We thank E.C. Liao for help with suturing, and R. Ethier and L. Gyr for zebrafish care. A.J.D. was supported by the Harvard Stem Cell Institute, the American Society of Nephrology, and NIH/NIDDK (P50DK074030). C.Q.D. was supported by the Massachusetts General Hospital-Fund for Medical Discovery.

1. Conditioning of non-transgenic recipient fish.

<ol>
 <li>Select the biggest male fish in the system for transplantation.</li>
 <li>Place the fish in 0.02% Tricaine (pH 7) for approximately 3 minutes or until it stops swimming, but not so long that the heart stops beating.</li>
 <li>Put the anesthetized fish on a damp paper towel on a bench top.</li>
 <li>Use an insulin syringe needle to inject 40 ug of gentamicin in 20 uL of water into the intraperitoneal cavity of the fish. For renal regeneration studies, this step is important for inducing a favorable environment for enhanced engraftment. </li>
 <li>Put the fish back into system water for recovery.</li>
 <li>The fish may be docile and very sensitive to external stimulus such as vibration. After 3 hours of recovery, treat the fish with 25 grey of gamma irradiation.</li>
 <li>Put the fish back into the system for 3 days without food prior to transplantation.</li>
 
</ol>
2. Preparation of progenitor donor cells genetically labeled with a fluorescent marker (EGFP or mCherry).
<ol>
 <li>Just prior to the surgical procedure, prepare the donor cells to be transplanted and keep them on ice. </li>
 <li>For renal studies, we use whole kidney marrow cells from the Tg (cdh17:EGFP) transgenic line, which contain renal and hematopoietic progenitor cells. These progenitor cells are genetically labeled and will only express EGFP if they engraft and differentiate into renal epithelial cells. Other types of labeled progenitor cells can also be used, such as Tg(scl:EGFP) for hematopoietic stem cell or Tg(fli1a:EGFP) for angioblast progenitors transplants.</li>
 <li>Euthanize 3 adult transgenic fish by placing them in 0.2% Tricaine for 5 minutes.</li>
 <li>Use a sterilized razor blade to remove the head and tail, and dissecting scissors to make a midline incision along the ventral side. </li>
 <li>Under a stereoscope, remove all of the internal organs in the body cavity including the swim bladder with tweezers. Be careful not to remove the kidney, which is a flat and pigmented organ attached to the dorsal wall of the cavity.</li>
 <li>Next, dissect out the kidney from the dorsal wall and place it into a 1.5 mL centrifuge tube containing 300 uL of cold phosphate buffer saline supplemented with 2% fetal calf serum (1X PBS/2% FCS).</li>
 <li>Use a pestle to shear the kidney until it becomes homogenized.</li>
 <li>Place a 40 uM cell strainer inside a 50 mL Falcon tube and transfer the cell solution on top of the cell strainer, allowing the loose cells to filter through into the Falcon tube.</li>
 <li>Use the pestle to gently smear the big pieces of tissue that are left on the strainer to further break up the cells. </li>
 <li>Leave the cell solution in the Falcon tube and wash the strainer with 1.2 mL of 1X PBS/2% FCS to collect residual cells from the strainer. </li> 
 <li>Transfer the cell solution into a new 1.5 mL tube and centrifuge at 4&deg;C at 300 rcf for 5 minutes.</li>
 <li>Wash the cell pellet with 1.5 mL of 1X PBS/2% FCS and pass it through a new strainer to filter out clumps of sticky cells and centrifuge again.</li>
 <li>Resuspend the cell pellet in 200 uL of of 1X PBS/2% FCS and transfer to a PCR tube.</li>
 <li>Centrifuge the PCR tube (4&deg;C, 300 rcf, 1 min) and remove most of supernatant.</li>
 <li>Resuspend the cells in the residual supernatant to get a 2-5 uL final volume and store it on ice. </li>
 <li>Final cellular yield should be approximately 2 million cells per kidney.</li>
 <li>Count the number of cells with a hemocytometer and adjust the concentration to 5 x 105 cells per uL.</li> 
</ol>
3. Load the cell solution into a Hamilton syringe. 
<ol>
	<li>Rinse the Hamilton syringe with sterile water 3 times.</li>
	<li>Rinse the syringe with 75% ethanol 3 times.</li>
	<li>Rinse the syringe again with 1X PBS/2% FCS 3 times.</li>
	<li>Then load the syringe with 1 uL of cell solution contain approximately 5 x 105 cells just prior to injection.</li>
</ol>
4. Transplantation of progenitor cells directly into the kidney.
<ol> 
	<li>Anesthetize the conditioned recipient fish in 0.02% Tricaine for about 3 minutes, or until the fish has stopped moving, but not too long that the heart stops beating. </li>
	<li>Under a stereomicroscope, lay the fish on a paper towel with the head towards the left, ventral side up, in order to dry off the transplanted side.</li>
	<li>Then roll the fish over to expose the dried side. </li>
	<li>Using sterilized tweezers, remove the scales in the region immediately posterior to the gills.</li>
	<li>Make a 1 cm lateral incision in this area at the midline with a scalpel while stabilizing the tissue with tweezers.</li>
	<li>Using the tweezers, expose the tissue while continuing to make the incision deep enough to expose the head kidney.</li>
	<li>If there is a lot of bleeding that prevents visualization of the kidney, euthenize the fish and start over with another recipient fish.</li>
	<li>While prying the tissue open to expose the head kidney, inject 1 uL of the cell solution directly into the head kidney.</li>
	<li>Then, at the same time as the syringe needle is being retrieved, release the tweezers so that the tissue falls back into place to prevent leaking of the injected cells.</li>
	<li>Hold a suture needle with a needle driver and pierce it through the muscle on each side of the incision.</li>
	<li>Tie 2 knots and cut off the excess suture. Only a single suture is needed for a 1 cm incision.</li>
	<li>Place the fish in fish water containing 10 parts per million of Tricaine for 5 hours to provide fish with a calming analgesic effect.</li>
	<li>Place the fish back into the system for recovery with normal feedings.</li>
	<li>After 7-18 days of recovery, we dissect the fish and analyze engraftment of renal progenitor cells by live EGFP fluorescence.</li>
</ol>
5. Representative Results: 
The overall strategy of the transplantation is illustrated in Figure 1. For our renal studies, the kidney is dissected and analyzed by EGFP fluorescence. Engraftment and differentiation of renal progenitor cells is detected as early as seven days post transplant. This is evident by the presence of multiple clusters of EGFP+ renal epithelial cells (Figure 2). By 18 days post transplant, we detect an average of 24 EGFP+ nephrons, indicating that donor cells have formed new kidney tissue (Figure 3). To demonstrate long-term engraftment, we dissected a recipient fish 59 days post transplant (Figure 4). This fish had many nephrons at the site of injection (red arrowhead) in addition to multiple nephrons at distant sites (white arrowheads), suggesting that renal progenitor cells are migratory. This technique is faster and more efficient compared to when cells are injected into the circulation via the heart, which takes approximately three months to detect engraftment.
<img src="/files/ftp_upload/2725/2725fig1.jpg" alt="Figure 1"/> 
Figure 1. Overall scheme of transplantation. Non-transgenic recipient fish are irradiated to suppress immune rejection of transplanted cells. Three days later, an incision is made on the flank of the fish and donor progenitor cells are injected into the exposed head kidney. The incision is closed up with one suture and fish are placed back into the system for recovery. Kidneys of recipient fish are dissected at later time points and analyzed for engraftment and progenitor activity.
<img src="/files/ftp_upload/2725/2725fig2.jpg" alt="Figure 2"/> 
Figure 2. Engraftment of progenitor cells after seven days. Engraftment of renal progenitor cells is detected on the injected side of the head kidney at seven days post transplant, as determined by the presence of clusters of EGFP+ renal epithelial cells (inset &#x2013; magnified view).
<img src="/files/ftp_upload/2725/2725fig3.jpg" alt="Figure 3"/> 
Figure 3. Engraftment of progenitor cells after 18 days. After 18 days post transplant, we detect an average of 24 EGFP+ donor-derived nephrons (insert &#x2013; magnified view of individual EGFP+ nephrons).
<img src="/files/ftp_upload/2725/2725fig4.jpg" alt="Figure 4"/> 
Figure 4. Engraftment and migration of progenitor cells after 59 days. We continued to detect engraftment of progenitor cells long after transplantation, even after 59 days (red arrow head). At this later time point, donor-derived nephrons are detected at sites away from the site of injection (white arrow heads), consistent with progenitor cell migration.

