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 JoVE Biology

Dissection of Organs from the Adult Zebrafish

1, 1

1Department of Cell and Developmental Biology, University of Pennsylvania-School of Medicine

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    Summary

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

    Date Published: 3/04/2010, Issue 37; doi: 10.3791/1717

    Cite this Article

    Gupta, T., Mullins, M. C. Dissection of Organs from the Adult Zebrafish. J. Vis. Exp. (37), e1717, doi:10.3791/1717 (2010).

    Abstract

    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.

    Protocol

    1. 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.
    2. 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).

      Figure 1

      Figure 1. Adult male fish with fins labeled.
    3. Pin the fish to the dissecting mat through the fleshy part of the tail and the ventral part of the eye socket.
    4. 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).

      Figure 2

      Figure 2. Steps in removing skin and body wall muscle to expose internal organs.
    5. 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).
    6. Carefully remove the skin and underlying muscle from the side of the fish. Many of the internal organs are now visible (Figure 3).

      Figure 3

      Figure 3. Adult male fish with body wall and muscle removed allowing the visualization of the internal organs.
    7. 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).

      Figure 4

      Figure 4. Dissection of a testis. The white roundish structures are the seminiferous tubules.
    8. 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.
    9. 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).
    10. Place a piece of the intestine in PBS and observe the epithelial folds using transmitted light.
    11. 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).
    12. 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.

      Figure 5

      Figure 5. Location of head, body, and tail kidney along the dorsal body wall.
    13. 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.
    14. 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.

      Figure 6

      Figure 6. Live stage I, II, and III oocytes observed under a dissecting microscope with transmitted light.
    15. 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.
    16. 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).

      Figure 7

      Figure 7. Dissected adult heart.
    17. 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.
    18. Identify the olfactory bulbs, telencephalon, habenula, optic tectum, cerebellum and medulla (Figure 8, Wullimann et al., 1996; Schilling, 2002).

      Figure 8

      Figure 8. Dissected adult zebrafish brain. Dorsal view, anterior to top.

    Disclosures

    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.

    Acknowledgements

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

    Materials

    Name Company Catalog Number Comments
    Reagents:
    0.2% Tricaine (ethyl 3-aminobenzoate)
    200 mg tricaine powder
    97.9 ml DD water
    ~1 ml 1 M Tris (pH 9)
    Adjust pH to 7.0
    Phosphate buffered saline (PBS)
    4.0 g NaCl
    0.1 g KCl
    100 ml 0.1 M PO4 Buffer, pH 7.3
    150 ml dH2O
    Ringer’s solution
    6.7g NaCl
    0.2g KCl
    0.2g CaCl2
    1.2g Hepes
    1 L H2O
    Adjust pH to 7.2
    Dissecting dish with mat Electron Microscopy Sciences 70540
    Vannas spring scissors Fine Science Tools 91500-09

    References

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

    Comments

    12 Comments

    Dear
    Tripti & Mary
    Thanks for such a nice publication. I will be highly obliged if you could send me a full text of this article. I am really impressed by your presentation here.
    I shall be thankful to you
    Cheers
    Avdesh
    Reply

    Posted by: AnonymousMarch 21, 2010, 4:42 AM

    Dear Avdesh,
    Thanks! The article is now OPEN Access, so you should be able to access everything online.
    Mary
    Reply

    Posted by: AnonymousJune 29, 2010, 9:57 PM

    Congratulation for this well done video. It will be very useful for my students.
    Carla Lucini
    Reply

    Posted by: carla l.June 9, 2010, 3:15 AM

    Thank you very much for this very useful presentation.
    Reply

    Posted by: AnonymousJuly 16, 2010, 11:57 AM

    Dear
    Tripti & Mary
    First of all congratulations for this well done video..
    My questions are:
    The microscopic aspects of the kidney are similar to human? glomeruli, tubules, interstitium? which part of the kidney should I analyze?
    Thanks
    Antonio
    Reply

    Posted by: michele b.August 24, 2010, 9:56 AM

    Dear colleagues Tripti and Mary,
    Thank you very much for your higly professional anatomical and histological investigation as video. It will be very usefull for one of my doctorants.
    But I have got a question: were they wild zebrafish or transgenic ones?
    Sincerely yours,
    Prof. Mikodina Ekaterina Victorovna, Russian Federal Research Institute of Fisheries and Oceanography, Moscow
    Reply

    Posted by: AnonymousAugust 2, 2011, 7:32 AM

    They were wild-type zebrafish.
    Mary
    Reply

    Posted by: AnonymousAugust 2, 2011, 12:22 PM

    Dear Tripti & Mary:
    Thanks for this great work, this is really usefull to me, because i begin my Phd Studies and i will work with zebrafish.
    Regards from Chile!.
    Mario Caruffo.
    Reply

    Posted by: AnonymousAugust 28, 2011, 1:51 AM

    Tripti and Mary,

    Thank you for posting this very helpful video. I am considering producing Zebrafish antibodies and this organ dissection study demonstrates that the process of tissue extraction can be done quickly and easily with trained hands.
    Many thanks for your detailed and thorough walk through.
    Reply

    Posted by: AnonymousSeptember 26, 2011, 5:28 PM

    Dear Tripti and Mary,
    Let me congratulate first...for this great job,
    Know I am confident that we can start our research with Zebra fish.
    Thanks a lot for sharing your knowledge and experience.
    regards
    Rakhi
    Reply

    Posted by: AnonymousFebruary 1, 2012, 6:43 AM

    MOHAMED ASIK R


    Thank you for posting this video, I need this video for dissection of zebra fish for toxicity studies. So i pleased to you for sent this video for my research work.
    Thank you.
    Reply

    Posted by: MOHAMED A.January 24, 2014, 7:23 AM

    Hello,

    I am trying to identify the gonads in zebrafish and I am wondering at what magnification are figures 4 (testis) and 5 (stages of oocyte) shown.

    Thank you.
    Reply

    Posted by: Jevi A.May 12, 2014, 9:59 PM

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