>
			<td>861171</td>
			<td>155 x 15 mm, LD-PE, transparent</td>
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			<td>Zeiss SteREO Discovery.V12 version 4.7.1.0.</td>
			<td>Zeiss</td>
			<td></td>
			<td>Equipped with Axiocam MRm camera and AxioVision sofware</td>
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two-photon laser-mediated ablation of osteoblasts in developing zebrafish larvae

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.

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...

Author Spotlight: Immune-Mediated Bone Regeneration &#8212; The Critical Role of Macrophages and Controlled Immunomodulation in Restorative Processes

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.

Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using ...

Our research focuses on bone regeneration. We try to understand the mechanisms that drive bone regeneration, in particular, how the immune system is involved in the restoration of bone. The immune response is essential for bone homeostasis and regeneration.The absence or excess of an inflammatory response after injury impairs the regeneration process. For that reason, immunomodulation must be tightly controlled to allow for successful regeneration. We have shown that macrophages, which are innate immune cells, are recruited to bone injuries, and that immunosuppression impair bone regeneration.

two-photon-laser-mediated-ablation-osteoblasts-developing-zebrafish

Research

JoVE Journal

Developmental Biology

326 Views.  CRTD - Center for Regenerative Therapies TU Dresden. Our research focuses on bone regeneration. We try to understand the mechanisms that drive bone regeneration, in particular, how the immune system is involved in the restoration of bone. The immune response is essential for bone homeostasis and regeneration.The absence or excess of an inflammatory response after injury impairs the regeneration process. For that reason, immunomodulation must be tightly controlled to allow for successful regeneration. We have shown that macrophages, which...

Video: Author Spotlight: Immune-Mediated Bone Regeneration — The Critical Role of Macrophages and Controlled Immunomodulation in Restorative Processes

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

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&#160;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.

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 ...

Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns.

Embryonic development requires large-scale coordination of cell motion. Two-photon excitation mediated laser ablation allows the spatially controlled 3-dimensional ablation of large groups of deep cells. In addition, this technique can probe the reaction of collectively migrating cells in vivo to perturbations in their mechanical environment.

Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.