Thanks to Marie Birne, Areti Tsiola, Jaymie Estevez and Arelys Uribe. This work was supported in part by CUNY Research Foundation and Howard Hughes Medical Institute Grant Summer Program for Undergraduate Students (SPUR) and NIH RO3 41702-00-01. Research was also supported by Queens College and some of the experiments were done on equipment from the Core Facility for Imaging, Cellular and Molecular Biology at Queens College.

Zebrafish were raised using standard protocols13 in the Queens College Animal Facility at 28&deg;C. Individual fish pairs or group matings should be preformed and the resulting embryos cleaned and stored in a 28&deg;C incubator. Fish health should be examined daily. At 5-day post fertilization (dpf), fish should be separated into tanks at a 10-15 fish density and entered into the fish facility nursery. For the protocol presented here zebrafish were collected at larva through adult stages: 15, 30, 45, 60, 90, 180 dpf. 
1. Zebrafish subject collection and fixation
<ol>
<li>Remove fish from habitat tank using net and place in 0.2% Tricaine to anesthetize.</li>
<li>Place anesthetized fish in ice water for 15 minutes to euthanize.</li>
<li>Make fresh 4% paraformaldehyde (PFA) in 1x Phosphate-buffered saline (PBS).</li>
<li>Incubate collected fish in 4% PFA for 1-2 hours at RT then overnight at 4&deg;C in a petri dish wrapped in parafilm. This keeps the fish flat and makes later dissection much easier.</li>
<li>After fixation, rinse twice in PBS with Tween (PBT). Fish can be keep in PBT at 4&deg;C for up to 10 days.</li>
</ol>
2. Measuring fish and dissection
<ol>
<li>Place single fish into petri dish half filled with fresh PBS and lay it on its right side.</li>
<li>Measure length of fish from snout to base of tail (the caudal peduncle), not including tail fins, using a digital mm caliper (Figure 1). This is the standard length (SL).</li>
<li>Each fish can be assigned a number/letter combination to allow an individual fish and the resulting dissected heart to be tracked for later analysis. Data, including parental stock number, age at fixation, SL measurements, were entered into a spreadsheet and organized based on these labels.</li>
<li>Before dissection, label PCR tube strips with assigned tracking number and fill with PBT or other solution appropriate for further analysis.</li>
<li>Orient the fish ventral side up in petri dish while stabilizing the body with forceps holding the head between the eyes and gills (Figure 2).</li>
</ol>
Smaller fish (smaller than 12mm SL):
<ol start="4">
<li>a) Using sharp forceps, or a micro-needle in a pin holder, remove the pectoral muscles and fins from the body to reveal the heart. The constant motion of the forceps during dissection can cause currents in the PBT that move the fish. This is particularly problematic for smaller fish, thus a micro-needle can be used to remove pectoral muscles and fins and open the body cavity.</li>
<li>a) Gently use the forceps to scoop the heart out of the cavity from under the atrium. Alternately, in some fish you may be able to remove the heart by gently pulling the bulbous out and the rest of the heart will follow. Be careful of the location of the atrium if using this technique as it can easily be damaged. If there is extra tissue that comes with the heart, that is fine and can be cleared after.</li>
<li>a) If the fish contains a fluorescent cardiac marker such as Tg(myl7:GFP)twu34, use fluorescent scope for dissection and verify removal of the heart by florescence (Figure 3).</li>
<li>a) Once the heart is removed from the cavity, use the forceps to hold the heart and a micro-needle to remove extra non-cardiac tissue and the epicardial lining from the outside of the heart.</li>
</ol>
Larger fish (greater than 12mm SL):
<ol start="4">
<li>b) Using spring handled micro-scissors, make three incisions of less than 1mm deep: 1- transverse cut through the gills 2- transverse cut at anterior belly 3- sagittal cut on ventral side connecting both cuts (Figure 4).</li>
<li>b) Using sharp forceps remove the pectoral muscles and fins from the body to open the body cavity and reveal the silvery tissue of the pericardium (Figure 5A). Remove this pericardial tissue and the heart will become visible (Figure 5B).</li>
<li>b) Use the micro-scissors to cut the artery connected to the bulbous arteriosus, located superior to the heart. Once this artery is cut, put the forceps tips under the atrium, use the forceps to scoop the heart out of the cavity. If there is extra tissue that comes with the heart, that is fine and can be removed later.</li>
<li>b) Once the heart is removed from the cavity, use the two forceps to hold the heart and remove extra tissue, or one pair of forceps to hold the heart and a micro-needle to remove extra, non-cardiac tissue. The epicardial lining, evident by its black pigmentation, can be removed from the outside of the heart if desired by gently scraping the tissue.</li>
</ol>
3. Photography of hearts
<ol>
<li>Prepare an 23g/L nutrient agar petri dish and cover solidified agar with PBS.</li>
<li>Using the forceps, make a well in the agar that allows the heart to rest in the desired orientation for photographing.</li>
<li>Remove the heart from the PCR tube by holding the tip of the bulbous with the forceps firmly. Place the heart in the agar dish.</li>
<li>Orient the heart in agar to photograph. The sample can be rotated after each picture to all possible orientations (Figure 6).</li>
<li>Use overhead light and short exposure time to photograph the hearts. Make adjustments for light quality using the exposure as necessary to obtain the best photograph.</li>
</ol>
4. Representative Results: 
An example of a well-dissected clean heart is shown in multiple orientations in Figure 6. Each heart will have some differences in overall morphology, but the heart should be complete, containing an intact ventricle, atrium and bulbous arteriosus. Some examples of this variation in overall morphology are shown in Figure 7. The black spotted epicardial tissue can also removed from the heart.
<img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Figure 1"/> Figure 1. Measure the fish from snout to caudal peduncle using a digital mm caliper. 
<img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Figure 2"/> Figure 2. Hold the fish with forceps between the eyes and gills.
<img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Figure 3"/> Figure 3. A cardiac specific fluorescent transgenic marker such as Tg(myl7:GFP)twu34, shown here, can aid in visualizing hearts dissected from smaller fish. 
<img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Figure 4"/> Figure 4. Three incisions to remove the pectoral muscles and skin: 1- transverse cut through the gills 2- transverse cut at anterior belly 3- sagittal cut on ventral side connecting both cuts. 
<img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Figure 5"/> Figure 5. Black letters label the chambers of the heart: bulbous arteriosus (ba), ventricle (v), and atrium (a). 
A-	The upper body cavity is open to show the silvery tissue of the pericardium. 
B-	After removing the pericardium, the heart is visible and easily removed. 

