This work was supported by the &#34;1000plan Youth&#34; program, the Startup Funds from Tongji University, and NSFC Grant# 21607115 and 21777116 (Lin).

1. Design and Fabrication of a Zebrafish Embryos Arraying Template
<ol>
	<li>Design the arraying template with a 12 by 8, 96-well layout that fits a standard 96-well plate. Use the dimensions listed in Figure 1A for the upper embryo entrapment chamber (see also the Supplemental File).
	<ol>
		<li>Use the dimensions shown in Figure 1B and 1D for the entrapment well.</li>
		<li>Use the dimensions in Figure 1C for the bottom vacuum chamber.</li>
		<li>Use the dimensions in Figure 1B for the air in/outlet.</li>
	</ol>
	</li>
	<li>Use a 3D-printer (with 0.1 mm precision) to print the template; see Table of Materials for recommended resin to be used for printing. 
	NOTE: 3D printers with a 0.1 mm precision are recommended for fabrication of the arraying template (see Table of Materials). The suggested color for the surface of the template is dark grey or black.</li>
</ol>
2. Zebrafish Embryo Spawning
<ol>
	<li>Place two pairs of male and female fish per mating box one day prior to spawning. Separate males and females by a clear plastic divider.</li>
	<li>Take off the dividers in the morning to mix male and female fish.</li>
	<li>Remove the male and female fish and collect zebrafish embryos using a fine-mesh strainer. Wash the embryos with 250 mL egg water (see Table of Materials).</li>
	<li>Transfer the collected embryos to petri dishes (90 mm in diameter) with Holtfreter&#39;s solution (see Table of Materials) and remove dead and unfertilized embryos using a stereomicroscope.</li>
	<li>Place the embryos in a 28.5 &#176;C incubator. At 4 h post fertilization (hpf), observe the embryos and remove any dead and unhealthy embryos. The embryos are now ready for the next step.</li>
</ol>
3. Preparation of Arraying Template
<ol>
	<li>Wash the template 2&#8211;3 times with 500 mL deionized water and put it into the drying oven (45 &#176;C) for 5 min.</li>
	<li>Tape the bottom chamber with a piece of sealing film (Figure 2, Step 1).</li>
	<li>Connect a vacuum pump through the air outlet at the bottom of the template. 
	NOTE: The recommended max vacuum for the vacuum pump is 0.1 Mpa. Be aware of the strength of the vacuum used. If the negative pressure is too strong, cut a cross-shaped hole on the sealing film to lower the pressure.</li>
</ol>
4. Arraying Zebrafish Embryos into a 96-well plate
<ol>
	<li>Using a plastic transfer pipette, place approximately 150 embryos into the template, as demonstrated in Figure 2, Step 2.</li>
	<li>Connect the vacuum pump to the air outlet to generate negative pressure in the chamber sealed by the sealing film in step 3.3.</li>
	<li>Shake the entire template horizontally until each well has one embryo entrapped (Figure 2, Step 3). 
	NOTE: If the Holtfreter&#39;s Solution dries up before the embryos are trapped in each well, add additional Holtfreter&#39;s Solution in the entrapment chamber and repeat this step.</li>
	<li>Discard extra Holtfreter&#39;s Solution and embryos that are not entrapped in the wells (Figure 2, Step 4).</li>
	<li>Turn off and disconnect the vacuum pump.</li>
	<li>Place a standard 96-well plate upside down against the template (Figure 2, Step 5) and rotate both at the same time (Figure 2, Step 6).</li>
	<li>Tap the bottom of the template or connect the air outlet to a compressed gas dusting can to transfer all trapped embryos from the template to the 96-well plate (Figure 2, Step 6).</li>
	<li>Repeat step 4.1 to 4.8 to prepare additional multi-well plates.</li>
	<li>Remove the sealing film and wash the template 3 times from top to bottom with 500 mL deionized water for future use. 
	NOTE: Do not use any organic solvents, like ethanol, to clean the template.</li>
</ol>

