The authors would like to thank David Langenau for generously providing aquarium space; Eric Stone, John C. Moore and Qin Tang for help with zebrafish maintenance and reagents, and Anne Robertson and Elliott Hagedorn from Leonard Zon&#39;s lab for procuring the zebrafish strain used here. They would also like to thank Octavio Hurtado for advice on photolithographic techniques. FE was funded by Fellowships from Shriner&#39;s Hospital for Children and the American Australian Association. This work was funded by NIH grant GM92804.

Microinjection of larvae was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care under Protocol 2011N000127.
1. Device Fabrication
NOTE: All computer assisted drawing (CAD) files used to design photolithography masks described here (Figure 1) are available for download. See Table of Materials for links.
<ol>
	<li>Fabricate the master mold wafer in a Class 1,000 clean room using standard photolithographi...

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Dang, M., Fogley, R., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+Novel+Cancer+Therapies+Using+Chemical+Genetics+and+Zebrafish.">Identifying Novel Cancer Therapies Using Chemical Genetics and Zebrafish.</a> Adv Exp Med Biol. 916, 103-124 (2016).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Benard, E. 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(119), (2017).</li><li>Shao, G., Wu, J., Cai, Z., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+elastomeric+high-aspect-ratio+microstructures+using+polydimethylsiloxane+(PDMS)+double+casting+technique.">Fabrication of elastomeric high-aspect-ratio microstructures using polydimethylsiloxane (PDMS) double casting technique.</a> Sens Actuators A Phys. 178, 230-236 (2012).</li><li>Bhuiyan, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinetobacter+baumannii+phenylacetic+acid+metabolism+influences+infection+outcome+through+a+direct+effect+on+neutrophil+chemotaxis.">Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.</a> Proc Natl Acad Sci U S A. 113 (34), 9599-9604 (2016).</li><li>Henry, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhagoSight:+an+open-source+MATLAB&#174;+package+for+the+analysis+of+fluorescent+neutrophil+and+macrophage+migration+in+a+zebrafish+model.">PhagoSight: an open-source MATLAB&#174; package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.</a> PloS one. 8 (8), e72636 (2013).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dynam. 203 (3), 253-310 (1995).</li><li>Hemmil&#228;, S., Cauich-Rodr&#237;guez, J. V., Kreutzer, J., Kallio, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+simple,+and+cost-effective+treatments+to+achieve+long-term+hydrophilic+PDMS+surfaces.">Rapid, simple, and cost-effective treatments to achieve long-term hydrophilic PDMS surfaces.</a> Applied Surface Science. 258 (24), 9864-9875 (2012).</li><li>Masselink, W., Wong, J. C., Liu, B., Fu, J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+silicone+imaging+casts+for+zebrafish+embryos+and+larvae.">Low-cost silicone imaging casts for zebrafish embryos and larvae.</a> Zebrafish. 11 (1), 26-31 (2014).</li><li>Yang, F., Gao, C., Wang, P., Zhang, G. J., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish-on-a-chip:+microfluidics+for+zebrafish+research.">Fish-on-a-chip: microfluidics for zebrafish research.</a> Lab Chip. 16 (7), 1106-1125 (2016).</li><li>Wielhouwer, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryo+development+in+a+microfluidic+flow-through+system.">Zebrafish embryo development in a microfluidic flow-through system.</a> Lab Chip. 11 (10), 1815-1824 (2011).</li><li>Shen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+student+team+in+a+University+of+Michigan+biomedical+engineering+design+course+constructs+a+microfluidic+bioreactor+for+studies+of+zebrafish+development.">A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development.</a> Zebrafish. 6 (2), 201-213 (2009).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+on+a+chip:+a+novel+platform+for+real-time+monitoring+of+drug-induced+developmental+toxicity.">Zebrafish on a chip: a novel platform for real-time monitoring of drug-induced developmental toxicity.</a> PLoS One. 9 (4), e94792 (2014).</li><li>Akagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+embryo+array+for+automated+trapping,+immobilization+and+microperfusion+of+zebrafish+embryos.">Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.</a> PLoS One. 7 (5), e36630 (2012).</li><li>Akagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+on+chips:+Microfluidic+living+embryo+array+for+accelerated+in+vivo+angiogenesis+assays.">Fish on chips: Microfluidic living embryo array for accelerated in vivo angiogenesis assays.</a> Sens Actuators B Chem. 189, 11-20 (2013).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+mapping+of+brain-wide+activity+in+awake+and+drug-responsive+vertebrates.">High-throughput mapping of brain-wide activity in awake and drug-responsive vertebrates.</a> Lab Chip. 15 (3), 680-689 (2015).</li><li>Noori, A., Selvaganapathy, P. R., Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+in+a+microfluidic+format+using+flexible+and+compliant+channels+and+electroosmotic+dosage+control.">Microinjection in a microfluidic format using flexible and compliant channels and electroosmotic dosage control.</a> Lab Chip. 9 (22), 3202-3211 (2009).</li></ol>

