剖析器官的成年斑马鱼

Published 3/04/2010
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Biology
 

Summary

本协议描述了识别和解剖成年斑马鱼的器官的过程。

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

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Abstract

在过去的20年中,斑马鱼已成为一个强大的模式生物,为了解脊椎动物的发育和疾病。虽然是广泛的胚胎和幼虫的实验分析和形态一直都有详细记载,成年斑马鱼的解剖和发展成人的结构和器官的研究说明,连同与成人工作的技术,所缺乏的。幼虫机关接受在其整体结构,形态,解剖位置,并在向成年过渡的幼虫的重大变化。从外部看,透明的幼虫发展其特点成人条纹色素模式和配对腹鳍,而在内部,机关经过大量增长和重塑。此外,bipotential性腺原基发展成为无论是睾丸或卵巢。该协议确定成人的许多器官和演示脑,性腺,肠胃系统,心脏,和成年斑马鱼的肾脏解剖的方法。可用于原位杂交,分子生物学技术,组织学,免疫组织化学法提取RNA,蛋白质分析,和其他解剖器官。该协议将有助于在斑马鱼在扩大研究范围,包括幼虫器官的重塑,成人器官系统的成年和其他调查机关的具体形态发生。

Protocol

  1. 将一只雄性斑马鱼解剖第一,其次是雌鱼。开始剥离之前,麻醉在0.2%Tricaine的鱼,然后安乐死在冰水中的潜伏期为15分钟。
  2. 首先,轻轻拍着干纸巾上的鱼放在解剖垫。对外,斑马鱼有单一的背鳍,尾鳍和臀鳍和成对的胸鳍和腹鳍(图1)。

    图1

    图1。成人男性标有鳍鱼类。
  3. 引脚通过解剖垫鱼尾巴的肉质部分和眼圈腹侧部分。
  4. 剪断腹鱼的臀鳍前的皮肤。剪切沿着腹部的皮肤和下面的肌肉从臀鳍到鳃盖(硬覆盖在鳃)(图2,第1步)。

    图2

    图2,在去除皮肤和体壁肌肉暴露内部器官的步骤。
  5. 接下来,删除的鳃盖和胸鳍,包括肩带(厚,鳍基骨的区域)(图2,第2步)。切沿一侧的鱼,从现在暴露的鳃后部皮肤及以上潜在的肌肉开始又回落到臀鳍(图2,第3步)。
  6. 小心取出从一侧的鱼的皮肤和下面的肌肉。现在许多内部器官可见(图3)。

    图3

    图3。成年雄鱼体壁肌肉除去,使机关内部的可视化。
  7. 睾丸长,白色,配对,连接到背体壁机关。取出睾丸和地方菜的PBS。反射光检查睾丸曲细精管(图4),其中包含各个阶段发展spermatagonia到精子的生殖细胞囊肿(莱亚尔等,2009)可视化。

    图4

    图4。睾丸解剖。白色圆形的结构,曲细精管。
  8. 下一步,清除肠胃系统从鱼的体腔。规模大,叶状形态,tannish颜色,和广泛的血管,肝脏,可确定。胆囊,一个绿色的半透明的充满液体的囊,脾,出现鲜红色,内脏内被发现。
  9. 从其他机关单独小肠和伸展出来。高度上皮褶皱(Wallace等人,2005年)的前,中,后肠道内的地区被定义。
  10. 将一块在PBS肠道和使用透射光观察上皮褶皱。
  11. 接下来,检查的鱼鳔。鳔后房型通过气动管,和前房通过韦伯器具(芬尼等人,2006年),它是连接到内耳,它是连接到食道。
  12. 删除和丢弃的鱼鳔。不固定的鱼,并重新针它的腹侧方解剖肾,这是位于沿背侧体壁。肾脏是一种半透明的粉红色的结构与背主动脉和色素细胞相关。肾脏分为头部,身体和尾部区域(图5)。解剖出一块肾脏和地方的PBS。梳理除了肾组织透露肾小管。

    图5

    图5。头部,身体和尾部沿肾脏背侧体壁的位置。
  13. 解剖雌鱼。如前所述,安乐死在冰水中的鱼和寄托解剖垫之前,帕特干燥。正如先前所表现出的鱼从一侧取出皮肤。卵巢是一个bilobed结构,悬浮体腔血管卵巢系膜。
  14. 取出卵巢叶,在PBS的地方,并与透射光检查。卵母细胞可以被戏弄除了使用细针,然后上演(图6中,塞尔曼等,1993年)。舞台上我的卵母细胞大约有10 - 150微米的大小和半透明。第二阶段的卵母细胞约150 - 350微米大小和皮质颗粒的外观定义。第三阶段卵母细胞有350 - 750微米大小,由于卵黄的积累,是不透明的。成熟阶段IV和V的卵母细胞是半透明的,一般不会在最近交配的女性卵巢中发现。

    图6

    图6现场舞台下观察我,二,三卵母细胞解剖显微镜透射光。
  15. 接下来,解剖鱼的心脏。心脏位于鳃后,腹。首先切出心脏和周围组织,并把它在PBS。仔细剖析了心脏周围的组织,小心不要损伤娇嫩的心房。
  16. 将解剖林格氏液,观察心脏跳动的心脏。
    确定的心房,心室和bulbus动脉(图7,胡锦涛等,2001)。

    图7

    图7。解剖成人心脏。
  17. 完成去除鱼的大脑解剖。 unpinning鱼,用刀片去除头部开始。尽可能从取出的头骨与腹侧方钳尽可能多的软组织。取出的眼睛,用小弹簧剪刀。破开的头骨和消除大脑的腹侧骨。现在放置在头菜的PBS和消除大脑背侧皮肤和头骨。
  18. 确定的嗅球,端脑,缰,视神经顶盖,小脑和延髓(图8,Wullimann等,1996;席林,2002)。

    图8

    图8。成年斑马鱼的大脑解剖。背视图,前顶。

Disclosures

动物实验是由宾夕法尼亚机构动物护理和使用委员会大学规定的准则和法规的规定执行。

Acknowledgements

这项工作是由美国国立卫生研究院授予R01 HD050901 MCM和美国癌症协会博士后奖学金#PF - 05 - 041 - 01 - DDC到TG的支持。

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

DOWNLOAD MATERIALS LIST

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

13 Comments

  1. 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: Anonymous
    March 21, 2010 - 4:42 AM
  2. Dear Avdesh,
    Thanks! The article is now OPEN Access, so you should be able to access everything online.
    Mary

    Reply
    Posted by: Anonymous
    June 29, 2010 - 9:57 PM
  3. 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
  4. Thank you very much for this very useful presentation.

    Reply
    Posted by: Anonymous
    July 16, 2010 - 11:57 AM
  5. 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
  6. 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: Anonymous
    August 2, 2011 - 7:32 AM
  7. They were wild-type zebrafish.
    Mary

    Reply
    Posted by: Anonymous
    August 2, 2011 - 12:22 PM
  8. 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: Anonymous
    August 28, 2011 - 1:51 AM
  9. 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: Anonymous
    September 26, 2011 - 5:28 PM
  10. 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: Anonymous
    February 1, 2012 - 6:43 AM
  11. 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
  12. 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
  13. thank you for posting this video
    , it was very helpful for my research work

    Reply
    Posted by: Anju t.
    June 15, 2015 - 1:32 PM

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