<ol>
	<li>Hopkins, C., Li, J., Rae, F., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+options+for+kidney+disease.">Stem cell options for kidney disease.</a> The Journal of Pathology. 217, 265-281 (2009).</li><li>Brittijn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+lethal+irradiation+in+zebrafish+and+rescue+by+hematopoietic+cell+transplantation.">Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation.</a> Blood. 104, 1298-1305 (2004).</li></ol>

No conflicts of interest declared.

Cell transplantation techniques are crucial for regenerative studies and are the gold standard assay for measuring stem cell activity. Here, we report a method for transplanting cells directly into the kidney of adult zebrafish to determine stem and progenitor cell activity. This procedure involves making an incision through the muscle to expose the head kidney, followed by injection of cells directly into the kidney. The incision is closed with a single suture and the operated fish have a 90-100% survival rate. In our experiments, engrafted renal progenitor cells are detected in 80-100% of the transplanted fish by seven days post transplantation. One limitation of this technique is that the incision is near the dorsal aorta, which can be easily ruptured by the scalpel. However, once mastered, the rate of bleeding can be reduced to less than one percent. Our method could be adapted to deliver other cell types (blood, stromal, or vascular) as well as small molecules to the kidney. The development of this technique will help advance regenerative studies and permit stem and progenitor cell potential to be assessed in vivo.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 
 </tr>
 <tr>
 	<td>Insulin syringe needle (28 gauge)</td>
 <td>Becton Dickinson </td>
 <td>329461</td>
 
 </tr>
 <tr>
 	<td>Cell strainer (40 uM)</td>
 <td>Becton Dickinson</td>
 <td>352340</td>
 
 </tr>
 <tr>
 	<td>Tricaine</td>
 <td>Sigma</td>
 <td>A5040</td>
 
 </tr>
 <tr>
 	<td>Hamilton syringe (26 gauge)</td>
 <td>Sigma</td>
 <td>20734</td>
 
 </tr>
 <tr>
 	<td>Ethilon suture (size 9-0)</td>
 <td>Ethicon</td>
 <td>2819G</td>
 
 </tr>
 
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Transplantation of Cells Directly into the Kidney of Adult Zebrafish at JoVE.com

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

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 ...

transplantation-of-cells-directly-into-the-kidney-of-adult-zebrafish

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JoVE Journal

Biology

15.0K ビュー.  Massachusetts General Hospital. 細胞移植は、組織の再生を研究するための、病気の細胞ベースの治療法を開発するために必要不可欠な技術です。ここでは、成人のゼブラフィッシュの魚の腎臓に直接遺伝的に標識された細胞の移植を可能に顕微鏡下手術を示しています。

ビデオ: Transplantation of Cells Directly into the Kidney of Adult Zebrafish

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 ...

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

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, ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

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.

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 ...

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 ...