Here, we describe the use of devices we recently developed to facilitate 2 dpf zebrafish microinjection5, and introduce a simple agarose-free mounting device for convenient imaging of embryos. These tools highlight the utility of photolithographic techniques for fabrication of devices useful for zebrafish techniques.
We have found MSA devices particularly useful for injection of cells or particles prone to aggregation within the microinjection needle, such as fungal con...

Zebrafish have emerged as a powerful model for many fields, from studies of fundamental developmental biology to large-scale genetic and chemical screens1,2. Routine genetic manipulations, such as gene overexpression, knockdown, CRISPR/Cas9 mutagenesis, and transgenesis rely on microinjection of genetic material into the single-cell zygote, which has led to the development of simple, easy-to-use, commercially available tools for orienting and stabilizing eggs for injection3. Other approaches, such as transplantation and infection, often require microinjection into later stage embryos and larvae using larger gauge capillary needles4. However, use of larger gauge needles presents significant technical challenges, as it is more difficult to penetrate the target tissue without pushing or rolling the embryo. Under these conditions, obtaining the appropriate water tension required to stabilize the embryo while avoiding drying during the procedure is difficult, and embryos may not be ideally oriented for injection into the target tissue.
Following microinjection, it is often useful to screen injected embryos to select those that have been successfully injected, and to capture images of the initial time point. To address these challenges, we have developed a range of microstructured devices that help to stabilize 2 dpf embryos in various orientations both for microinjection5, and for rapid image-based screening post-injection.
To obtain sufficient structural resolution in these devices, we utilized photolithographic techniques. Commonly used in microelectronic industries and more recently extrapolated to microfluidic fabrication, these approaches can achieve vertical structures ranging from 1-1,000 &#181;m, a scale well suited to manipulation of zebrafish embryos and larvae. All devices were fabricated using polydimethylsiloxane (PDMS), which is cheap, physically robust, biologically inert, and transparent.
Microstructured surface arrays (MSAs) were formatted as blocks of PDMS with a patterned top surface, analogous to the simple channels in agarose blocks commonly used for egg microinjection. For post-injection screening, 6 imaging devices can be arrayed in a standard glass-bottomed 6-well plate. These devices are designed for easy loading of embryos, while the unloading procedure conveniently allows rescue of specific embryos, facilitating image-based screening approaches in a more user-friendly manner than those devices previously developed by the Beebe laboratory6.

