Name: dissection of the adult zebrafish kidney
Uploaded: 2011-08-29T12:00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Dissection of the Adult Zebrafish Kidney at JoVE.com

R.A.W. wishes to thank members of the Zon and Davidson research laboratories for their innumerable contributions to helping to develop strategies that have progressively shaped this dissection technique in adult zebrafish over the past decade. In addition, we wish to express our gratitude to the staff members of the Notre Dame Center for Zebrafish Research for providing excellent ongoing husbandry care for our zebrafish colony. R.A.W. garnered research funding from the NIH-NIDDK grant award DK083512, and generous laboratory start-up funding from the University of Notre Dame.

1. Dissection of the adult zebrafish kidney from an unfixed animal sample
<ol>
	<li>Select an adult zebrafish between 3-6 months in age for dissection.</li> 
	<li>Euthanize the fish by placing it into a dish with 0.2% Tricaine pH 7.0 for approximately 4-5 minutes, watching carefully to see that the gills stop moving and the heart stops beating.</li>
	<li>Using a spoon, lift the fish from the Tricaine bath in a small amount of solution, decant the solution and gently place the animal on a paper towel.</li> 
	<li>Use a sharp pair of dissection scissors to remove the head of the animal, making a cut just behind the gill operculum (Figure 1, path A). Discard the head in a biohazard waste container.</li>
	<li>Use the dissection scissors to open the body of the animal, by making a long ventral incision, starting from the head and terminating at the tail at the base of the caudal fin (Figure 1, path B).</li>
	<li>Remove the internal organs of the animal using a pair of dissection tweezers/forceps, and place them in biohazard waste. Be careful not to brush the dorsal body wall, as this is the location of the kidney (Figure 2). If you have selected a female fish, you will need to scoop developing eggs out of the body cavity, which can be most easily done using an angled pair of forceps or tweezers.</li>
	<li>Use dissection needles to pin open the body walls to a dissection tray, and angle the pins so that the kidney can be visualized. The kidney will possess a characteristic shape (head, trunk/saddle, tail) and color, being interspersed with black hued pigmentation (Figure 2). You may also see the aorta filled with peripheral blood, which runs down the midline of the organ structure.</li> 
	<li>Use fine forceps to detach the kidney from the dorsal body wall, using one pair of forceps to lift the kidney as you separate the organ from the connective tissues that underlie it.</li> 
	<li>Gently place the kidney into the desired vehicle for further live cell studies, such as a microcentrifuge tube containing 1x PBS for FACS preparation.</li> 
</ol>
2. Preparation and dissection of the adult zebrafish kidney from a fixed animal sample
<ol>
	<li>Prepare a solution of 4% paraformaldehyde/1X PBST. Boil the solution to dissolve the PFA into solution, then cool the mixture to 4&deg;C and add 0.1% dimethylsulfoxide. We have had the most success with tissue preservation from using freshly made PFA solutions as opposed to frozen aliquots. Make enough solution to submerge the adult zebrafish during dissection.</li>
	<li>Fill a dissection tray with enough fixation solution to submerge the adult zebrafish during the dissection. We perform the subsequent steps with this tray positioned under the microscope, which can be placed in a chemical hood to minimize exposure to fixation odors.</li>
	<li>Select an adult zebrafish between 3-6 months in age for dissection.</li> 
	<li>Euthanize the fish by placing it into a dish with 0.2% Tricaine pH 7.0 for approximately 5 minutes, watching carefully to see that the gills and heart stop beating.</li>