<ol>
	<li>Howe, K., 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Leslie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+larvae+could+help+to+personalize+cancer+treatments.">Zebrafish larvae could help to personalize cancer treatments.</a> Science. 357 (6353), 745-745 (2017).</li><li>Lin, 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=Understanding+the+Transformation,+Speciation,+and+Hazard+Potential+of+Copper+Particles+in+a+Model+Septic+Tank+System+Using+Zebrafish+to+Monitor+the+Effluent.">Understanding the Transformation, Speciation, and Hazard Potential of Copper Particles in a Model Septic Tank System Using Zebrafish to Monitor the Effluent.</a> ACS Nano. 9 (2), 2038-2048 (2015).</li><li>Lin, 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=Aspect+ratio+plays+a+role+in+the+hazard+potential+of+ceo2+nanoparticles+in+mouse+lung+and+zebrafish+gastrointestinal+tract.">Aspect ratio plays a role in the hazard potential of ceo2 nanoparticles in mouse lung and zebrafish gastrointestinal tract.</a> ACS Nano. 8 (5), 4450-4464 (2014).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nature Communications. 4, (2013).</li><li>Lin, 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=High+content+screening+in+zebrafish+speeds+up+hazard+ranking+of+transition+metal+oxide+nanoparticles.">High content screening in zebrafish speeds up hazard ranking of transition metal oxide nanoparticles.</a> ACS Nano. 5 (9), 7284-7295 (2011).</li><li>Kuipers, J., Kalicharan, R. D., Wolters, A. H. G., van Ham, T. J., Giepmans, B. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+Scanning+Transmission+electron+microscopy+(nanotomy)+of+healthy+and+injured+zebrafish+brain.">Large-scale Scanning Transmission electron microscopy (nanotomy) of healthy and injured zebrafish brain.</a> Journal of Visualized Experiments. (111), (2016).</li><li>Liu, R., 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=Automated+Phenotype+Recognition+for+Zebrafish+Embryo+Based+In+vivo+High+Throughput+Toxicity+Screening+of+Engineered+Nano-Materials.">Automated Phenotype Recognition for Zebrafish Embryo Based In vivo High Throughput Toxicity Screening of Engineered Nano-Materials.</a> PLoS One. 7 (4), (2012).</li><li>Tu, 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=Automatic+Categorization+and+Scoring+of+Solid,+Part-Solid+and+Non-Solid+Pulmonary+Nodules.">Automatic Categorization and Scoring of Solid, Part-Solid and Non-Solid Pulmonary Nodules.</a> in CT Images with Convolutional Neural Network. Scientific Reports. 7, 8533 (2017).</li><li>Mandrell, D., 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=Automated+zebrafish+chorion+removal+and+single+embryo+placement:+optimizing+throughput+of+zebrafish+developmental+toxicity+screens.">Automated zebrafish chorion removal and single embryo placement: optimizing throughput of zebrafish developmental toxicity screens.</a> Journal of Laboratory Automation. 17 (1), 66-74 (2012).</li><li>Lin, S., Zhao, Y., Nel, A. E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+An+in+vivo+model+for+nano+EHS+studies.">Zebrafish: An in vivo model for nano EHS studies.</a> Small. 9 (9-10), 1608-1618 (2013).</li></ol>

The authors have filled a patent on the described 3D-printed template.

There are two critical steps in this protocol that require close attention for a successful implementation of 3D-printed template for arraying zebrafish embryos.
The most important factor on the design of the arraying template is the entrapment well. To makes sure there is only one embryo trapped in each well, one should pay close attention to the diameter and the depth of the entrapment well, and the diameter of the through hole. The recommended diameter is within 1.5 to 2 times of the diamet...

As a popular model organism, zebrafish is widely used in the fields of medicine and toxicology1,2,3,4. Compared to in vitro platforms, zebrafish offer much greater biological complexities that one or two cell types could not offer. Besides being a whole organism model, the zebrafish&#39;s large fecundity, rapid and simultaneous embryonic development, and high organ translucency have given this model unique advantages to be used for large scale toxicity or drugs/compound screening5. The hundreds of embryos produced by one pair of adult zebrafish each week surpass any other whole animal models and have made it suitable for high throughput screening.
A typical screening procedure using zebrafish involves a significant amount of manual work, such as adult zebrafish spawning, embryo selection, and arraying embryos into suitable containers where they are subjected to exposure through water immersion. The development of the embryos is monitored and observable endpoints such as mortality, hatchability and abnormality are often evaluated manually and used as the preliminary identifications of the toxicity of chemicals or indications of the effectiveness of drugs or compounds. To speed up the screening procedure, approaches such as automated imaging and computer-assisted image analysis have been explored previously. For example, microscopes with high content imaging capabilities have been adapted to perform automated bright-field or fluorescence imaging on zebrafish embryos at various developmental stages from 96/384 well plates6. Microfluidic devices coupled with microscopes were used to position zebrafish larvae through current manipulation for imaging of brain neurons7. These approaches could significantly improve the efficiency of image acquisitions compared to traditional manual operation. Moreover, with large number of images being generated, image analysis tools have also been developed to speed up the data processing, as demonstrated by Liu et al. and Tu et al.8,9.
As the throughput level of imaging and image analysis increases, it became clear that the rate-limiting step for screening lies in the process of preparing zebrafish embryos for exposure, which typically means arraying them into 96- or 384-well plates. To solve this bottleneck step, vision-guided robotics were developed by Mandrell et al.10 and us11 previously to replace manual handling but the instruments were rather sophisticated and there is a deep learning curve to implement such techniques. Therefore, to provide an easy-to-use approach becomes one important factor to further improve the throughput level of zebrafish screening and is the main objective of this work.
In this work, we designed and fabricated an embryo arraying template by 3D printing. Such an arraying template was designed to entrap zebrafish embryo into wells that fit with a standard 96-well plate. Instead of selecting embryos and arraying them into individual well one by one, one could perform embryo entrapment and array all 96 embryos into a multiwall plate at once. Using this template and the following protocol, one could significantly increase the efficiency of arraying embryos into multiwall plates, which would in term boost the screening capacity at least tenfold, compared to manual operation. The protocol described below includes an overall design for the arraying template, zebrafish spawning, embryo collection, and arraying. Figure 1 shows the overall design of the arraying template. Figure 2 shows an overview of the step-by-step protocol on using the template described in Parts 3 and 4.

