Name: Dissection of Organs from the Adult Zebrafish (Video) | JoVE Journal
Uploaded: 2010-03-04T16:45:00.000+00:00
Duration: 10 min 18 s
Description: Learn dissection of adult zebrafish organs for developmental biology and comparative anatomy research. This protocol demonstrates techniques for isolating gonads, heart, liver, brain, and other major organ systems.

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5 minutes

This work was supported by NIH grant R01 HD050901 to MCM and American Cancer Society Postdoctoral Fellowship #PF-05-041-01-DDC to TG.

<ol>
<li>A male zebrafish will be dissected first, followed by a female fish. Before beginning the dissection, anesthetize a fish in 0.2% Tricaine and then euthanize it by incubation in ice water for 15 minutes.
</li>
<li>Begin by lightly patting the fish dry on a paper towel and placing it on a dissecting mat. Externally, zebrafish have single dorsal, caudal and anal fins and paired pectoral and pelvic fins (Figure 1). 
 <img src="/files/ftp_upload/1717/1717fig1.jpg" alt="Zebrafish anatomy diagram showing fin locations: dorsal, caudal, pectoral, pelvic, anal." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Adult male fish with fins labeled. </li>
<li>Pin the fish to the dissecting mat through the fleshy part of the tail and the ventral part of the eye socket.</li>
<li>Snip the skin on the belly of the fish just anterior to the anal fin. Cut the skin and underlying muscle along the belly from the anal fin to the operculum (the hard covering over the gill) (Figure 2, step 1). <img src="/files/ftp_upload/1717/1717fig2.jpg" alt="Zebrafish anatomical diagram; labeled circulatory system path; educational biology study." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Steps in removing skin and body wall muscle to expose internal organs. </li>
<li>Next, remove the operculum and the pectoral fin, including the pectoral girdle (the thick, bony region at the base of the fin) (Figure 2, step 2). Cut the skin and underlying muscle beginning from above the now exposed gill posteriorly along the side of the fish and then down to the anal fin (Figure 2, step 3).</li>
<li>Carefully remove the skin and underlying muscle from the side of the fish. Many of the internal organs are now visible (Figure 3). 
<img src="/files/ftp_upload/1717/1717fig3.jpg" alt="Zebrafish anatomy diagram, showing heart, liver, intestine, testis, and swim bladder for study." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Adult male fish with body wall and muscle removed allowing the visualization of the internal organs. </li>
<li>The testes are long, white, paired organs that are attached to the dorsal body wall. Remove a testis and place it in a dish of PBS. Examine the testis with reflected light to visualize the seminiferous tubules (Figure 4), which contain cysts with various stages of developing germ cells from spermatagonia to spermatids (Leal et al., 2009). <img src="/files/ftp_upload/1717/1717fig4.jpg" alt="Microscope image of plant starch granules; microscopy technique; cellular structure study." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Dissection of a testis. The white roundish structures are the seminiferous tubules. </li>
<li>Next, remove the gastrointestinal system from the body cavity of the fish. The liver can be identified by its large size, lobed morphology, tannish color, and extensive vascularization. The gallbladder, a green translucent fluid-filled sac, and the spleen, which appears bright red, are found within the viscera.</li>
<li>Separate the intestine from the rest of the organs and stretch it out. The anterior, mid, and posterior regions of the intestine are defined by the height of the epithelial folds (Wallace et al., 2005).</li>
<li>Place a piece of the intestine in PBS and observe the epithelial folds using transmitted light.</li>
<li>Next, examine the swim bladder. The swim bladder consists of a posterior chamber, which is connected to the esophagus via the pneumatic duct, and an anterior chamber, which is connected to the inner ear through the Weberian apparatus (Finney et al., 2006).</li>
<li>Remove and discard the swim bladder. Unpin the fish and re-pin it ventral side up to dissect the kidney, which is located along the dorsal body wall. The kidney is a translucent pink structure associated with the dorsal aorta and pigmented cells. The kidney is divided into head, body, and tail regions (Figure 5). Dissect out a piece of the kidney and place it in PBS. Tease apart the kidney tissue to reveal the renal tubules. <img src="/files/ftp_upload/1717/1717fig5.jpg" alt="Flatworm anatomy diagram showing labeled head, body, tail; educational biology use." data-altupdatedat="1788235205000" data-alt="Figure 5"> Figure 5. Location of head, body, and tail kidney along the dorsal body wall. </li>
<li>Dissect a female fish. As described previously, euthanize the fish in ice water and pat it dry before pinning it to the dissecting mat. Remove the skin from the side of the fish as previously demonstrated. The ovary is a bilobed structure that is suspended in the body cavity by a vascularized mesovarium.</li>
<li>Remove one lobe of the ovary, place it in PBS, and examine it with transmitted light. The oocytes can be teased apart using fine needles and then staged (Figure 6, Selman et al., 1993). Stage I oocytes are approximately 10 - 150 microns in size and translucent. Stage II oocytes are about 150 - 350 microns in size and defined by the appearance of cortical granules. Stage III oocytes are 350 - 750 microns in size and are opaque due to the accumulation of yolk. Mature stage IV and V oocytes are translucent and generally not found in the ovaries of females that have recently mated. 
<img src="/files/ftp_upload/1717/1717fig6.jpg" alt="Colony morphology analysis, microscope image, shows distinct bacterial growth phases labeled I, II, III." data-altupdatedat="1788235205000" data-alt="Figure 6"> Figure 6. Live stage I, II, and III oocytes observed under a dissecting microscope with transmitted light. </li>
<li>Next, dissect the heart from the fish. The heart is located posterior and ventral to the gill. Begin by cutting out the heart and all of the surrounding tissue and placing it in PBS. Carefully dissect away the tissue surrounding the heart, being careful not to damage the delicate atrium.</li>
<li>Place the dissected heart into Ringer's solution to observe the heart beat. Identify the atrium, ventricle, and bulbus arteriosus (Figure 7, Hu et al., 2001). <img src="/files/ftp_upload/1717/1717fig7.jpg" alt="Fish heart anatomy diagram with labeled atrium, ventricle, and bulbus arteriosus." data-altupdatedat="1788235205000" data-alt="Figure 7"> Figure 7. Dissected adult heart. </li>
<li>Complete the dissection by removing the brain from the fish. Begin by unpinning the fish and removing the head with a razor blade. Remove as much soft tissue as possible from the ventral side of the skull with forceps. Remove the eyes using small spring scissors. Break open the skull and remove the bone from the ventral side of the brain. Now place the head in a dish of PBS and remove the skin and skull bones from the dorsal side of the brain.</li>
<li>Identify the olfactory bulbs, telencephalon, habenula, optic tectum, cerebellum and medulla (Figure 8, Wullimann et al., 1996; Schilling, 2002). <img src="/files/ftp_upload/1717/1717fig8.jpg" alt="Zebrafish brain anatomy diagram; labeled regions include olfactory bulb, cerebellum, medulla." data-altupdatedat="1788235205000" data-alt="Figure 8"> Figure 8. Dissected adult zebrafish brain. Dorsal view, anterior to top. </li>
</ol>

