Notochord Injury Assays that Stimulate Transcriptional Responses in Zebrafish Larvae
诱导幼年期斑马鱼转录响应的脊索损伤实验    

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eLIFE
Feb 2018

 

Abstract

Zebrafish have become an increasingly important model organism in the field of wound healing and regenerative medicine, due to their high regenerative capacity coupled with high-resolution imaging in living animals. In a recent study, we described multiple physical and chemical methods to induce notochord injury that led to highly specific transcriptional responses in notochord cellular subpopulations. The notochord is a critical embryonic structure that functions to shape and pattern the vertebrae and spinal column. Here, we describe precision needle injury, tail-notochord amputation, and chemical inhibition of caveolin that trigger a wound-specific wt1b expression response in the notochord sheath cell subpopulation. We propose that these procedures can be used to study distinct cell populations that make up the cellular processes of notochord repair.

Keywords: Zebrafish (斑马鱼), Notochord (脊索), Tail fin (尾鳍), Injury (损伤), Amputation (切断术), Tissue repair (组织修复), Tungsten wire (钨丝), Nystatin (制霉菌素)

Background

The notochord is a transient embryonic structure that provides axial support and signaling information to the developing embryo (Ellis et al., 2013). It is comprised of two structurally distinct cell populations: the inner vacuolated cells that provide embryo support and structure, and the outer sheath cells that maintain turgor pressure for the vacuolated cells as well as patterning the developing vertebrate spine (Wopat et al., 2018; Lleras Forero et al., 2018; Figure 1A). We have recently discovered that wilms' tumor 1b (wt1b) is specifically expressed in a notochord sheath cell subpopulation that emerges at the site of damage and is maintained throughout repair and formation of adult vertebra structure in zebrafish (Figures 1B and 1C; Lopez-Baez et al., 2018). WT1 is a zinc-finger transcription factor involved in mesodermal tissue development, adult tissue homeostasis, and becomes reactivated during epicardial tissue damage (Hastie et al., 2017). Our discovery that wt1b becomes expressed at the notochord wound may have important implications for the development of therapies for vertebrae spinal injuries or degenerative processes.

In zebrafish wounding and regeneration models, injury is induced by a variety of methods such as amputation, surgical resection, irradiation, laser ablation and genetic ablation (Gemberling et al., 2013). For example, in larval zebrafish, syringe needles of various sizes have been used for tail fin amputation and spinal cord injury experiments (Lisse et al., 2015; Wehner et al., 2017).

We have conducted notochord injury assays in zebrafish larvae using physical and chemical approaches (Lopez-Baez et al., 2018). Electrolysis-sharpened tungsten wire and insect pins described here and in our recent paper induce precise, localized injury and trigger wound-specific wt1b expression (Figures 1B and 1C). The structural integrity of the notochord can also be disrupted chemically by treating embryos with nystatin, a small molecule which binds sterols and disassembles caveolae (Rothberg et al., 1992), which are particularly abundant in the notochord (Lim et al., 2017). We detected increased wt1b expression in nystatin treated notochords suggesting changes in caveolae caused by non-physical damage and stress may also induce wt1b expression.


Figure 1. Cell populations of the notochord and the wt1b notochord wound response. A. Schematic of the cell populations of the notochord. The notochord is comprised of two physically distinct cell populations: an epithelial-like notochord sheath cell population (outer cells; red) and a large vacuolated notochord cell population (inner cells, green), which are tightly wrapped by a thick, elastic extracellular basement membrane (peri-notochordal sheath). B. Schematic of the zebrafish embryo and the site of the notochord wound at the end of the yolk sac (YS). C. Needle injury triggers localized wt1b:gfp expression in the notochord at the site of damage by 24 h post injury (hpi; arrow). Scale bars = 100 µm in Panel C.

Materials and Reagents

  1. Ø 0.25 mm Tungsten wire (Alfa Aesar, catalog number: 010073.G2)
  2. Metal needle holder (VWR International Ltd. UK, catalog number: MURRL110/01)
  3. Sterile scalpel blade (Swann Morton UK, catalog number: 11708353)
  4. 0.10 mm Austerlitz insect pins, stainless steel (Fine Science Tools, catalog number: 26002-10)
  5. Glass Pasteur Pipettes length: 145 mm (Brand, catalog number: 7477 15)
  6. Petri dish (Thermo Scientific, catalog number: 15370366)
  7. 96-well plate
  8. Zebrafish larvae (3-7 days post fertilization)
    Non-pigmented mitfa mutant (nacre allele) (Lister et al., 1999) may be preferable for ease of imaging. For our experiments, we used the Tg(R2col2a1a:mCherry) transgenic line to visualize notochord sheath cells (Dale and Topczewski, 2011), and the Tg(wt1b:gfp) line to study the wound response in the notochord (Perner et al., 2007).
    Important: Zebrafish older than 5 days post fertilization are protected animals by UK and EU law and require proper animal procedure licenses and approval from institutional ethics committees.
  9. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650-100ML)
  10. Nystatin (Sigma-Aldrich, catalog number: N6261-500KU)
  11. Agarose (Invitrogen, catalog number: 15510-027)
  12. Tricaine (MS-222, 3-amino benzoic acid ethyl ester, Sigma, catalog number: A-5040)
  13. NaOH pellets (Sigma-Aldrich, catalog number: S8045-500G)
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-1KG)
  15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G)
  16. Calcium chloride dihydrate (CaCl2•2H2O) (Sigma-Aldrich, catalog number: 223506-500G)
  17. Magnesium chloride hexahydrate (MgCl2•6H2O) (Sigma-Aldrich, catalog number: M2393-500G)
  18. E3 embryo medium (60x stock solution) (see Recipes)
  19. Tricaine (MS-222) (1x working solution) (see Recipes)
  20. NaOH (5 M) (see Recipes)