	<li>Using a spoon, lift the fish from the Tricaine bath in a small amount of solution, decant the solution and gently place the animal on a paper towel.</li> 
	<li>Use a sharp pair of dissection scissors to remove the head of the animal, making a cut just behind the gill operculum (Figure 1, path A). Discard the head in a biohazard waste container, and immediately place the animal into the PFA-containing dissection tray.</li>
	<li>Use the dissection scissors to open the body of the animal, by making a long ventral incision, starting from the head and terminating at the tail at the base of the caudal fin (Figure 1, path B), and keep the body partially submerged during the procedure.</li> 
	<li>Remove the internal organs of the animal using a pair of dissection tweezers/forceps, and place them in biohazard waste. Be careful not to brush the dorsal body wall, as this is the location of the kidney (Figure 2).</li>
	<li>Use dissection needles to pin open the body walls, and angle the pins so that the kidney can be visualized. The kidney will possess a characteristic shape (head, saddle, tail) and color, being interspersed with black hued pigmentation (Figure 2). You may also see the aorta filled with peripheral blood, which runs down the midline of the organ structure.</li> 
	<li>Fix the sample overnight, placing the tray at 4&deg;C when you are done with arranging the desired samples.</li> 
	<li>The next day, remove the PFA solution from the tray and replace with 1x PBST.</li> 
	<li>Use fine forceps to carefully detach the kidney from the dorsal body wall, using one pair of forceps to lift the kidney as you separate the organ from the connective tissues that underlie it. The kidney tissue will be soft and pliable from the DMSO in the fixative solution, making it possible to dissect the entire kidney organ in one piece.</li>
	<li>Use a wide-bore transfer pipet to place the kidney into a microcentrifuge tube.</li> 
	<li>The kidney can now be processed further to conduct histological or whole mount in situ gene expression studies based on the desired study.</li> 
</ol>
3. Representative results: 
The steps that are diagrammed in Figure 1 indicate the dissection procedure. The kidney can be identified based on its characteristic shape and coloration, and anatomical location on the dorsal wall of the animal&#x2019;s body cavity, as shown in Figure 2. After dissection of a fixed kidney sample, whole mount in situ hybridization can be used to localize the expression of a gene of interest, as shown in Figure 3.
<img src="/files/ftp_upload/2839/2839fig1.jpg" alt="Figure 1"/> Figure 1: Procedure that enables direct access for dissecting the zebrafish adult kidney. (A) Euthanized zebrafish is dissected in a stepwise fashion (indicated by numbers and arrows) to give the researcher the easiest access to the complete kidney organ. Images of the abdominal cavity before (B) and after (C) removal of the abdominal organs.
<img src="/files/ftp_upload/2839/2839fig2.jpg" alt="Figure 2"/> Figure 2: Visualization of the zebrafish kidney in an unfixed sample. Following removal of organs in the body cavity, the kidney appears as a single, flattened organ that is adherent to the dorsal body wall via connective tissues (A), and has been schematized (B) to show its anatomical shape.
<img src="/files/ftp_upload/2839/2839fig3.jpg" alt="Figure 3"/> Figure 3: Whole mount in situ hybridizations performed on the whole adult kidney organ. (A) cadherin17 transcripts are detected in the tubule of adult kidney nephrons, enlarged in (A&#x2019;). (B) mafba transcripts are localized proximal nephron cell types in the adult kidney (compare to entire tubule in panel A). (B&#x2019;) An enlarged view of mafba expression reveals that transcripts are specifically localized to the podocyte (P) cells of the blood filter (glomerulus) and proximal convoluted tubule (PCT).

