This work was supported by the Deutsche Forschungsgemeinschaft (DFG), we wish to thank Ayele Tsedeke Taddese for technical support.

1. Preparation of the Glass Needles (Figure 1)
<ol>
	<li>Use glass capillaries with a 0.75 mm diameter to prepare injection needles (Figure 1A).</li>
	<li>Place the glass capillary into a needle puller and pull the needle with the following parameter: heating cycle value: 463; pulling cycle value: 230; velocity: 150 msec; time: 150 msec (Figure 1B).</li>
	<li>Break the pulled glass capillary with watchmaker tweezers to produce a 20 &#181;m diameter needle under a stereoscope with a micrometer eyepiece.</li>
	<li>Use a lathe with a wetted rubber spinning wheel to sharpen the needle and produce a 20 &#181;m bevel (Figures 1C and 1D). Note: Sharp, beveled needles improve the ease of insertion into the ventricle and thus minimize the damage to the tissue and allow the perforated muscle to reseal after removal of the needle (Figures 1E-G).</li>
</ol>
2. Preparation of the Morpholino Solution
<ol>
	<li>Prepare the morpholino stock by dissolving the lyophilized morpholino in 1x Phosphate-Buffered Saline (PBS) to a final concentration of 7.5 mM. [See manufacturer&#39;s instructions for details (Materials table)].</li>
	<li>Prepare the morpholino injection solution by mixing 2.5 &#181;l morpholino stock solution (7.5 mM) with 2.8 &#181;l Endo-Porter stock solution (1 mM) (See&#160;Materials table) for a final concentration of 3.5 mM morpholino and 0.5 mM Endo-Porter.</li>
</ol>
3. Preparation of the Morpholino Injection (Figure 2)
<ol>
	<li>Load the beveled glass needle with 5 &#181;l of this solution using a micropipette with a 10 &#181;l microloader pipette tip.</li>
	<li>Insert the glass needle into the needle holder of the micromanipulator connected to the pneumatic pico pump (Figures 2A-C).</li>
	<li>Place the needle holder next to the microscope so that the needle only needs to be moved in one planar direction to insert it into the cardiac ventricle of the larva (Figure 2A).</li>
	<li>Adjust the angle for the injections at around 45&#176;.</li>
	<li>Set the microinjector values as follows: hold pressure: 20 pounds per square inch (psi); ejection pressure: 15 psi; 100 msec range of gating, period value of 1.9 (corresponds to 10.9 msec).</li>
	<li>Melt agarose in 1x PBS using a standard microwave to produce 20 ml of a 1.5% (w/v) gel. Pour melted gel into a 10 cm Petri dish. Place a grooved injection form into the warm agarose so that once hardened, the agarose will have furrows in which the sedated larva will be placed11 (Figures 2D and 2E).</li>
</ol>
4. Injections (Figure 3)
<ol>
	<li>Anesthetize larva in 100 ml of aquarium water with 20 mg/L tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate) until they stop responding to touch.</li>
	<li>Use a plastic Pasteur pipette to carefully transfer the sedated larva into a groove of the wet agarose mold so that the ventral side is facing against the vertical agarose wall of the groove (Figure 3A).</li>
	<li>Place the agarose plate under the stereomicroscope so that the ventricle is facing away from the injection needle (Figure 3A).</li>
	<li>Lower the needle to insert it into the heart ventricle. Insert the needle only 1-2 &#181;m into the ventricle taking care not to insert the needle too deeply (Figures 3B and 3C).</li>
	<li>Once the needle is inserted, inject the morpholino solution into the ventricle with 4-6 pulses, each delivering 3 nl of the solution, with waiting intervals to allow clearing of the heart (Figures 3D and 3E).</li>
	<li>After injection, remove the needle (parallel to the plane of insertion) and then carefully transfer the larva using a plastic Pasteur pipette filled with E3 medium back into a Petri dish containing fresh E3 medium.</li>