Equipment

  1. 250 ml glass bottle
  2. Forceps
  3. Microwave oven
  4. Incubator
  5. Bunsen burner
  6. Microscopes:
    Upright Stereomicroscope (e.g., Nikon, model: SM2645)
    Light microscope (e.g., Olympus, model: SZX16)
    Confocal microscope (e.g., Nikon, model: A1R)
  7. Electrolysis device for tungsten wire sharpening (the device shown in this protocol is custom-made and no longer in production; Figure 2A)
    However, users can set up their own equipment.
    1. Parts required are: a) DC power supply (3-20 V, such as Bosch C3 smart car battery charger, catalog number: 0092C35000), b) carbon electrode rod (Lasec, catalog number: ERDI9470), c) crocodile clips (optional, DC power supply may come with leads with crocodile clips), d) a 200 ml glass container (jar) with lid. 
    2. Assemble the equipment: Plug the leads into the charger. Using crocodile clips, connect the carbon electrode rod to the negative terminal (black lead), connect the metal holder to the positive terminal (red lead). Set the voltage to 6 V and fill up the jar with 100 ml of NaOH solution. The equipment is now ready for use. 


    Figure 2. Preparation of electrolysis-sharpened needle. A. Electrolysis-based apparatus, consisting of a rectified DC transformer, an anode with crocodile clip (red), a cathode with carbon electrode (yellow), electrolysis chamber with 5 M NaOH electrolyte. B-C. Lateral and dorsal views of a tungsten wire needle being sharpened. With the power on (6 V), the mounted needle is held vertically and dipped into and out of the electrolyte steadily and slowly until the desired tip is achieved. D. A finished electrolysis-sharpened tungsten wire needle.

Procedure

  1. Notochord needle injury of zebrafish larvae
    1. Prepare electrolysis-sharpened tungsten wire (adapted from Brady, 1965) (Figure 2 and Video 1)
      1. Cut off 3 cm of tungsten wire and mount it into a needle holder. 
      2. Connect the metal handle of the needle holder to the (+) terminal of the transformer using a crocodile clip, connect the cathode with carbon plate to the (-) terminal.
      3. Place the carbon electrode in the glass chamber with 100 ml of 5 M NaOH solution and switch on the transformer, set the output voltage to 6 V. With the mounted needle held vertically, dip the needle into and out of NaOH, slowly and steadily until desired tip is produced. Faster movement = Longer slope on needle, Slower movement = Shorter tip with more angled slope. The dial gauge of current reads between 0 and 1 A when the needle moves up and down. It takes about 2.5 min to sharpen a needle from 0.25 mm to 0.02 mm in diameter. 

      Video 1. Sharpening tungsten wire

    2. Alternatively, 0.1 mm insect pins can also be used to injure the notochord (Figure 3 and Video 2)
      1. Take a clean glass pipette and using a Bunsen burner, bend the thin side in the middle in order to create a 45-degree angle. This will help with the injury manipulation procedure.
      2. Close the hole of the thin side by about three-quarters using the Bunsen burner. This is done by placing the tip in the strongest part of the flame and rotating it in a circular manner. 
      3. Take the insect pin with forceps and place it in the hole, taking care that the sharp side is facing the outside.
      4. Carefully continue burning the tip of the glass pipette in the weakest part of the flame, until the hole is closed and the insect pin secure. The insect pin will burn if exposed to too much heat. Try to close the hole as much as possible before inserting the pin.


      Figure 3. Preparation of insect pin. A. The required equipment. B. The finished instrument. C. A close up image of the tip.

      Video 2. Preparing insect pin

    3. Prepare 1.5% agarose
      Weight 1.5 g agarose and melt in 100 ml E3 embryo medium in a 250 ml glass bottle using a microwave oven, pour a thin layer into a Ø 90 mm Petri dish. About 20 ml of 1.5% agarose is used. Let the agarose solidify.
    4. Anaesthetize larvae in tricaine solution
      Prepare 1:10,000 tricaine solution using E3 embryo medium (please see Recipes), and pour 30 ml in a separate Ø 90 mm Petri dish. Transfer one larva into the tricaine solution and wait until it is anesthetized.
    5. Under a stereomicroscope, place one larva on its side on a Petri dish coated with agarose so that the lateral side can be accessed with needle from above. Remove as much liquid as possible so that the surface tension adheres the larvae to the dish and prevents it from slipping. Gently insert the tip of the tungsten wire into the notochord vertically at the level of the end of the yolk sac (Figure 1B), then withdraw the wire. (Video 3)
    6. Transfer injured larvae to a Petri dish with fresh E3 medium to recover and place the dish at 28.5 °C to grow the larvae to the desired stages. Keep uninjured age-matched larvae as non-injured controls.