<table><tbody><tr><td>Zebrafish Facility</td><td>Shanghai Haisheng Biotech Co., Ltd.</td><td>Z-A-S5</td><td></td></tr><tr><td>Mating box</td><td>Shanghai Haisheng Biotech Co., Ltd.</td><td></td><td></td></tr><tr><td>Wash Bottle, 500 ml</td><td>Sangon Biotech</td><td>F505001-0001</td><td></td></tr><tr><td>Sodium chloride</td><td>Vetec</td><td>V900058-500G</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10016318</td><td></td></tr><tr><td>Calcium chloride</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>20011160</td><td></td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Vetec</td><td>v900182-500G</td><td></td></tr><tr><td>Methylene Blue Hydrate</td><td>TCI</td><td>M0501</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10011008</td><td></td></tr><tr><td>Sea Salts</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Pipetter</td><td>Fisherbrand</td><td>13-675M</td><td></td></tr><tr><td>Controlled Drop Pasteur Pipet</td><td>Fisherbrand</td><td>13-678-30</td><td></td></tr><tr><td>Microscope</td><td>OLYMPUS</td><td>SZ61</td><td></td></tr><tr><td>Biochemical incubator</td><td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td><td>LRH-250</td><td></td></tr><tr><td>3D printer</td><td>UnionTech</td><td>Lite600</td><td></td></tr><tr><td>Photosensitive resin</td><td>UnionTech</td><td>UTR9000</td><td></td></tr><tr><td>Vacuum pump</td><td>Shanghai Yukang Scientific Instrument Co., Ltd.</td><td>SHB-IIIA</td><td></td></tr><tr><td>Adhesive PCR Plate Seals</td><td>Solarbio</td><td>YA0245</td><td></td></tr><tr><td>96 well plate</td><td>Costar</td><td>3599</td><td></td></tr><tr><td>Multi 8-channel pipette 30 - 300 &amp;mu;l</td><td>Eppendorf</td><td>3122000.051</td><td></td></tr><tr><td>Compressed Gas Duster</td><td>Shanghai Zhantu Chemical Co., Ltd.</td><td>ST1005</td><td></td></tr><tr><td>DI Water</td><td>Thermo</td><td>GenPure Pro UV/UF</td><td></td></tr><tr><td>Drying oven</td><td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td><td>BPG-9106A</td><td></td></tr><tr><td>System water</td><td></td><td></td><td>Water out of the facility&amp;rsquo;s water system</td></tr><tr><td>Egg water</td><td></td><td></td><td>Dilute 60mg &amp;ldquo;Instant Ocean&amp;rdquo; sea salts and 0.25 mg/L methylene blue in 1 L DI water</td></tr><tr><td>Holtfreter&amp;rsquo;s solution</td><td></td><td></td><td>Dissolve 7.0 g Sodium chloride (NaCl), 0.4 g Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;), 0.1 g Potassium Chloride (KCl), 0.235 g Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O) in 1.9 L DI water. Adjust pH to 7 using HCl and adjust volume to 2 L using Di water</td></tr></tbody></table>

a 3-dimensional (3d)-printed template for high throughput zebrafish embryo arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

Figure 3 shows a typical 3D-printed arraying template. This template uses photosensitive resin as raw material and was made by a 3D printer; a layer of black paint was applied to provide a better contrast to the color of embryos. The position of 96 wells (12 by 8) was designed to fit with a standard 96-well plate. Similarly, a 384 (24 by 16) well template could also be designed and fabricated using the same method. The upside chamber was slightly bigger than ...

Watch this Scientific Journal Video about A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying at JoVE.com

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

a-3-dimensional-3d-printed-template-for-high-throughput-zebrafish

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6.7K Views.  Tongji University. Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

Video: A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

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 ...

Developmental Biology

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.&#160;

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Biology

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Neuroscience

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

Summary

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Chapters in this video

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Development of automated imaging and analysis for zebrafish chemical screens.

A Simple and Effective Transplantation Device for Zebrafish Embryos

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos