Os autores gostariam de agradecer a Laura Anne Lowery, que desenvolveu essa técnica no laboratório. HS é suportada por NIHMH70926. Jennifer Gutzman foi apoiado pelo companheirismo CSBI / Merck pós-doutorado.

Protocolo Parte 1: Preparando-se para microinjeção <ol><li> Prepare-se para a injeção puxando agulhas capilar utilizando Sutter extrator de agulhas Instruments. </li><li> Preencha a agulha com um corante fluorescente (como Texas Red-Dextran). </li><li> Montar a agulha em um aparelho de microinjeção e micromanipulador. </li><li> Cortar a agulha para o tamanho apropriado em um ângulo para criar uma ponta chanfrada. </li><li> Medir o tamanho de gota e verifique se ele está entre 1-2nl por injecção de óleo. </li></ol> Parte 2: Preparando os embriões <ol><li> Injetar os embriões 30 minutos mais cedo do que o estágio de interesse. Por exemplo, para estudar os ventrículos menos 24 horas após a fertilização (hpf), injetar os embriões em 23,5 hpf. Estágio os embriões de acordo com Kimmel et al. (4). </li><li> Sob o estereomicroscópio, remova cuidadosamente o córion dos embriões usando uma pinça. </li><li> Usando um prato de plástico revestidas com agarose 1%, poke buracos na agarose com uma ponteira de plástico 100-200 mL. Remova cuidadosamente os plugues de agarose dos furos usando uma pinça. </li><li> Transferência de embriões em meios embrião no prato agarose com buracos. </li><li> Adicionar tricaina (feita de acordo com Westerfield (5)) para a mídia para anestesiar os embriões e evitar qualquer movimento durante o experimento. </li><li> Delicadamente, coloque a cauda do embrião para dentro do buraco, e orientá-la de modo que o aparelho de injecção está no lado posterior do embrião. </li></ol> Parte 3: A injeção do ventrículo cerebral <ol><li> Para injetar o ventrículo cerebral, providenciar a instalação de micromanipulação de modo que a ponta da agulha está no mesmo campo de visão como o embrião em potência alta. </li><li> Cuidadosamente coloque a agulha através da roofplate fina do rombencéfalo apenas posterior à hingepoint r0/r1 sem bater o tecido cerebral abaixo. </li><li> Apenas o suficiente injetar corante para encher completamente os ventrículos. Pode levar várias injeções para preencher o espaço ventrículo, dependendo do estágio do embrião. </li></ol> Parte 4: Imaging <ol><li> Usando um limpa 1% agarose prato revestido, poke novos buracos no prato, retire as velas, e adicione tricaina para anestesiar os embriões e impedir o movimento. </li><li> Coloque a cauda do embrião ventrículo-injetado no buraco e posição para uma imagem dorsal. </li><li> Tirar uma foto usando luz transmitida. </li><li> É muito crítico para não mover o embrião ou microscópio. </li><li> Alterar as configurações no microscópio e ter uma imagem com luz fluorescente. </li><li> Reposicionar o embrião para tirar fotos com os dois laterais luz transmitida e luz fluorescente. </li><li> Guardar as imagens como arquivos TIFF para processamento de imagens em Photoshop. </li></ol> Parte 5: Processamento de Imagem <ol><li> Para processar as imagens no Photoshop, abra a luz transmitida e luz fluorescente imagens do embrião mesmo tomadas na mesma posição. Certifique-se de todas as configurações são as mesmas para cada imagem. </li><li> Arraste a imagem fluorescentes para a imagem de luz transmitida e alinhá-las com exatidão. </li><li> Com a imagem selecionada fluorescentes, selecione &quot;Imagem&quot;, &quot;Ajustes,&quot; e depois &quot;Substituir