      Video 3. Notochord Needle Injury

  2. Chemically-induced disruption of notochord
    1. Cross fish carrying the Tg(wt1b:GFP) and the notochord-marking Tg(R2-col2a1a:mCherry) transgenes in an unpigmented nacre-/- background, to obtain Tg(wt1b:GFP;R2-col2a1a:mCherry);nacre-/- embryos.
    2. Prepare fresh 5 mg/ml nystatin stock solution (5.4 mM) before each use by dissolving in DMSO. 
    3. Dilute nystatin stock solution in E3 to obtain 20 μM final working concentration. Add this to dechorionated 48 hpf embryos in a 6-well plate. Add 0.4% DMSO to control embryos.
    4. Incubate embryos at 28.5 °C for up to 48 h. After 24 h of nystatin treatment, lesions appear along the length of the notochord. They tend to appear first in regions that are naturally compressed as the embryo moves, and then spread along the length of the notochord. The majority of embryos acquire notochord lesions, however their size and severity can be variable. Therefore, regular screening for lesions and/or the onset of wt1b:GFP expression is recommended in order to identify embryos with the desired level of notochord damage.
    5. For imaging, anesthetize embryos in tricaine (1:10,000), and mount sagittally in 1% low-melt agarose. Brightfield images are taken using a light microscope (Figures 4A and 4B). Expression of the R2-col2a1a:mCherry transgene, which marks the notochord, and the induction of the wt1b:GFP transgene at sites of notochord damage is visualized using confocal microscopy (Figures 4A’ and 4B’’).


      Figure 4. Disrupting notochord structure using nystatin (modified from Lopez-Baez, 2018). Nystatin is a small molecule which binds sterols and leads to the disassembly of caveolae, a component abundant in the notochord (Lim et al., 2017). Tg(wt1b:GFP;R2-col2a1a:mCherry);nacre-/- zebrafish embryos are treated with either DMSO or 20 μM nystatin from 48 hpf to 72 hpf. When observed under a light microscope, the notochord structure of (A) DMSO-treated embryos appears normal, however lesions can be observed in (B) nystatin-treated embryos. (A’ and B’) wt1b:GFP expression is induced at lesion sites, but not in control notochords. R2-col2a1a:mCherry expression in notochord sheath cells also shows increased cellularity at (B’’) lesion sites of nystatin-treated embryos compared to (A’’) DMSO controls. Scale bars are 50 μm.

  3. Tail amputation
    1. Prepare 1.5% agarose using E3 embryo medium and pour a thin layer into a Petri dish. Let the agarose solidify.
    2. Anaesthetize larvae in tricaine solution.
    3. Under a stereomicroscope, place one larva on its side onto the solidified agarose. Remove as much as liquid as possible, so the surface tension adheres the larvae to the dish and prevents it from slipping, then amputate the tail with a sterile scalpel blade with slight pressure. Amputation sites are dependent on experiments being performed (Figure 5). Amputations at the tail fin and tip of notochord site do not include notochord tissue, and do not stimulate a wt1b:gfp notochord injury response. Amputations beyond the tail fin and into the notochord (before caudal vein, past caudal vein) stimulate a wt1b:gfp expression.


      Figure 5. Selected tail amputations uncover the notochord specificity of the response (modified from Lopez-Baez, 2018). A. Illustration of tail amputations at different tail sites and time points when images are taken. Tail fin (TF), tip of the notochord (TN), before caudal vein (BCV), past caudal vein (PCV), somite (S), notochord (N), caudal vein (CV). B. TF and TN amputated larvae showed no GFP upregulation in their notochord after the injury, but show marked fin regeneration (arrow head). BCV and PCV amputated groups both showed strong GFP upregulations by 72 hpa (arrows), with PCV amputated larvae showing an overall stronger and faster upregulation than BCV amputated larvae.

    4. Transfer injured larvae to a Petri dish with fresh E3 medium to recover, and place the dish at 28.5 °C to grow the larvae to the desired stages. Keep uninjured age-matched larvae as non-injured controls.

Notes

  1. In the UK and EU, all animal procedures need to be approved by the Home Office (UK) or its equivalent. Appropriate Personal Project License (PPL) and Personal individual License (PIL) are required.
  2. The procedure of notochord needle injury requires a fair amount of practice, and care should be taken to not cause injury outside the needle injury site. The appearance of a small bulge structure at the site of injury within the notochord about 5 min post-surgery indicates a successful operation. It is achievable to injure 30 larvae during a period of an hour.
  3. Optimizing nystatin dosage for the first-time use is recommended, as there is batch-to-batch variation. A longer nystatin incubation period can be attempted however the adverse off-target effects of nystatin cause gross developmental abnormalities and embryos do not survive long-term. In our hands, treatment with 20 μM nystatin from 48 hpf for 24 h gives the most consistent results. Treatment with nystatin before 48 hpf is possible, however, due to its off-target effects, more toxicity is seen. Embryos tolerate later nystatin treatment from 72 and 96 hpf much better, with 20 μM nystatin producing notochord lesions in 60%-80% of embryos after 24 h, although these lesions are smaller in size and fewer per embryo compared those shown in Figure 4.