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No conflicts of interest declared.

Adult stem cells are dynamic and essential components that maintain the adult body form1. Adult stem cells can also serve to counteract damage that the body incurs during disease states, and the dysfunction or loss of adult stem cells can lead to the decline of organ function and has been implicated to drive cancer malignancies and contribute to aging. Adult stem cells exhibit cellular properties that distinguish them from differentiated cell types. Upon cell division, adult stem cells are capable of self-renewal and the production of progenitor cells that in turn can give rise to distinct differentiated offspring. There is great interest in understanding the pathways that regulate the cell fate decisions and potency of adult stem cells because of their important roles in tissue homeostasis1. For example, many scientists currently seek to identify the signals that maintain various adult stem cell populations in their niche, or specialized microenvironment, and how this niche is impacted by humoral factors. 
Adult hematopoietic stem cells (HSCs) are pluripotent cells that fuel the continual rejuvenation of blood cells in animals8. Hematopoiesis is a vibrant process that is constantly ongoing during the life of a vertebrate organism because the mature differentiated blood cell types are short-lived and hence must be replenished regularly. As they divide, HSCs supply this crucial demand by self-renewing and producing a series of progenitors that give rise to the erythroid, megakaryocyte, lymphoid and myeloid lineages. Each of these blood cell types performs unique functions in the circulation, including providing gas exchange to tissue, a way for sealing injuries to blood vessels by means of clot formation, or defense mechanisms against invading pathogens. While HSCs have been recognized for many years, a plethora of fascinating questions remain unanswered about these amazing cells. Recent studies have identified subclasses of HSCs with different long-term self-renewal properties, and the elucidation of niche components and signals that modulate HSC behavior8. The zebrafish is now among the cornerstones of hematopoiesis research paradigms, based on the genetic and molecular attributes of this animal model8. Research utilizing the zebrafish has led to novel insights about HSC biology that are conserved with higher vertebrates like mammals, emphasizing the biomedical relevance of fish blood investigations8.
Historically, knowledge about HSCs has paved the way for the expeditions seeking to discover if other body organs are maintained by adult stem cells. Numerous adult stem cell populations are now appreciated to exist, ranging across diverse structures from the brain to the intestine, skin, and skeletal muscle. Continued efforts in the stem cell field seek to identify how adult stem cells and even other differentiated cell types can display enhanced potency in various settings.
With respect to the kidney, there has been great debate among nephrologists as to whether renal stem cells exist16. Independent findings have documented populations of kidney cells in mammals that possess varying degrees of regenerative potential, but a cohesive understanding of these reports has yet to be achieved. Interestingly, there is strong evidence to support the notion that many fish species possess cells that can robustly fuel regeneration of damaged nephrons, as well as grow entirely new nephrons during adulthood9. Recent work has identified the molecular hallmarks of adult kidney stem cells in the zebrafish10,11, and demonstrated the self-renewal potency of these so-called renal stem/progenitor cells (RPCs)11. Future studies are needed to ascertain whether analogous kidney cells are present in mammals. Nevertheless, continued investigations about the cellular and molecular mechanisms that regulate fish RPCs may provide insight into how regenerative feats may be stimulated in the mammalian kidney. It is evident that much remains to be understood about RPCs, and that the many tools now available to the zebrafish researcher can be implemented to tackle these intriguing questions. 
In this protocol, we describe our method for the anatomical identification and dissection of the adult zebrafish kidney. Alternative steps can be used to dissect the kidney, such as opening the abdominal cavity with a ventral incision using dissection scissors; however, we have encountered the most success in accessing the entire kidney when the abdominal wall is pinned open and the head is removed. Blood and kidney researchers alike who wish to isolate and work with these cell types can use either dissection method. It should be noted that the method we describe for isolating and working with the zebrafish adult kidney is certainly not limited to scientists who wish to pursue questions of adult stem cell biology, as the dissection of this organ can facilitate studies for those researchers pursuing investigations on topics ranging from physiology to aging. Further, this method can be coupled with other technologies such as transgenics, so as to molecularly label and then purify and study discrete subpopulations of blood or kidney cells from wildtype or genetically mutant zebrafish11. Looking forward, such detailed purification procedures of stem and progenitor cell types is vital to gaining knowledge about how their behavior is regulated, as operational tests such as transplantation or progenitor culturing constitute the basis by which the potency and identity of these cells is defined. Recent advances in hematopoietic progenitor culture methods open many new avenues for study, and may be adapted to culturing of renal populations17. Zebrafish chemical genetics have been successful in the identification of pathways that modulate HSCs and renal progenitors in various contexts18-20, and will continue to be a useful avenue to test in combination with the above cell biology techniques. Taken together, there have been a number of groundbreaking contributions to the fields of hematology and nephrology using the zebrafish model, and continued research in these arenas promises to wield further transformative insights in the coming years.

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 <td>Dissection scissors</td>
 <td>Roboz</td>
 <td>RS-5882</td>
 </tr>
 <tr>
 <td>Dissection forceps</td>
 <td>Roboz</td>
 <td>RS-5010</td>
 </tr>
 <tr>
 <td>Dissection angled forceps</td>
 <td>Roboz</td>
 <td>RS-5058</td>
 </tr>
 <tr>
 <td>Tricaine</td>
 <td>Sigma</td>
 <td>E10521</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Electron Microscopy Services</td>
 <td>19210</td>
 </tr>
 </tbody>
</table>

dissection of the adult zebrafish kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

Watch this Scientific Journal Video about Dissection of the Adult Zebrafish Kidney at JoVE.com

Dissection of the Adult Zebrafish Kidney

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Research

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Biology

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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.

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, ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...

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.

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

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...