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Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin
再生斑马鱼鳍中间充质细胞(包括芽基)的移植   

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Abstract

Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful approach for tracking and manipulating the blastema cells during fish fin regeneration.

Keywords: Zebrafish(斑马鱼), Fin regeneration(鳍再生), Blastema(芽基), Transplantation(移植), Cell lineage(细胞谱系)

Background

Cell transplantation studies are routinely performed during the early embryonic stage in animal models such as fish, amphibians and mammals, but targeting the transplanted cells to specific tissues has been difficult. Blastema transplantation developed in our studies is an efficient method for introducing mesenchymal donor cells into host fin ray. It enables us to track cell fate and/or manipulate cell signaling such as fibroblast growth factor (Fgf) during fish fin regeneration. Actually, in our recently published work, we transplanted blastema cells, which carried the hsp70l:dominant-negative fgf receptor and the β-actin:dsRed2 transgenes, into a wild-type blastema region and mosaically inhibited Fgf signaling in a subset of fin ray mesenchymal cells (Shibata et al., 2016). This method is applicable for analyzing other cell signals and for tracking cell fate by live cell imaging.

Materials and Reagents

  1. 50 ml disposable syringe (Terumo, catalog number: SS-50ESZ )
  2. Stainless steel surgical blade, No.10 (FEATHER Safety Razor, catalog number: No. 10 )
  3. Plastic dish (9 cm diameter) (As One, catalog number: GD90-15 )
  4. Glass capillary (1 x 90 mm, without filament) (Narishige, catalog number: G-1 )
  5. Dissection needle: This is made by attaching 30 G needle (BD, catalog number: 305106 ) to a P1000 pipette chip (BM Equipment, catalog number: BIO1000RF ) whose tip is truncated (Figure 1)
    Note: A handmade tool for removing the wound epidermis from the regenerate and for dissecting the blastema. This needle is different from that for transplantation (see Procedure B).


    Figure 1. Dissection needle

  6. Syringe filter unit (0.22 µm) (EMD Millipore, catalog number: SLGV033RB )
  7. Zebrafish transplantation donor strain: Tg(Olactb:loxP-dsred2-loxP-egfp), which constitutively expresses the DsRed2 ubiquitously (Yoshinari et al., 2012). For simplicity, we refer the line as Tg(β-actin:dsred2) in this manuscript
  8. Zebrafish transplantation host: a wild-type strain, which has been maintained in our facility by inbred breeding
  9. Agarose (Nacalai Tesque, catalog number: 01028-85 )
  10. Tricaine (3-aminobenzoic acid ethyl ester) (Sigma-Aldrich, catalog number: A5040 )
  11. Fetal bovine serum (FBS) (any brand can be used)
  12. Leibowitz’s L-15 medium (powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 41300070 )
  13. 1 M Tris-HCl (pH 9)
  14. L-15 medium (see Recipes)
  15. 20x tricaine stock solution (see Recipes)

Equipment

  1. Microwave oven
  2. Micropipette puller (Sutter Instrument, model: P-97 )
  3. Forceps
  4. Glass dish (5 cm diameter)
  5. Surgical blade handle (FEATHER Safety Razor, catalog number: No. 3 )
  6. Microforge (Narishige, model: MF-900 )
  7. Three-dimensional coarse manual manipulator (Narishhige, model: M-152 ) installed on a magnet stand
  8. P20 micropipette
  9. Fluorescence stereomicroscope

Procedure

  1. Preparation of agar gel stage (Figure 2)
    1. Mix agarose and water to make a 2% agarose solution and melt in a microwave oven.
    2. Pour the melted agarose into a plastic dish up to a half of dish depth and leave at room temperature for 20-30 min.
    3. Further fill the dish with the melted 2% agarose solution to approximately full depth and leave at room temperature for 20-30 min.
    4. Make a rectangular well (2 x 4 cm square, 0.5 cm depth) at the center of gel stage by cutting and removing upper layer of the gel.


      Figure 2. Agarose gel stage

  2. Preparation of transplantation needle
    1. Pull the glass capillary with the micropipette puller to make a needle with a thin and long shaft. We routinely pull the glass capillary using the same conditions as for microinjection needles (our settings: heat 570, pull 150, velocity 120, time 60).
    2. Set the pulled needle on the microforge, break the needle with forceps at a site where the inside diameter is approximately 20 μm. Make the end as blunt as possible with forceps. Using the microforge heater, make the needle end smooth and further make a harpoon.


      Figure 3. Transplantation needle. Scale bar = 50 μm.

  3. Preparation of donor mesenchymal cells of whole regenerate including blastema (Video 1)

    Video 1. Transplantation of mesenchymal cells including the blastema in regenerating zebrafish fin

    1. Two days before transplantation, anaesthetize the donor zebrafish with 1x tricaine solution (~30 ml or enough volume to cover the fish body) in a plastic dish and cut their caudal fins with a scalpel. Note that the fins of host fish need to be amputated simultaneously. The amputated fish were returned to aquarium and normally fed until transplantation.
    2. On the day of transplantation (2 days post amputation [dpa]), anaesthetize the donor fish with tricaine and cut the fin regenerate with a scalpel.
    3. Transfer the fin regenerate with forceps to a glass dish containing 5-10 ml L-15 medium supplemented with 0.01% FBS.
    4. Remove the wound epidermis from the regenerate (mesenchymal part) using the dissection needles as forceps. Peel the epidermis from the proximal side and remove the whole epidermis by gently pulling the epidermis from the edge as if taking off a T-shirt.
    5. Divide the mesenchymal part of the regenerate to respective fin ray units. If necessary, the blastema can be further cut into smaller sizes.

  4. Transplantation (Video 1)
    1. Set the transplantation needle into the needle holder of manipulator.
    2. Anaesthetize the host zebrafish with tricaine and place it on the agarose stage (Figure 4 and Figure 5).



      Figure 4. Zebrafish fin and the transplantation needle


      Figure 5. Microscope and manipulator set up

    3. Make an opening on the surface of wound epidermis using the transplantation needle. Push the needle onto the regenerate surface and tap the head of manipulator knob to break the epidermis.
    4. Transfer a piece of donor mesenchymal tissue including the blastema to site of the epidermal opening using a P20 micropipette.
    5. Push the donor mesenchymal tissue with the transplantation needle to insert into the host regenerate. Hook the donor mesenchymal tissue with harpoon of the needle and force to insert into the host regenerate. Because proximal side of the donor mesenchyme is rigid, it is easier to hook the proximal side. Note that it is difficult to push the tissue with thinner needles. Transplantation can be performed for several fin rays at a time, but either of dorsal or ventral side of the fin should be blank as a regeneration control.
    6. Put the operated fish back to aquarium (Figure 6). 
      Notes:
      1. Only regenerating fins are amenable to transplantation. Removing the epidermis from the regenerate is easily done with 1.5-2.5 dpa regenerating fin. The host tissue should also be 1.5-2.5 dpa. Only the thick region in the regenerating fin can be used as a host tissue. The transplanted cells in the blastema proliferate to produce lots of progeny cells within fin ray, and this is a hallmark that the transplanted cells incorporated as a part of fin mesenchyme.
      2. Though we have not traced the transplanted cells beyond 14 dpa, in some cases we observed a significant decrease of donor cells, which could be due to the transplant rejection by host immune system.


        Figure 6. A representative example of blastema transplantation. The fin of the Tg(β-actin:dsred2) was amputated 2 days before transplantation, and the blastema tissue was transplanted into the blastema region of wild-type host. Operated fish were anaesthetized and placed on the agarose gel stage, and the pictures were taken under a fluorescence stereomicroscope. Arrowheads, amputation sites; dpt, days post transplantation. Scale bars = 200 μm.

Recipes

  1. L-15 medium
    1. Dissolve L-15 powder according to the manufacturer’s instruction
    2. Sterilize through a 0.22-μm filter
  2. 20x tricaine stock solution
    1. Dissolve 2 g of tricaine in 489.5 ml of distilled water
    2. Add 10.5 ml 1 M Tris-HCl (pH 9) to bring the pH to 7
    3. Store the solution in aliquots at -30 °C
    4. Dilute the tricaine stock to 20 times with fish water to make 1x tricaine solution, which is used for anesthesia

Acknowledgments

This protocol was adapted from our previous works (Yoshinari et al., 2012; Shibata et al., 2016). The works were supported by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS).

References

  1. Shibata, E., Yokota, Y., Horita, N., Kudo, A., Abe, G., Kawakami, K. and Kawakami, A. (2016). Fgf signalling controls diverse aspects of fin regeneration. Development 143(16): 2920-2929.
  2. Yoshinari, N., Ando, K., Kudo, A., Kinoshita, M. and Kawakami, A. (2012). Colored medaka and zebrafish: transgenics with ubiquitous and strong transgene expression driven by the medaka beta-actin promoter. Dev Growth Differ 54(9): 818-828.

简介

鱼鳍和尿le肢的再生通过形成大气泡,其是在截肢位点处形成的质量的间充质细胞并且对于再生是必需的。胚胎移植是我们以前研究(Shibata等人,2016; Yoshinari等人,2012)开发的一种新技术,是一种有用的方法,用于跟踪和操纵鱼翅再生期间的blastema细胞。

背景在诸如鱼,两栖动物和哺乳动物之类的动物模型的早期胚胎阶段常规进行细胞移植研究,但将移植细胞靶向特定组织是困难的。在我们的研究中开发的Blastema移植是将间充质供体细胞引入宿主翅片射线的有效方法。它使我们能够在鱼翅再生期间跟踪细胞命运和/或操纵细胞信号,如成纤维细胞生长因子(Fgf)。实际上,在我们最近出版的作品中,我们将携带hsp701:显性负性fgf受体和β-肌动蛋白:dsRed2转基因的blastema细胞移植到野生型类型blastema区域并且在鳍状线间充质细胞的子集(Shibata等人,2016)中马齿抑制Fgf信号传导。该方法适用于分析其他细胞信号和通过活细胞成像跟踪细胞命运。

关键字:斑马鱼, 鳍再生, 芽基, 移植, 细胞谱系

材料和试剂

  1. 50ml一次性注射器(Terumo,目录号:SS-50ESZ)
  2. 不锈钢手术刀,10号(FEATHER安全剃刀,目录号:10号)
  3. 塑料盘(直径9厘米)(一号,目录号:GD90-15)
  4. 玻璃毛细管(1 x 90 mm,无长丝)(Narishige,目录号:G-1)
  5. 解剖针:这是通过将30 G针(BD,目录号:305106)连接到尖端被截断的P1000移液器芯片(BM设备,目录号:BIO1000RF)上制成的(图1)
    注意:一种手工工具,用于从再生中去除伤口表皮,并用于解剖胚泡。这种针与移植不同(见程序B)。


    图1.解剖针

  6. 注射器过滤器(0.22μm)(EMD Millipore,目录号:SLGV033RB)
  7. 斑马鱼移植供体菌株:Tg(ioctb:loxP-dsred2-loxP-egfp),其组成式表达DsRed2无处不在(Yoshinari等人,2012)。为了简单起见,在本手稿中我们将该行称为Tg(β-actin:dsred2 )
  8. 斑马鱼移植宿主:野生型菌株,通过近交育种在我们的设施中维持
  9. 琼脂糖(Nacalai Tesque,目录号:01028-85)
  10. 三甲基(3-氨基苯甲酸乙酯)(Sigma-Aldrich,目录号:A5040)
  11. 胎牛血清(FBS)(任何品牌都可以使用)
  12. Leibowitz的L-15介质(粉末)(Thermo Fisher Scientific,Gibco TM,目录号:41300070)
  13. 1M Tris-HCl(pH 9)
  14. L-15培养基(见食谱)
  15. 20x三氯生酮储备溶液(见配方)

设备

  1. 微波炉
  2. 微量拔取器(Sutter Instrument,型号:P-97)
  3. 镊子
  4. 玻璃皿(直径5厘米)
  5. 手术刀柄(FEATHER安全剃须刀,目录号:3号)
  6. Microforge(Narishige,型号:MF-900)
  7. 安装在磁铁架上的三维粗略手动操纵器(Narishhige,型号:M-152)
  8. P20微量移液器
  9. 荧光立体显微镜

程序

  1. 琼脂凝胶阶段的制备(图2)
    1. 混合琼脂糖和水,制成2%琼脂糖溶液,并在微波炉中熔化。
    2. 将融化的琼脂糖倒入塑料盘中至深达一半的深度,并在室温下放置20-30分钟。
    3. 进一步用熔化的2%琼脂糖溶液将盘充满大致全深度,并在室温下放置20-30分钟。
    4. 通过切割和去除凝胶上层,在凝胶阶段的中心形成矩形井(2×4cm见方,0.5cm深)。


      图2.琼脂糖凝胶阶段

  2. 准备移植针
    1. 用微量吸管拔出器拉动玻璃毛细管,使针头长而细。我们通常使用与显微注射针相同的条件(我们的设置:热570,拉150,速度120,时间60)拉玻璃毛细管。
    2. 将拉针设置在微型锻造件上,用内径约为20μm的位置用镊子打破针头。尽可能使用镊子尽可能的钝。使用微型加热器,使针头平滑,进一步做一个鱼叉。


      图3.移植针。 比例尺=50μm。

  3. 包括囊胚在内的全再生供体间质细胞的制备(视频1)

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    1. 在移植前两天,在塑料盘中麻醉供体斑马鱼,用1克三卡因溶液(约30毫升或足够的体积覆盖鱼体),并用解剖刀切割尾鳍。请注意,宿主鱼的翅片需要同时截肢。截肢鱼回到水族箱,通常喂养直到移植。
    2. 在移植当天(截肢后2天[dpa]),用三叉戟麻醉供体鱼,用解剖刀切割翅片再生。
    3. 用镊子将翅片再生物转移到含有5-10毫升含有0.01%FBS的L-15培养基的玻璃皿中。
    4. 使用解剖针作为镊子从再生(间充质部分)中除去伤口表皮。从近端剥离表皮,通过从边缘轻轻拉扯表皮,去除整个表皮,就好像脱掉T恤一样。
    5. 将再生的间充质部分分成各自的翅片射线单元。如有必要,可以进一步将小卵泡切割成较小尺寸。

  4. 移植(视频1)
    1. 将移植针插入机械手的针座。
    2. 麻醉三叉戟的宿主斑马鱼,并将其置于琼脂糖平台上(图4和图5)。



      图4.斑马鱼和移植针头


      图5.显微镜和机械手设置

    3. 使用移植针在伤口表皮表面开口。将针推到再生表面上,然后敲击操纵手柄的头部以破坏表皮。
    4. 使用P20微量移液管将包括胚泡发生的供体间充质组织转移到表皮开口部位。
    5. 用移植针推动给体间充质组织插入宿主再生。用针头的钩子钩住供体间充质组织,并将其插入宿主再生中。由于供体间质的近端是刚性的,因此更容易钩住近侧。请注意,使用较薄的针很难推动组织。一次可以进行几个鳍状射线的移植,但翅片的背侧或腹侧均应为空白,作为再生控制。
    6. 将操作的鱼放回水族馆(图6)。 
      注意:
      1. 只有再生翅片才适合移植。使用1.5-2.5 dpa的再生翅片很容易地从再生中去除表皮。宿主组织也应为1.5-2.5 dpa。只有再生翅片中的厚区才能用作宿主组织。胚泡中的移植细胞在翅片射线内增殖以产生许多后代细胞,这是移植细胞作为翅片间质的一部分结合的标志。
      2. 尽管我们尚未追踪超过14 dpa的移植细胞,但在某些情况下,我们观察到供体细胞显着减少,这可能是由于宿主免疫系统的移植排斥反应。


        图6. blastema移植的代表性实例。在移植前2天截留Tg(β-肌动蛋白:dsred2)的鳍,并且胚泡将组织移植到野生型宿主的blastema区域。操作的鱼被麻醉并置于琼脂糖凝胶阶段,并且在荧光立体显微镜下拍摄照片。箭头,截肢点; dpt,移植后的几天。比例尺= 200μm

食谱

  1. L-15培养基
    1. 根据制造商的说明书溶解L-15粉末
    2. 通过0.22μm过滤器进行杀菌
  2. 20x三氯生酮储备溶液
    1. 将2克三奈因溶于489.5毫升蒸馏水中
    2. 加入10.5ml 1M Tris-HCl(pH9)使pH达到7
    3. 将溶液以-30℃等分试样储存
    4. 用鱼水稀释三氯生酮20倍,制成1×三氯苯胺溶液,用于麻醉。

致谢

这个协议是从我们以前的作品(Yoshinari等人,2012; Shibata等人,2016)改编而来的。这些作品得到了日本科学促进会(JSPS)的科学研究资助。

参考文献

  1. Shibata,E.,Yokota,Y.,Horita,N.,Kudo,A.,Abe,G.,Kawakami,K.and Kawakami,A。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27402707"target ="_ blank"> Fgf信号控制鳍再生的各个方面。 开发 143(16 ):2920-2929。
  2. Yoshinari,N.,Ando,K.,Kudo,A.,Kinoshita,M。和Kawakami,A.(2012)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/23157381"target ="_ blank">彩色的medaka和斑马鱼:由medakaβ-肌动蛋白启动子驱动的无处不在和强大的转基因表达的转基因。发展成长差异 54(9):818-828。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shibata, E., Ando, K. and Kawakami, A. (2017). Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin. Bio-protocol 7(2): e2109. DOI: 10.21769/BioProtoc.2109.
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