Recipes

  1. E3 embryo medium (60x stock solution)
    17.4 g NaCl
    0.8 g KCl
    2.9 g CaCl2•2H2O
    4.89 g MgCl2•6H2O
    Dissolve in 1 L H2O
  2. Tricaine (MS-222) (1x working solution)
    Dissolve 0.1 g of Tricaine powder in 1 L of 1x E3 medium, adjust pH to 7.0
  3. NaOH (5 M)
    Dissolve 200 g NaOH pellets in 1 L H2O

Acknowledgments

Funding sources: MRC Human Genetics Unit Programme (MC_PC_U127585840, MC_PC_U127527180), European Research Council (ZF-MEL-CHEMBIO-648489) and L'Oreal-Melanoma Research Alliance (401181), Cells in Motion - Cluster of Excellence (EXC 1003-CiM). These procedures are adapted from our recent eLife paper (Lopez-Baez et al., 2018)

Competing interests

The authors have no competing interests.

Ethics

Procedures presented here were approved by the University of Edinburgh Ethics Committee, and performed under the Home Office Project License 70/8000 to EEP at the University of Edinburgh, United Kingdom; and by the Animal Experimentation Committee (DEC) of the Royal Netherlands Academy of Arts and Sciences to SSM at the Hubrecht Institute and the Institute of Cardiovascular Organogenesis and Regeneration WWU Münster, Germany.

References

  1. Brady, J. (1965). A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically. Bull World Health Organ 32(1): 143-144.
  2. Dale, R. M. and Topczewski, J. (2011). Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene. Dev Biol 357(2): 518-531.
  3. Ellis, K., Hoffman, B. D. and Bagnat, M. (2013). The vacuole within: how cellular organization dictates notochord function. Bioarchitecture 3(3): 64-68.
  4. Gemberling, M., Bailey, T. J., Hyde, D. R. and Poss, K. D. (2013). The zebrafish as a model for complex tissue regeneration. Trends Genet 29(11): 611-620.
  5. Hastie, N. D. (2017). Wilms' tumour 1 (WT1) in development, homeostasis and disease. Development 144(16): 2862-2872.
  6. Lim, Y. W., Lo, H. P., Ferguson, C., Martel, N., Giacomotto, J., Gomez, G. A., Yap, A. S., Hall, T. E. and Parton, R. G. (2017). Caveolae protect notochord cells against catastrophic mechanical failure during development. Curr Biol 27(13): 1968-1981 e1967.
  7. Lisse, T. S., Brochu, E. A. and Rieger, S. (2015). Capturing tissue repair in zebrafish larvae with time-lapse brightfield stereomicroscopy. J Vis Exp (95): 52654.
  8. Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. and Raible, D. W. (1999). Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126(17): 3757-3767.
  9. Lleras Forero, L., Narayanan, R., Huitema, L. F., VanBergen, M., Apschner, A., Peterson-Maduro, J., Logister, I., Valentin, G., Morelli, L. G., Oates, A. C. and Schulte-Merker, S. (2018). Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock. Elife 7: e33843.
  10. Lopez-Baez, J. C., Simpson, D. J., L, L. L. F., Zeng, Z., Brunsdon, H., Salzano, A., Brombin, A., Wyatt, C., Rybski, W., Huitema, L. F. A., Dale, R. M., Kawakami, K., Englert, C., Chandra, T., Schulte-Merker, S., Hastie, N. D. and Patton, E. E. (2018). Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair. Elife 7: e30657.
  11. Perner, B., Englert, C. and Bollig, F. (2007). The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros. Dev Biol 309(1): 87-96.
  12. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R. and Anderson, R. G. (1992). Caveolin, a protein component of caveolae membrane coats. Cell 68(4): 673-682.
  13. Wehner, D., Tsarouchas, T. M., Michael, A., Haase, C., Weidinger, G., Reimer, M. M., Becker, T. and Becker, C. G. (2017). Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish. Nat Commun 8(1): 126.
  14. Wopat, S., Bagwell, J., Sumigray, K. D., Dickson, A. L., Huitema, L. F. A., Poss, K. D., Schulte-Merker, S. and Bagnat, M. (2018). Spine patterning is guided by segmentation of the notochord sheath. Cell Rep 22(8): 2026-2038.