<ol><li>Finney, J. L., Robertson, G. N., McGee, C. A., Smith, F. M., Croll, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+Autonomic+Innervation+of+the+Swim+Bladder+in+the+Zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Structure and Autonomic Innervation of the Swim Bladder in the Zebrafish (Danio rerio).</a> J Comp Neurol. 495, 587-606 (2006).</li>
<li>Hu, N., Yost, H. J., Clark, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+morphology+and+blood+pressure+in+the+adult+zebrafish%5BTitle%5D)+AND+%22Anat+Rec%22%5BJournal%5D)">Cardiac morphology and blood pressure in the adult zebrafish.</a> Anat Rec. 264, 1-12 (2001).</li>
<li>Leal, M. C., Cardoso, E. R., Nóbrega, R. H., Batlouni, S. R., Bogerd, J., França, L. R., Schulz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histological+and+stereological+evaluation+of+zebrafish+%28Danio+rerio%29+spermatogenesis+with+an+emphasis+on+spermatogonial+generations%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.</a> Biol Reprod. 81, 177-187 (2009).</li>
<li>Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+morphology+of+larval+and+adult+zebrafish%5BTitle%5D)+AND+%22Zebrafish%3A+A+Practical+Approach+%28The+Practical+Approach+Series%29%22%5BJournal%5D)">The morphology of larval and adult zebrafish.</a> Zebrafish: A Practical Approach (The Practical Approach Series). Nusslein-Volhard, C., Dahm, R. , Oxford University Press. (2002).</li>
<li>Selman, K., Wallace, R., Sarka, A., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stages+of+oocyte+development+in+the+Zebrafish%2C+Brachydanio+rerio%5BTitle%5D)+AND+%22J.+Morphol%22%5BJournal%5D)">Stages of oocyte development in the Zebrafish, Brachydanio rerio.</a> J. Morphol. 218, 203-224 (1993).</li>
<li>Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+growth+and+differentiation+in+zebrafish%5BTitle%5D)+AND+%22Mech+Dev%22%5BJournal%5D)">Intestinal growth and differentiation in zebrafish.</a> Mech Dev. 122, 157-173 (2005).</li>
<li>Wullimann, M. F., Rupp, B., Reichert, H. Neuroanatomy of the zebrafish brain : a topological atlas. , Birkhäuser Verlag. (1996).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Pennsylvania Institutional Animal Care and Use Committee.