<table>
	<tbody>
		<tr>
			<td>Dow Corning Sylgard 184&#160; Polydimethylsiloxane (PDMS)&#160;</td>
			<td>Ellsworth Adhsives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>For casting the devices. Kit includes PDMS monomer and Initiator</td>
		</tr>
		<tr>
			<td>Low gelling temperature agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9414-10G</td>
			<td>For casting agarose devices</td>
		</tr>
		<tr>
			<td>PFDTS silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td>For casting of negative PDMS molds</td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma Aldrich</td>
			<td>E10521-10G</td>
			<td>To anesthetize&#160; zebrafish&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine Dextran 70,000 Da</td>
			<td>ThermoFisher</td>
			<td>D1818</td>
			<td>To trace microinjections</td>
		</tr>
		<tr>
			<td>Leukotriene B4 (LTB4)</td>
			<td>Cayman Chemicals</td>
			<td>20110</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td>
			<td>Sigma Aldrich</td>
			<td>F3506-50MG</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>15 cm Petri dish</td>
			<td>Fisher scientific</td>
			<td>08-757-148</td>
			<td>For Casting from the master wafer</td>
		</tr>
		<tr>
			<td>Glass-bottom 6-well plates</td>
			<td>MatTek</td>
			<td>P06G-0-20-F</td>
			<td>For imaging devices</td>
		</tr>
		<tr>
			<td>Borosilicate glass microcapillaries</td>
			<td>World Scientific Instruments</td>
			<td>TW-100-4</td>
			<td>For microinjection needles</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sigma Aldrich</td>
			<td>Z350796</td>
			<td>For transferring zebrafish embryos</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Fisher scientific</td>
			<td>E5242956003</td>
			<td>For loading the microinjection needles</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 1.5 mm punch</td>
			<td>Ted Pella Inc.</td>
			<td>15111-15</td>
			<td>To punch ports in PDMS imaging devices</td>
		</tr>
		<tr>
			<td>No.&#160;11 Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For cutting PDMS&#160;</td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-10</td>
			<td>For dechorionating embryos and breaking microinjection needle tips</td>
		</tr>
		<tr>
			<td>Marzhauser Micromanipulator</td>
			<td>ASI&#160;</td>
			<td>MM33-R</td>
			<td>For manipulating microinjection needle</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>MSC</td>
			<td>SPI - 87242624</td>
			<td>For mounting micromanipulator</td>
		</tr>
		<tr>
			<td>MPPI-3 Picopump controller</td>
			<td>ASI</td>
			<td>MPPI-3</td>
			<td>To control microinjection volume and timing</td>
		</tr>
		<tr>
			<td>EVOS inverted fluorescent microscope</td>
			<td>ThermoFisher</td>
			<td>EVOS FL</td>
			<td>To image injected embryos</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>For visualizing microinjecion</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td></td>
			<td>Download AutoCAD files from: https://dx.doi.org/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &#160;</td>
		</tr>
	</tbody>
</table>

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.

The approach described here demonstrates the design (Figure 1) and fabrication of devices for use with 2 dpf zebrafish, using photolithographic (Figure 2) and soft-lithographic (Figure 3) techniques. This method allows rapid testing of many design iterations and modifications, and alterations and optimization of microstructure dimensions for use with zebrafish at other stages of development may exten...

Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

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 ...