cor&quot; e mudar o fundo preto ao branco. </li><li> No &quot;Substituir cor&quot; janela ajustar a &quot;imprecisão&quot; fator para a direita até as imagens fluorescentes parece uniforme em cor. </li><li> No &quot;Layers&quot; janela, selecione &quot;Multiply&quot; para ver as imagens de sobreposição. Sua imagem de espaço ventrículo deve corresponder à imagem de luz transmitida com exatidão. </li><li> Salve este arquivo e repita para quaisquer outras imagens. </li></ol> Resultados representante / Resultado Adequada imagens injeção ventrículo são mostrados na Figura 1 AD painéis, enquanto um exemplo de imagens de injeção incorreta são mostrados na EH. Injeções adequada têm arestas vivas e não-difusa corante. Injeção incorreta, que ocorre quando a agulha é inserida longe demais para o ventrículo e atinge o tecido cerebral abaixo, pode resultar em corante que é visível fora do espaço ventrículo e na gema. <img src="/files/ftp_upload/1218/1218figure1.png" alt="Figura 1" width="600" height="742" /> Figura 1. Ventrículo injeções de 25 hpf embriões de tipo selvagem. (AD) método de injeção correta e (EH) método de injeção incorreta são mostrados como exemplos. (A, E) imagens brightfield dorsal com correspondentes imagens fluorescentes (B, F) ea sobreposição das imagens fluorescentes e brightfield (C, G) para métodos de injeção corretas e incorretas mostrado. Setas indicam regiões onde corante foi além do espaço do ventrículo cerebral devido a uma injeção ventrículo incorreta (consulte &quot;Solução de problemas&quot;). Anterior é para a esquerda em todas as imagens. Solução de problemas: <table border="1"><tbody><tr style="font-weight:bold; font-size:16px;"><td> Problema </td&gt; <td> Causa </td><td> Remédio </td></tr><tr><td> Você está esmagando o embrião com a agulha. </td><td> Sua agulha é demasiado brusco ou a ponta é muito grande. </td><td> Comece com uma agulha nova e não quebrá-lo, tanto para trás. Bisel da agulha para torná-lo seu afiada como uma agulha de seringa. </td></tr><tr><td> O corante é em todo o embrião e da gema (ver Fig. 1E-H). </td><td> Sua agulha atravessou o tecido e não ficar no espaço ventrículo. </td><td> A agulha não pode ser afiado o suficiente, então você é incapaz de controlar a velocidade de punção. </td></tr><tr><td> O corante é muito fraco ou não na parte frontal do cérebro. </td><td> Você não usar uma pressão alta o suficiente para a injeção, ou a agulha não foi longe o suficiente para alcançar o anterior do prosencéfalo quando injetado o corante. </td><td> Aumentar a sua pressão de injeção ou colocar a agulha um pouco mais durante a injeção anterior. Você pode colocar a agulha através da fronteira mesencéfalo rombencéfalo-se mais detalhe é necessário no prosencéfalo. Você também pode simplesmente esperar um pouco para o corante para difundir ainda mais antes de imagem. </td></tr><tr><td> Suas imagens fluorescentes são escuras em torno das bordas após a substituição de cores no Photoshop. </td><td> Você se esqueceu de mudar a &quot;confusão&quot; na janela de configuração substitui a cor. </td><td> Mover a &quot;confusão&quot; por todo o caminho à direita na janela de substituição de cor. </td></tr><tr><td> A agulha não perfurar a roofplate rombencéfalo. </td><td> A agulha não é cortado corretamente. </td><td> Faça uma nova agulha. Se você estiver fazendo um monte de injeções que você pode precisar de várias agulhas como eles podem obter maçante. </td></tr></tbody></table>

