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

Research

JoVE Journal

Biology

15.0K Views.  Massachusetts General Hospital. 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.

Video: Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Developmental 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 ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Cancer Research

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Immunology and Infection

A Simple and Effective Transplantation Device for Zebrafish Embryos

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

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

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

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

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 ...

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

Author Spotlight: Mapping Cellular Connectivity in the Zebrafish Nervous System During Development and Regeneration

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

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 ...