<img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Figure 6"/> Figure 6. Different orientations of the heart are shown. Black letters label the chambers of the heart: bulbous arteriosus (ba), ventricle (v), and atrium (a). Grey arrows indicate location and orientation of the heart in reference to its normal location in the body: anterior (A), posterior (P), dorsal (D), ventral (V), left (L), and right (R). Scale bar indicates 400&mu;m. 
A-	ventral view of the heart 
B-	dorsal view of the heart 
C-	lateral view from the left side 
D-	lateral view from the right side 

<img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Figure 7"/> Figure 7. Morphology of hearts of different sizes is shown. Black letters label the chambers of the heart: bulbous arteriosus (ba), ventricle (v), and atrium (a). Scale bar indicated 400 &mu;m. 
	A-C- Hearts form fish with SL less than 12mm 
	D-F- Hearts form fish with SL greater than 12mm

<ol>
	<li>Ahmad, N., Long, S., Rebagliati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+zebrafish+heart:+from+left-right+axis+specification+to+epithelial+tissue+morphogenesis.">Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis.</a> Dev Biol. 330, 213-220 (2009).</li><li>Glickman, N. S., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+development+in+zebrafish:+coordination+of+form+and+function.">Cardiac development in zebrafish: coordination of form and function.</a> Semin Cell Dev Biol. 13, 507-513 (2002).</li><li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</li><li>Schoenebeck, J. 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A., Holdway, J. E., Major, R. J., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+addition+of+new+myocardial+and+epicardial+cells+fosters+homeostatic+cardiac+growth+and+maintenance+in+adult+zebrafish.">Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish.</a> Development. 135, 183-192 (2008).</li><li>Parichy, D. M., Elizondo, M. R., Mills, M. G., Gordon, T. N., Engeszer, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Dev Dyn. 238, 2975-3015 (2009).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book,+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio).</a> , (1995).</li></ol>

No conflicts of interest declared.

These methods for dissection of the zebrafish heart of post-embryonic fish allows for the complete removal of an undamaged heart. While this technique is straightforward and provides guides for external cuts that will prevent damaging the heart, a steady hand is essential for smaller fish.
A key step during the dissection is to identify the silvery pericardium as the heart is directly within this sac. Visualizing the pericardium provides an important marker for finding and ultimately removing ...

<table border="1">
 <tbody>
 <tr>
 <th>Material</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Paraformaldehyde </td>
 <td>EMD</td>
 <td>PX0055 </td>
 <td>.04g/mL in PBS</td>
 </tr>
 <tr>
 <td>Forceps #55 </td>
 <td>FST</td>
 <td>11295-51</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro-scissors </td>
 <td>FST</td>
 <td>15005-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pin holder </td>
 <td>FST </td>
 <td>26016-12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro-needles </td>
 <td>FST </td>
 <td>10130-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nutrient Agar </td>
 <td>BBL</td>
 <td>11472</td>
 <td>23g/L in distilled H2O</td>
 </tr> 
 <tr>
 <td>Petri dishes</td>
 <td>BD Falcon</td>
 <td>351029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PCR 8-tube strips </td>
 <td>USA Scientific</td>
 <td>1402-2500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Caliper</td>
 <td>Fisher Scientific</td>
 <td>14-648-17</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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.

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 ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

21.4K Views.  Queens College, City University of New York. שיטה ברורה, תקנית לנתיחה ובידוד של הלב דג הזברה בשלבים התפתחותיים מרובים מתוארים. טכניקות ביאור וכימות הם דנו גם.

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

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 ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

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 ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

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 ...