	<li>Place the needle into a Petri dish containing 1x PBS to prevent the drying of the needle when transferring the injected larva.</li>
	<li>Repeat steps 4.1-4.6 every 12-24 hr&#160;for the duration of the experiment. Note: Repeating the injections ensures the uptake and maintenance of the morpholino in cells.</li>
</ol>
5. Analyses of Injections (Figure 4)
<ol>
	<li>Anesthetize the fish in 100 ml of aquarium water with 20 mg/L tricaine until they stop responding to touch.</li>
	<li>Place the larva laterally on a flat wet 1.5% (w/v) agarose plate covered with E3 medium.</li>
	<li>Image larvae using a stereomicroscope with brightfield and fluorescence imaging. Note: As the larvae are transparent, the fluorescein-tagged morpholino should be visible as fluorescent green in the blood vessels throughout the animal directly after injection. This fluorescence will further disperse into the vascularized tissues by 15 min&#160;(Figures 4A-4C).</li>
</ol>
6. Assessment of Regenerative Outgrowth
<ol>
	<li>Assess captured images using Fiji Image J free software (http://fiji.sc/Fiji). For investigating regeneration, use the line-tracing tool to determine the amount of regenerative growth that has occurred on control and morpholino-injected larva.</li>
	<li>To calibrate images, open one of the original image files in Fiji by dragging and dropping the file into the main Fiji window.</li>
	<li>To set the scale for all following images, go to &#34;Analyze&#34; in the main menu.</li>
	<li>In the drop down menu click on &#34;Set scale&#34;.</li>
	<li>In this window, turn on the &#34;Global&#34; option by clicking the check box.</li>
	<li>Now open the image to measure the amount of regenerative growth again by drag and drop (see step 6.1).</li>
	<li>Choose the &#34;freehand selections&#34; tool in the tool bar.</li>
	<li>Encircle the area to be measured (Figures&#160;4J-4L).</li>
	<li>Press Ctrl+M. This will open a new &#34;Results&#34; window showing the value of the encircled area in square pixels.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+in+zebrafish+reverse+genetics:+moving+forward.">Current perspectives in zebrafish reverse genetics: moving forward.</a> Developmental Dynamics. 237, 861-882 (2008).</li><li>Zu, 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=TALEN-mediated+precise+genome+modification+by+homologous+recombination+in+zebrafish.">TALEN-mediated precise genome modification by homologous recombination in zebrafish.</a> Nature Protocols. 10, 329-332 (2013).</li><li>Bedell, V. 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R., 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=.">.</a> Zebrafish: a practical approach. 261, 121-143 (2000).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Eisen, J. S., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+morpholino+experiments:+don't+stop+making+antisense.">Controlling morpholino experiments: don't stop making antisense.</a> Development. 135, 1735-1743 (2008).</li><li>Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+and+techniques+for+ectopic+gene+expression+in+zebrafish.">Vectors and techniques for ectopic gene expression in zebrafish.</a> Methods Cell Biol. 59, 117-126 (1999).</li><li>Hans, S., Kaslin, J., Freudenreich, D., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+controlled+site-specific+recombination+in+zebrafish.">Temporally controlled site-specific recombination in zebrafish.</a> PLoS One. 4, (2009).</li><li>Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K. 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The authors have nothing to disclose.