<table><tbody><tr><td colspan="4">Reagents:</td></tr><tr><td colspan="4">&lt;strong&gt;&lt;em&gt;0.2% Tricaine (ethyl 3-aminobenzoate)&lt;/em&gt;&lt;/strong&gt;&lt;br/&gt;200 mg tricaine powder&lt;br/&gt;97.9 ml DD water&lt;br/&gt;~1 ml 1 M Tris (pH 9)&lt;br/&gt;Adjust pH to 7.0</td></tr><tr><td colspan="4">&lt;strong&gt;&lt;em&gt;Phosphate buffered saline (PBS)&lt;/em&gt;&lt;/strong&gt;&lt;br/&gt;4.0 g NaCl&lt;br/&gt;0.1 g KCl&lt;br/&gt;100 ml 0.1 M PO&lt;sub&gt;4&lt;/sub&gt; Buffer, pH 7.3&lt;br/&gt;150 ml dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td colspan="4">&lt;strong&gt;&lt;em&gt;Ringer&amp;rsquo;s solution&lt;/em&gt;&lt;/strong&gt;&lt;br/&gt;6.7g NaCl&lt;br/&gt;0.2g KCl&lt;br/&gt;0.2g CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt;1.2g Hepes&lt;br/&gt;1 L H&lt;sub&gt;2&lt;/sub&gt;O &lt;br/&gt;Adjust pH to 7.2</td></tr><tr><td>Dissecting dish with mat</td><td>Electron Microscopy Sciences</td><td>70540</td><td></td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr></tbody></table>

dissection of organs from the adult zebrafish

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 ...