The overall goal of this methodology is to make microinjection of zerbrafish larvae easier and more accurate. This method helps researchers using zebrafish model systems to immobilize zebrafish larvae in specific orientations in large arrays. The technique allows for easier access to specific tissues for microinjection and imaging, and is very effective when large numbers of larvae are required for applications like infection or xenograft cancer models.My fabrication strategies in zebrafish models are highly complementary. One is man-made and simple. The other one is live and complex.Both share the same size scale. They are both transparent. And both enable observations at single-cell resolution.As you show here, they can be easily combined, and they could have applications to the study of virtually any major disease. After preparing a PDMS block in a Petri dish covered in a thin film of E-3, according to the text protocol, use a transfer pipet to transfer the required number of embryos onto the surface of the PDMS block. Using a hair loop or similar tool, push the embryos into the microstructure divots, particularly for dorsal and ventral orientations.When loading larvae into the divots, it's important to have enough water covering the PDMS block to allow easy manipulation. It's also important to push them into the divots so that they remain in place during the injection. To orient zebrafish on microstructure channels previously prepared according to the text protocol, use a transfer pipet to draw E-3 off the patterned PDMS block.The level of E-3 should drop below the edge of the block, but still be present in the reservoir and channels, and a thin layer remains on the PDMS. Using a transfer pipet, transfer 10 anesthetized embryos into the reservoir, being careful not to overflow the edges. Using a hair loop, manipulate each embryo into the funnel head, first at the entrance of the appropriate channel, ensuring that the embryo has the appropriate orientation as it enters the channel.Then, use a hair loop to slide each embryo down the channel, until it reaches the orienting microfeatures in the channel walls that keep it in place. Following the preparation of microinjection needles and microinjection solution, use a finely tapered pipet tip to load five microliters of the solution into the needle. Mount the needle into a micromanipulator mounted on a magnetic stand, and connect it to a PICO pump microinjector.Then, use forceps to break the tip of a needle at a 45 degree angle by pinching the tapered tip. Adjust the injection bolus size to one nanoliter by adjusting the injection time on the PICO pump controller. Adjust the angle of the microinjection needle on the micromanipulation controller, beginning with 45 degrees with the needle parallel to the length of the embryo.A steeper angle may be used to minimize lateral movement of the embryo during the microinjection. Controlling the Petri dish with the left hand, and the need micromanipulator with the right, bring the needle as close as possible to the intended site of microinjection, such as the otic vesicle. Using the micromanipulation controller, insert the needle into the target tissue, and use the PICO pump foot switch to inject the preset volume.If the tissue resists penetration, gently tap the micromanipulation controller knob. To release the embryos, use a transfer pipet to flow E-3 over the surface from top to bottom, or by swirling the dish such that the embryos are released into the surrounding E-3. Transfer the embryos to a recovery dish of E-3 without MS-222.To screen the embryos using the PDMS device in the wells of a six well plate, use a 1, 000 microliter pipet or a narrow-tipped transfer pipet to prime the device by flowing E-3 through the port. Use E-3 and MS-222 to fill the well until the device is covered. Then, use a transfer pipet to deliver four previously injected embryos into each well.Using a hair loop or similar tool, position the embryos at the entrances of the imaging channels in the desired orientation, and partially push them into the device. This will help draw them into the device evenly. Using a 1, 000 microliter pipet or transfer pipet, draw E-3 through the device from the port until the embryos are drawn into the channels in the correct orientation for imaging.Transfer the well plate to an inverted fluorescent microscope. To image the embryos, adjust the magnification by selecting the appropriate lens, such as 10x for imaging zebrafish neturaohil migration. Adjust the light source intensity and exposure time for each channel, so that each signal is clear, but not saturated.Finally, capture images for each fluorescent and transmitted channel. Using the microstructured channel device to stabilize embryos in the lateral orientation, the ability of neutrophils to respond to the standard chemoattractants fMLP and LTB4 microinjected into the otic vesicles, of two days post-fertilization zebrafish embryos was tested. To visualize neutrophil recruitment, injections were traced with high molecular weight rhodamine dextran, and performed in transgenic embryos with green fluorescent neutrophils.Uninjected embryos and embryos injected with rhodamine alone were used as negative controls. Following microinjection, embryos were transferred into the imaging channel device and imaged using an EVOS fluorescent microscope at 30 minutes and five hours post injection. As expected, a small number of fluorescent neutrophils were recruited to the mock injected otic vesicle at the early time point, likely due to damaged mediated recruitment.Interestingly, the rhodamine dextran tracer was observed to accumulate in the otoliths during the experiment. For both chemoattractants, neutrophils were recruited in higher numbers, particularly in response to LTB4. Once mastered, this technique can greatly improve the speed and accuracy of zebrafish microinjection.Development of this technique was driven by specific challenges in our lab. We refined this approach to fit the needs of our projects on fungal infection and tumor xenografts in zebrafish models. After watching this video, you should have a good understanding of how to use microstructured surface arrays for zebrafish microinjection and for extended live imaging.

microstructured-devices-for-optimized-microinjection-imaging

Research

JoVE Journal

Developmental Biology

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 ...

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 ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

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. ...

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 ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...