简介

斑马鱼已经成为伤口愈合和再生医学领域中越来越重要的模式生物,因为它们具有高再生能力以及活体动物的高分辨率成像。 在最近的一项研究中,我们描述了多种物理和化学方法来诱导脊索损伤,从而在脊索细胞亚群中产生高度特异性的转录反应。 脊索是一种关键的胚胎结构,其功能是塑造和模拟椎骨和脊柱。 在这里,我们描述精确针头损伤,尾部 - 脊索截肢和小窝蛋白的化学抑制,其触发脊索鞘细胞亚群中的伤口特异性 wt1b >表达反应。 我们建议这些程序可用于研究构成脊索修复细胞过程的不同细胞群。

【背景】脊索是一种短暂的胚胎结构,为发育中的胚胎提供轴向支持和信号传递信息(Ellis et al。>,2013)。它由两个结构不同的细胞群组成:内部空泡细胞提供胚胎支持和结构,外鞘细胞维持空泡细胞的膨胀压力以及发育脊椎动物脊柱的模式(Wopat 等。 >,2018; Lleras Forero et al。>,2018;图1A)。我们最近发现 wilms'肿瘤1b >( wt1b >)特异性表达于脊髓鞘细胞亚群中,该亚群出现在损伤部位并在整个修复和形成过程中得到维持。斑马鱼中的成年椎骨结构(图1B和1C; Lopez-Baez 等人>,2018)。 WT1是锌指转录因子,参与中胚层组织发育,成体组织稳态,并在心外膜组织损伤期间重新激活(Hastie et al。>,2017)。我们发现 wt1b >在脊索伤口中表达可能对椎骨脊髓损伤或退行性过程的治疗发展具有重要意义。

在斑马鱼创伤和再生模型中,通过各种方法诱发损伤,例如截肢,手术切除,照射,激光消融和基因消融(Gemberling et al。>,2013)。例如,在幼虫斑马鱼中,各种大小的注射器针头已被用于尾鳍截肢和脊髓损伤实验(Lisse et al。>,2015; Wehner et al。> ,2017)。

我们使用物理和化学方法对斑马鱼幼虫进行了脊索损伤检测(Lopez-Baez et al。>,2018)。在此处和我们最近的论文中描述的电解尖锐的钨丝和昆虫针诱导精确的局部损伤并触发伤口特异性的 wt1b >表达(图1B和1C)。脊索的结构完整性也可以通过用制霉菌素处理胚胎进行化学破坏,制霉菌素是一种结合甾醇和分解细胞膜穴样内陷的小分子(Rothberg et al。>,1992),它们在脊索中特别丰富( Lim et al。>,2017)。我们检测到制霉菌素处理的脊索中 wt1b >的表达增加,表明由非物理损伤和应激引起的细胞膜穴样内陷的变化也可能诱导 wt1b >表达。


图1.脊索的细胞群和 wt1b >脊索伤口反应。 A.脊索细胞群的示意图。脊索由两个物理上不同的细胞群组成:上皮样的脊索鞘细胞群(外细胞;红色)和大的空泡脊索细胞群(内细胞,绿色),由厚的弹性细胞外基底紧紧包裹膜(peri-chochordal鞘)。 B.斑马鱼胚胎的示意图和卵黄囊末端的脊索伤口部位(YS)。 C.针损伤在损伤后24小时(hpi;箭头)处在损伤部位的脊索中触发局部的 wt1b:gfp >表达。面板C中的比例尺=100μm

关键字:斑马鱼, 脊索, 尾鳍, 损伤, 切断术, 组织修复, 钨丝, 制霉菌素

材料和试剂

  1. Ø0.25毫米钨丝(Alfa Aesar,目录号:010073.G2)
  2. 金属针座(VWR International Ltd. UK,目录号:MURRL110 / 01)
  3. 无菌手术刀片(Swann Morton UK,目录号:11708353)
  4. 0.10 mm Austerlitz昆虫针,不锈钢(精细科学工具,目录号:26002-10)
  5. 玻璃巴斯德吸管长度:145 mm(品牌,目录号:7477 15)
  6. 培养皿(Thermo Scientific,目录号:15370366)
  7. 96孔板
  8. 斑马鱼幼虫(受精后3-7天)
    非着色的 mitfa >突变体( nacre >等位基因)(Lister et al。>,1999)可能是优选的,以便于成像。对于我们的实验,我们使用Tg(R2 col2a1a:mCherry >)转基因品系来显示脊索鞘细胞(Dale和Topczewski,2011)和Tg( wt1b:gfp > )研究脊索中的伤口反应(Perner et al。>,2007)。
    重要提示:受精后5天以内的斑马鱼是受英国和欧盟法律保护的动物,需要获得适当的动物程序许可并获得机构伦理委员会的批准。>
  9. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650-100ML)
  10. 制霉菌素(Sigma-Aldrich,目录号:N6261-500KU)
  11. 琼脂糖(Invitrogen,目录号:15510-027)
  12. 三卡因(MS-222,3-氨基苯甲酸乙酯,Sigma,目录号:A-5040)
  13. NaOH颗粒(Sigma-Aldrich,目录号:S8045-500G)
  14. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653-1KG)
  15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  16. 氯化钙二水合物(CaCl 2 •2H 2 O)(Sigma-Aldrich,目录号:223506-500G)
  17. 氯化镁六水合物(MgCl 2 •6H 2 O)(Sigma-Aldrich,目录号:M2393-500G)
  18. E3胚胎培养基(60x储备液)(见食谱)
  19. 三卡因(MS-222)(1x工作溶液)(见食谱)
  20. NaOH(5 M)(见食谱)

设备

  1. 250毫升玻璃瓶
  2. 钳子
  3. 微波炉
  4. 恒温箱
  5. 本生灯
  6. 显微镜:
    直立式立体显微镜(例如>,尼康,型号:SM2645)
    光学显微镜(例如>,奥林巴斯,型号:SZX16)
    共聚焦显微镜(例如>,尼康,型号:A1R)
  7. 用于钨丝磨刀的电解装置(本协议中所示的装置是定制的,不再生产;图2A)
    但是,用户可以设置自己的设备。
    1. 所需零件为:a)直流电源(3-20 V,如Bosch C3智能车充电器,目录号:0092C35000),b)碳棒电极(Lasec,目录号:ERDI9470),c)鳄鱼夹(可选) ,直流电源可能带有带鳄鱼夹的导线),d)带盖的200毫升玻璃容器(罐)。 
    2. 组装设备:将引线插入充电器。使用鳄鱼夹,将碳电极棒连接到负极端子(黑色引线),将金属支架连接到正极端子(红色引线)。将电压设置为6 V,并在罐中加入100 ml NaOH溶液。设备现在可以使用了。 


    图2.电解尖锐针的制备。 :一种。基于电解的装置,包括整流的DC变压器,带有鳄鱼夹的阳极(红色),带有碳电极的阴极(黄色),带有5M NaOH电解质的电解室。公元前。钨丝针的侧视图和背视图被锐化。打开电源(6 V)后,将安装好的针头垂直固定,然后缓慢地缓慢地浸入和流出电解液,直到达到所需的尖端。 D.完成电解削尖的钨丝针。

程序

  1. 斑马鱼幼虫的脊索针损伤
    1. 准备电解削尖的钨丝(改编自Brady,1965)(图2和视频1)
      1. 切掉3厘米的钨丝并将其安装到针座中。 
      2. 使用鳄鱼夹将针座的金属手柄连接到变压器的(+)端子,将阴极与碳板连接到( - )端子。
      3. 将碳电极放入玻璃室中,加入100 ml 5 M NaOH溶液并打开变压器,将输出电压设置为6 V.将安装好的针头垂直固定,将针头缓慢稳定地浸入和滴出NaOH。产生了所需的尖端。更快的运动=针上的斜率越长,运动越慢=尖端越短,斜率越倾斜。当针上下移动时,电流表盘的读数介于0和1A之间。将针头直径从0.25毫米锐化到0.02毫米需要大约2.5分钟。 


      视频1.锐化钨丝

    2. 或者,0.1毫米昆虫针也可用于伤害脊索(图3和视频2)
      1. 拿一个干净的玻璃吸管和使用本生灯,弯曲中间的薄边,以形成45度角。这将有助于伤害操纵程序。
      2. 使用本生灯将薄侧孔封闭约四分之三。这是通过将尖端放在火焰的最强部分并以圆形方式旋转来完成的。 
      3. 用镊子取出昆虫针并将其放入孔中,注意锋利的一面朝向外面。
      4. 小心地继续在火焰最薄弱的部分燃烧玻璃吸管的尖端,直到孔关闭并且昆虫针固定。如果暴露在过多的热量下,昆虫针会燃烧。在插入引脚之前,尽量关闭孔。


      图3.昆虫针的准备。 A.所需设备。 B.完成的仪器。 C.尖端的近距离图像。


      视频2.准备昆虫针

    3. 准备1.5%琼脂糖
      将1.5g琼脂糖重量并在250ml玻璃瓶中的100ml E3胚胎培养基中使用微波炉熔化,将薄层倒入Ø90mm培养皿中。使用约20ml的1.5%琼脂糖。让琼脂糖凝固。
    4. 用特里卡因溶液麻醉幼虫
      使用E3胚胎培养基制备1:10,000三卡因溶液(请参见食谱),并将30 ml倒入单独的Ø90mm培养皿中。将一只幼虫转移到三卡因溶液中,等待它被麻醉。
    5. 在立体显微镜下,将一只幼虫放在涂有琼脂糖的培养皿上,以便从上方用针接近侧面。尽可能多地去除液体,使表面张力将幼虫粘附在培养皿上并防止其滑动。将钨丝尖端轻轻地垂直插入卵黄囊末端水平的脊索(图1B),然后取出金属丝。 (视频3)
    6. 将受损幼虫转移到具有新鲜E3培养基的培养皿中以恢复并将培养皿置于28.5℃以使幼虫生长至期望的阶段。将未受伤害的年龄匹配的幼虫保持为未受伤的对照。


      视频3.脊索针伤

  2. 化学诱导的脊索破坏
    1. 携带 Tg(> wt1b:GFP > )>和脊索标记 Tg(R2-col2a1a:mCherry)的交叉鱼 >未染色的 nacre - / - >背景中的转基因,以获得 Tg(> > GFP > ; R2-col2a1a:mCherry); nacre - / - >胚胎。
    2. 每次使用前,先将新鲜的5 mg / ml制霉菌素储备液(5.4 mM)溶解于DMSO中即可。 
    3. 在E3中稀释制霉菌素储备溶液以获得20μM的最终工作浓度。将其加入到6孔板中的去离子48hpf胚胎中。加入0.4%DMSO以控制胚胎。
    4. 将胚胎在28.5°C孵育长达48小时。制霉菌素处理24小时后,沿着脊索长度出现病变。它们倾向于首先出现在胚胎移动时自然压缩的区域,然后沿着脊索的长度扩散。大多数胚胎获得脊索病变,但其大小和严重程度可以变化。因此,建议定期筛查病变和/或 wt1b:GFP >表达的发作,以便鉴定具有所需脊索损伤水平的胚胎。
    5. 对于成像,用三卡因(1:10,000)麻醉胚胎,并在1%低熔点琼脂糖中进行矢状位。使用光学显微镜拍摄明场图像(图4A和4B)。使用共聚焦显微镜观察 R2-col2a1a:mCherry >转基因的表达,其标记脊索,并且在脊索损伤部位诱导 wt1b:GFP >转基因(图4A'和4B'')。


      图4.使用制霉菌素破坏脊索结构(改编自Lopez-Baez,2018)。制霉菌素是一种小分子,可与甾醇结合并导致细胞膜穴样内陷的解体。在脊索中很多(Lim et al。>,2017)。 Tg( wt1b:GFP > ; R2-col2a1a:mCherry); nacre - / - >斑马鱼胚胎用DMSO或20μM制霉菌素来自48 hpf至72 hpf。当在光学显微镜下观察时,(A)DMSO处理的胚胎的脊索结构看起来是正常的,但是在(B)制霉菌素处理的胚胎中可以观察到损伤。 (A'和B') wt1b:GFP >表达在损伤部位诱导,但在对照脊索中不诱导。与(A'')DMSO对照相比,脊索鞘细胞中的 R2-col2a1a:mCherry >表达还显示制霉菌素处理的胚胎的(B'')损伤部位的细胞性增加。比例尺为50μm。

  3. 尾截肢
    1. 使用E3胚胎培养基制备1.5%琼脂糖,并将薄层倒入培养皿中。让琼脂糖凝固。
    2. 在tricaine溶液中麻醉幼虫。
    3. 在立体显微镜下,将一只幼虫放在凝固的琼脂糖上。尽可能多地去除液体,因此表面张力将幼虫粘附在培养皿上并防止其滑动,然后用无菌手术刀刀片轻轻压迫尾部。截肢部位取决于正在进行的实验(图5)。尾鳍和脊索部位尖端的截肢不包括脊索组织,并且不刺激 wt1b:gfp >脊索损伤反应。尾鳍以外的截肢和脊索(尾静脉,尾静脉前)刺激 wt1b:gfp >表达。


      图5.选择的尾部截肢揭示了反应的脊索特异性(改编自Lopez-Baez,2018)。 A.尾部截肢在不同尾部和时间点的插图拍摄图像时尾鳍(TF),脊尾(TN),尾静脉(BCV),尾静脉(PCV),体节(S),脊索(N),尾静脉(CV)。 B.TN和TN截肢的幼虫在损伤后的脊索中没有显示GFP上调,但显示出明显的鳍再生(箭头)。 BCV和PCV截肢组均表现出强烈的GFP上调72 hpa(箭头),PCV截肢幼虫表现出比BCV截肢幼虫整体更强和更快的上调。

    4. 将受损的幼虫转移到具有新鲜E3培养基的培养皿中以恢复,并将培养皿置于28.5℃以使幼虫生长至期望的阶段。将未受伤害的年龄匹配的幼虫保持为未受伤的对照。

笔记

  1. 在英国和欧盟,所有动物程序都需要得到内政部(英国)或同等机构的批准。需要适当的个人项目许可(PPL)和个人个人许可(PIL)。
  2. 脊索针损伤的程序需要相当多的练习,并且应注意不要在针伤部位外造成伤害。在手术后约5分钟内脊柱内损伤部位出现小凸起结构表明手术成功。在一小时内伤害30只幼虫是可以实现的。
  3. 建议首次使用制霉菌素剂量,因为存在批次之间的差异。可以尝试更长的制霉菌素孵育期,然而制霉菌素的不利脱靶效应导致严重的发育异常并且胚胎不能长期存活。在我们的手中,用来自48 hpf的20μM制霉菌素处理24小时给出最一致的结果。在48hpf之前用制霉菌素治疗是可能的,然而,由于其脱靶效应,可以看到更多的毒性。胚胎在72和96 hpf后耐受较晚的制霉菌素治疗,24小时后60%-80%胚胎中产生20μM制霉菌素产生脊索病变,尽管这些病变体积较小,每个胚胎较图4所示。

食谱

  1. E3胚胎培养基(60x储备液)
    17.4克NaCl
    0.8克KCl
    2.9克CaCl 2 •2H 2 O
    4.89g MgCl 2 •6H 2 O
    溶于1L H 2 O.
  2. 三卡因(MS-222)(1x工作溶液)
    将0.1克三卡因粉末溶于1升1x E3培养基中,调节pH至7.0
  3. NaOH(5 M)
    将200g NaOH颗粒溶解在1L H 2 O中

致谢

资金来源:MRC人类遗传学部门计划(MC_PC_U127585840,MC_PC_U127527180),欧洲研究理事会(ZF-MEL-CHEMBIO-648489)和欧莱雅黑色素瘤研究联盟(401181),运动中的细胞 - 卓越集群(EXC 1003-CiM) )。这些程序改编自我们最近的eLife论文(Lopez-Baez et al。>,2018)

利益争夺

作者没有竞争利益。

伦理

此处提供的程序由爱丁堡大学伦理委员会批准,并根据英国爱丁堡大学的内政部项目许可证70/8000进行;并由荷兰皇家艺术与科学学院的动物实验委员会(DEC)在德国的Hubrecht研究所和心血管器官发生与再生研究所进行SSM研究。

参考

  1. 布拉迪,J。(1965)。 通过电解磨削钨丝制作非常精细,耐用的解剖针的简单技术。 公牛世界卫生机构> 32(1):143-144。
  2. Dale,R。M.和Topczewski,J。(2011)。 鉴定斑马鱼 col2a1a >基因的进化保守调控元件。< / a> Dev Biol > 357(2):518-531。
  3. Ellis,K.,Hoffman,B。D.和Bagnat,M。(2013)。 内部空泡:细胞组织如何决定脊索功能。 Bioarchitecture > 3(3):64-68。
  4. Gemberling,M.,Bailey,T。J.,Hyde,D。R. and Poss,K.D。(2013)。 斑马鱼作为复杂组织再生的模型。 Trends Genet > 29(11):611-620。
  5. Hastie,N.D。(2017)。 Wilms肿瘤1(WT1)在发育,体内平衡和疾病方面的作用。 发展> 144(16):2862-2872。
  6. Lim,Y.W。,Lo,H.P.,Ferguson,C.,Martel,N.,Giacomotto,J.,Gomez,G.A.,Yap,A.S.,Hall,T.E。和Parton,R.G。(2017)。 Caveolae保护脊索细胞免受发育过程中的灾难性机械故障的影响。 Curr Biol 27(13):1968-1981 e1967。
  7. Lisse,T。S.,Brochu,E。A.和Rieger,S。(2015)。 使用延时明场立体显微镜捕捉斑马鱼幼虫的组织修复。 J Vis Exp >(95):52654。
  8. Lister,J.A.,Robertson,C.P.,Lepage,T.,Johnson,S.L。和Raible,D.W。(1999)。 Nacre编码斑马鱼小眼病相关蛋白,调节神经嵴衍生的色素细胞命运。 发展> 126(17):3757-3767。
  9. Lleras Forero,L.,Narayanan,R.,Huitema,LF,VanBergen,M.,Apschner,A.,Peterson-Maduro,J.,Logister,I.,Valentin,G.,Morelli,LG,Oates,AC and Schulte-Merker,S。(2018)。 斑马鱼轴向骨骼的分割依赖于脊索鞘细胞,而不依赖于分割时钟。 Elife > 7:e33843。
  10. Lopez-Baez,JC,Simpson,DJ,L,LLF,Zeng,Z.,Brunsdon,H.,Salzano,A.,Brombin,A.,Wyatt,C.,Rybski,W.,Huitema,LFA,Dale, RM,Kawakami,K.,Englert,C.,Chandra,T.,Schulte-Merker,S.,Hastie,ND和Patton,EE(2018)。 Wilms Tumor 1b定义了与脊索修复相关的伤口特异性鞘细胞亚群。 Elife > 7:e30657。
  11. Perner,B.,Englert,C。和Bollig,F。(2007)。 Wilms肿瘤基因wt1a和wt1b控制斑马鱼前列腺形成过程中的不同步骤。 Dev Biol > 309(1):87-96。
  12. Rothberg,K.G.,Heuser,J.E.,Donzell,W.C.,Ying,Y.S。,Glenney,J.R。和Anderson,R.G。(1992)。 Caveolin,caveolae膜包衣的蛋白质成分。 Cell > 68(4):673-682。
  13. Wehner,D.,Tsarouchas,T.M.,Michael,A.,Haase,C.,Weidinger,G.,Reimer,M.M.,Becker,T。和Becker,C.G。(2017)。 Wnt信号控制斑马鱼功能性脊髓再生中促再生胶原蛋白XII。 em> Nat Commun > 8(1):126。
  14. Wopat,S.,Bagwell,J.,Sumigray,K.D.,Dickson,A.L.,Huitema,L.F.A.,Poss,K.D.,Schulte-Merker,S。和Bagnat,M。(2018)。 通过脊索鞘的分割来指导脊柱花样。 Cell Rep 22(8):2026-2038。
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Copyright Zeng et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zeng, Z., Lopez-Baez, J. C., Lleras-Forero, L., Brunsdon, H., Wyatt, C., Rybski, W., Hastie, N. D., Schulte-Merker, S. and Patton, E. E. (2018). Notochord Injury Assays that Stimulate Transcriptional Responses in Zebrafish Larvae. Bio-protocol 8(23): e3100. DOI: 10.21769/BioProtoc.3100.
  2. Lopez-Baez, J. C., Simpson, D. J., L, L. L. F., Zeng, Z., Brunsdon, H., Salzano, A., Brombin, A., Wyatt, C., Rybski, W., Huitema, L. F. A., Dale, R. M., Kawakami, K., Englert, C., Chandra, T., Schulte-Merker, S., Hastie, N. D. and Patton, E. E. (2018). Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair. Elife 7: e30657.
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