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease. Although the morphology of the embryo and larva has been well-documented, descriptions of adult zebrafish, anatomy and techniques for working with adults are lacking. Mary Mullin's lab at the University of Pennsylvania studies gonad development and routinely dissects ovaries and testes from adult fish.This protocol demonstrates dissection of the gonads, as well as identification and dissection of the other major organ systems. To begin, a fish is anesthetized and euthanized, and then pinned to a dissecting mat. The skin and underlying muscle are removed from the fish to expose the heart, liver, intestine, gonad, and swim bladder.The internal organs are removed from the body cavity. For further examination or experimentation, Tripti Gupta from the laboratory of Mary Mullins in the cell and developmental biology department at the University of Pennsylvania will identify many of the organs of the adult and demonstrate how to dissect them from an adult zebra fish. For clarity and ease of dissection, the testes gastrointestinal system and kidney will be dissected from a male zebra fish and then the ovaries, heart and brain will be dissected from a female.To reduce the amount of food in the intestine. Do not feed the fish the day prior to the dissection. A male zebra fish will be dissected.First, you'll need a dissecting mat, Venice Spring scissors forceps, dissecting pins, a dish of PBS and a fish that has been anesthetized and euthanized. Lightly pat the fish dry on a paper towel and place it on its side. On a dissecting mat, zebra fish have single dorsal coddle and anal fins and paired pelvic and pectoral fins.Use dissecting pins to pin the fish to the dissecting mat through the fleshy part of the tail and the ventral part of the eye socket. Using the spring scissors snip the skin on the belly of the fish just anterior to the anal fin. Cut the skin and underlying muscle along the belly from the anal fin to the hard covering over the gill called the operculom.Use the scissors and tweezers to cut out the operculom and the pectoral fin, including the thick bony region at the base of the fin called the pectoral girdle. Remove them to expose the gills. Cut the skin and underlying muscle beginning from above the exposed gill posteriorly along the side of the fish, and then down to the anal fin.Carefully remove the skin and underlying muscle from the side of the fish to expose the internal organs. The testes are long, white, paired organs that are attached to the dorsal body wall. Remove a testis and place it in a dish of room Temperature PBS Remove the fat that is associated with the testis.Examine the testis with reflected light under a dissecting microscope to visualize the seminiferous tubules, which contain cysts with various stages of developing germ cells from spermatogonia to spermatids. Next, remove the gastrointestinal system from the body cavity of the fish, the gallbladder, a green translucent fluid-filled sac, and the spleen, which appears bright red are found within the viscera. The liver can be identified by its large size lobed morphology, tanish color, and extensive vascularization.Separate the intestine from the rest of the organs and stretch it out. The anterior, mid and posterior regions of the intestine are defined by the height of the epithelial folds with the anterior being the highest. Place a piece of the intestine in PBS and observe the epithelial folds under the microscope using transmitted light.Now examine the swim bladder. The swim bladder consists of a posterior chamber, which is connected to the esophagus via the pneumatic duct, and an anterior chamber, which is connected to the inner ear through the Arian apparatus. Remove and discard the swim bladder, unpin the fish and rein it ventral side up to dissect the kidney.The kidney is a translucent pink structure located along the dorsal body wall and is associated with the dorsal aorta and pigmented cells. The kidney is divided into head, body, and tail regions. Okay, so there's the head, kidney, body, and the tail.This is the aorta And these are the pigmented cells. Dissect out a piece of the kidney and place it in PBS under the microscope. Tease apart the kidney tissue with the needles to reveal the renal tubules.For the dissection of a female fish, you will need the same tools as before with the addition of some fine needles, a razor blade, and a dish of ringer solution. For the heart dissection, pat the euthanized fish dry and pin it to the dissecting mat. As before, use the same techniques as before to remove the skin from the side of the female fish.The ovary is a bi lobe structure that is suspended in the body cavity by a vascularized me verium. Remove one lobe of the ovary and place it in PBS. Examine the ovary with incident light under the microscope.The cytes can be teased apart using fine needles, and then assigned to stages given their size and appearance. Next, dissect the heart from the fish. The heart is located posterior and ventral to the gill.Begin by cutting out the heart and all of the surrounding tissue and placing it in PBS under the microscope. Gently dissect away the tissue surrounding the heart, being careful not to damage the delicate atrium. Place the dissected heart into ringer solution and put it under the microscope.To observe the heartbeat, identify the atrium, ventricle, and bulbus arteriosis. To access the brain, unpin the fish and remove the head with the razor blade. Remove as much soft tissue as possible from the ventral side of the skull with forceps.Identify the optic chiasm and then remove the eyes using small spring scissors. Using the forceps, break, open the skull and remove the bone from the ventral side of the brain. Cut the cranial nerves with spring scissors.If necessary, move the head to a dish of PBS. Turn the head over and remove the skin and skull bones from the dorsal side of the brain under the microscope, identify the olfactory bulbs. Tell Cephalon, ULA optic tum, cerebellum, and magula.So here are the olfactory bulbs, The teal cephalon, the ula, the optic tum, the cerebellum and medulla, The heart, gastrointestinal system, gonads, swim, bladder, kidney, and brain. Were dissected from adult zebra fish. These organs can now be used for in C two hybridization, immunohistochemistry, histology, RNA, extraction or other molecular techniques.

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Learn dissection of adult zebrafish organs for developmental biology and comparative anatomy research. This protocol demonstrates techniques for isolating gonads, heart, liver, brain, and other major organ systems.

Dissection of Organs from the Adult Zebrafish

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