The intraventricular injection provides a quick and reliable assessment method for testing gene function at later stages of development or of body homeostasis without affecting the gene function during embryogenesis. To ensure the success of this technique, one should be aware of four critical points: 1) needle size, 2) drying out of the needle, 3) minimizing volume, and 4) minimal exposure time in the sedation solution. Needles that are too small&#160;will clog frequently, while needles that are too large will damage th...

Regeneration of organs and appendages is fundamentally important for survival and fitness; however, several vertebrates including man have limited regenerative abilities. While several animal models exist that have extensive regenerative capacity, reverse genetic techniques to assess gene function during organ and appendage regeneration remain very limited or non-existent. Therefore, new approaches are required to dissect the molecular biology of regeneration in these model organisms.
The zebrafish is a well-established model for understanding the cell and molecular biology of organ and appendage regeneration1, not only because of its significant ability to regenerate multiple organs, tissues and appendages, but also because several transgenic fish lines exist to track cells and to overexpress gene constructs2,3. However, gene inhibition in larval zebrafish is mainly limited to the overexpression of dominant-negative constructs, which are not available to all genes of interest or whose transgene product can acquire gain-of-function effects that do not reflect the endogenous activity of the gene. Thus, an alternative method to specifically remove gene expression by knockout or knockdown is needed to overcome these issues.
Gene-specific targeting using TALENS exists as a reverse genetic means to knockout gene function; however, this knockout strategy is very frequently limited to functional assessments during early embryogenesis, because initial requirements of the gene prevent further progression of embryonic development. Thus, studying later phenomena such as regeneration or organ homeostasis after development using TALENS is precluded4,5. Therefore, an alternate gene removal strategy is needed that targets gene function after early development to assess gene requirements in fully formed organs and structures.
Morpholino injection has been shown to be effective in targeting genes in a few adult organs and the adult regenerating fin6-8, but these methods require electroporation and many internal organs are difficult to electroporate either due to their location or due to their sensitivity to electrical disruption. Furthermore, some tissues in the larva are difficult to inject directly, because direct injection may disrupt their structural integrity or because their size is limiting. The caudal fin of the larva is one such structure, because direct injection into the finfold is not possible. Thus, an alternative to electroporation and direct injection was needed to target genes in tissues that are either too small to inject or can&#39;t be electroporated.
In order to target and inhibit the function of specific genes during the regeneration of the larval caudal fin, we have modified existing morpholino technologies allowing the assessment of gene function during caudal fin regeneration in late-staged larva. This method employs intraventricular delivery9 of fluorescein-tagged morpholinos together with Endo-Porter transfection reagent10. Once in the ventricle, the morpholino-Endo-Porter mixture quickly spreads throughout the larva via the vasculature and enters tissues that have been previously impossible to target. This injection method may be modified to target genes in specific tissues and quite possibly can be applied in other animal models that currently lack reverse genetic methods to inhibit gene function. Thus, it offers a quick and easy method with the potential for broad range use to immediately study gene function during general organ homeostasis and regeneration at larval stages.

<table><tbody><tr><td>Morpholino with 3&amp;acute;fluorescein tag</td><td>Gene Tools Inc.</td><td>Customized</td><td>More information at www.gene-tools.com</td></tr><tr><td>Endo-Porter</td><td>Gene Tools Inc.</td><td></td><td>More information at www.gene-tools.com</td></tr><tr><td>Agarose</td><td>Serva</td><td>120623</td><td>For pouring plates to place the fish during injections and imaging</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzate-methane sulfonate)</td><td>Applichem</td><td></td><td>For anesthesia</td></tr><tr><td>E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;; 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td></td><td></td><td>For larval husbandry</td></tr><tr><td>Petri Dishes</td><td>Sarstedt</td><td>821.472</td><td>For placing the fish during injections and imaging</td></tr><tr><td>Thin Wall Glass Capillaries</td><td>World Precision Instruments, Inc.</td><td>TW100F-3</td><td>For preparing injection needles</td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>5,242,956,003</td><td>For loading the needle</td></tr><tr><td>Pasteur Pipettes (3 ml/1 ml)</td><td>Brand</td><td>747760/ 74775</td><td>For transfering larva</td></tr><tr><td>Flaming/Brown needle puller</td><td>Sutter</td><td></td><td>More information at www.sutter.com</td></tr><tr><td>Watchmaker (Dumont) Tweezers (size 5)</td><td>World Precision Instruments, Inc.</td><td>501985</td><td>To brake the needle before sharpening</td></tr><tr><td>Dissecting microscope</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td>Part of the whole injection setup</td></tr><tr><td>Fluorescence camera</td><td>Olympus, Leica, Zeiss</td><td>Varies with the manufacturer</td><td>To image the fluorescence after injeciton</td></tr><tr><td>PicoNozzle kit (microinjector holder)</td><td>World Precision Instruments</td><td>5430-12</td><td>For microinjections</td></tr><tr><td>Pneumatic PicoPump (microinjector)</td><td>World Precision Instruments</td><td>SYS-PV820</td><td>For microinjections</td></tr></tbody></table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

The sharp, beveled injection needle is easily placed in the cardiac ventricle of the zebrafish larva when approached dorsally (Figure 3A). The heart continues pumping and blood flow is maintained despite the presence of the needle (Figures 3B and 3C). Careful injections do not disrupt the morphology of the ventricle or cardiac contractions (Figure 3D) despite injection of the morpholino into the heart (Figure 3E).
<p class="jove_cont...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

10.5K Views.  Technische Universität Dresden. An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Video: Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Neuroscience

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gene Silencing with Morpholinos

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.
This video reviews proper experimental design when using these oligonucleotides. Following that, an explanation of morpholino microinjection techniques in zebrafish and the analysis of resulting phenotypes will be discussed. Finally, we showcase examples of specific applications where morpholino technology is used to model developmental disorders or to study tissue regeneration.

Morpholino Technology: Gene Silencing in Zebrafish

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Summary

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Chapters in this video

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Gene Silencing with Morpholinos