<ol>
	<li>Gutzman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech. Dev. 125, 974-983 (2008).</li><li>Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Classification+of+Zebrafish+Brain+Morphology+Mutants.">Characterization and Classification of Zebrafish Brain Morphology Mutants.</a> Anat. Rec. (Hoboken). 292, 94-106 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Kimmel, C. B. <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. Dyn. 203, 253-310 (1995).</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. , (1995).</li></ol>

Neste vídeo demonstramos como injetar corante fluorescente (Texas-Red Dextran) no desenvolvimento de ventrículos cerebrais zebrafish. Este método é usado para visualizar o espaço do ventrículo cerebral em contraste com a neuroepitélio circundante e é extremamente útil para determinar a forma do espaço ventrículo, bem como a forma do tecido cerebral circundante. Ela nos permite compreender melhor o processo de formação do cérebro e morfogênese do ventrículo cerebral ao longo do tempo no embrião vivo. Est...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Tubes (needles)</td>
<td>Tools</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Agarose-Coated dishes</td>
<td>Tools</td>
<td>Agarose (Seakem GTG; Lonza) Dishes (Corning) </td>
<td>Agarose (50027) Dishes (430166) </td>
<td>Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting</td>
</tr>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Zeiss Stereomicroscope Stemi SV 6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Camera with Fluorescence capabilities and with image capture software</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-200 &mu; pipette tips</td>
<td>Tools</td>
<td>Tip One, US Scientific</td>
<td>1111-0806</td>
<td>These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.</td>
</tr>
<tr>
<td>Photoshop</td>
<td>Image Analysis</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Loop</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.</td>
</tr>
<tr>
<td>Microinjector</td>
<td>Tools</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PL1-100</td>
<td>&nbsp;</td>
<tr>
<td>Needle Puller</td>
<td>Tools</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>Settings (Heat 785, Pull 70, Velocity 40, Time 70).</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tools</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent dye Texas-Red Dextran 70,000MW</td>
<td>Reagent</td>
<td>Molecular Probes</td>
<td>D1830</td>
<td>Other Molecular weights are available if desired.</td>
</tr>
<tr>
<td>Tricaine (3-aminobenzoic acid ethyl ester)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Media</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>According to Westerfield (5)</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish brain ventricle injection

Formação adequada do ventrículo do cérebro durante o desenvolvimento embrionário do cérebro é necessário para a função cerebral normal. Ventrículos cerebrais são as cavidades altamente conservadas dentro do cérebro que são preenchidos com fluido cerebrospinal. Em peixes-zebra, após a formação do tubo neural, o neuroepitélio sofre uma série de constrições e dobras, enquanto ele se enche de fluido, resultando na formação de ventrículo cerebral. A fim de compreender o processo de formação do ventrículo, e as mudanças que ocorrem neuroepiteliais forma, ao mesmo tempo, precisávamos de uma forma de visualizar o espaço do ventrículo em comparação com o tecido cerebral. No entanto, a natureza de embriões zebrafish transparente torna difícil diferenciar o tecido do espaço ventrículo. Por isso, desenvolvemos uma técnica de injeção ventrículo cerebral onde o espaço de ventrículo é preenchido com um corante fluorescente e fotografada pela claro e microscopia de fluorescência. O claro e as imagens fluorescentes são então processadas e sobrepostos no Photoshop. Esta técnica permite a visualização do espaço do ventrículo com o corante fluorescente, em comparação com a forma do neuroepitélio na imagem de campo claro. Injeção cérebro ventrículo no peixe-zebra pode ser empregado a partir de 18 horas após a fecundação até o início fases larval. Temos utilizado esta técnica extensivamente em nossos estudos de formação do cérebro do ventrículo e morfogênese, bem como na caracterização de mutantes cérebro morfogênese (1-3).

Watch this Scientific Journal Video about Zebrafish Brain Ventricle Injection at JoVE.com

Zebrafish Brain Ventricle Injection

Após a formação do tubo neural, o neuroepitélio contrai e dobras, enquanto o tubo se enche de líquido cefalorraquidiano embrionárias (eCSF) para formar os ventrículos do cérebro embrionário. Nós desenvolvemos esta técnica de injeção do ventrículo para visualizar melhor o espaço cheio de líquido, em contraste com a forma neuroepiteliais em um embrião vivo.

Zebrafish Injection Ventrículo Cerebral

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Research

JoVE Journal

Biology

21.1K visualizações.  Whitehead Institute for Biochemical Research. Após a formação do tubo neural, o neuroepitélio contrai e dobras, enquanto o tubo se enche de líquido cefalorraquidiano embrionárias (eCSF) para formar os ventrículos do cérebro embrionário. Nós desenvolvemos esta técnica de injeção do ventrículo para visualizar melhor o espaço cheio de líquido, em contraste com a forma neuroepiteliais em um embrião vivo.

Vídeo: Zebrafish Brain Ventricle Injection

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biologia

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-orbital de injeção no Zebrafish Adulto

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Transplante de GFP-expressando Blastômeros for Imaging ao vivo de Desenvolvimento da retina e do cérebro em embriões Zebrafish Quimérico

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Injeção intraperitoneal em Zebrafish Adulto

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Entrega direta da MIF Morpholinos Into the Otocyst Zebrafish por injeção e Eletroporação afeta o desenvolvimento da orelha interna

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

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.

Na eletroporação in vivo de Morpholinos no Adulto Regeneração Zebrafish Cauda Fin

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

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish Adulto

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

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;

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.

Reverter Genética Abordagem Morfolino Usando Injeção Cardíaca Ventricular para transf múltiplos tecidos de difícil alvo do Zebrafish Zergling

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...