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Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals or receptor inhibitors can be implanted in selective embryo regions at precise developmental stages activating or blocking different signaling pathways. Here, we describe the manipulation of Wnt signaling pathway using Dkk-1-soaked micro-beads, implanted in ovo in the anterior part of the developing neural tube of chicken embryos; specifically at the prospective zona limitans intrathalamica at stage HH10.

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Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos
移植Dkk-1浸润磁珠到鸡胚神经管

神经科学 > 发育 > 神经元
作者: Almudena Martinez-Ferre
Almudena Martinez-FerreAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
Bio-protocol author page: a967
Maria Navarro-Garberí
Maria Navarro-GarberíAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
Bio-protocol author page: a968
Carlos Bueno
Carlos BuenoAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
Bio-protocol author page: a969
 and Salvador Martinez
Salvador MartinezAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
For correspondence: smartinez@umh.es
Bio-protocol author page: a970
Vol 3, Iss 21, 11/5/2013, 4502 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.963

[Abstract] Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals or receptor inhibitors can be implanted in selective embryo regions at precise developmental stages activating or blocking different signaling pathways. Here, we describe the manipulation of Wnt signaling pathway using Dkk-1-soaked micro-beads, implanted in ovo in the anterior part of the developing neural tube of chicken embryos; specifically at the prospective zona limitans intrathalamica at stage HH10.
Keywords: Experimental embryology(实验胚胎学), Neutral tube(神经管), Wnt signaling regulation(Wnt信号通路的调控), Microbeads implant(微种植体), Ni vivo experimentos(镍体内experimentos)

[Abstract]

Materials and Reagents

  1. Fertilized chick (Gallus gallus)
  2. Recombinant mouse Dkk-1 (R&D Systems, catalog number: 1765-DK )
  3. Heparin Acrylic beads (Sigma-Aldrich, catalog number: H5263 )
  4. Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A2153 )
  5. Pelikan Drawing Ink Black
  6. Silikon Peroxid (IDEX Health & Science, catalog number: SC0083ST )
  7. Tungsten wire (0.380 mm, 10 FT) (World Precision Instruments, catalog number: TGW1510 )
  8. Grid with concentric circles (NE42-21 mm) (Graticules LTD, Tonbridge Kent)
  9. Penicillin-Streptomycin (10,000 U/ml) (Gibco, catalog number: 15140-122 )
  10. NaCl (Sigma-Aldrich, catalog number: S-3014 )
  11. KCl (Sigma-Aldrich, catalog number: P-9541 )
  12. CaCl2.2H2O (Prolabo, catalog number: 22317297 )
  13. NaH2PO4.2H2O (Scharlab, catalog number: SO0334 )
  14. MgCl2.6H2O (Prolabo, catalog number: 27778293 )
  15. D(+) glucose anhydre (Prolabo, catalog number: 24370294 )
  16. 10x Phosphate-buffer saline (PBS) (see Recipes)
  17. 4% Paraformaldehyde (PFA) from 20% PFA (see Recipes)
  18. PBS/0.1% BSA (see Recipes)
  19. Tyrodes buffer (see Recipes)
  20. 0.5% Bicarbonate (see Recipes)
  21. 0.5% Glucose (see Recipes)
  22. Tyrodes supplemented (see Recipes)

Equipment

  1. 37 °C, 5% CO2 forced-air incubator (Novital, model: Covatutto 120-4V )
  2. Dissecting microscope (Leica Microsystems, model: MS5 )
  3. Butane burner (Butsir)
  4. Glass micropipet (homemade)
  5. Pasteur pipets (Deltalab, catalog number: 200001 )
  6. Silicon tube (IDEX Health & Science, catalog number: SC0083ST )

Procedure

  1. Fertilized chick (Gallus gallus) was incubated at 37 °C in a forced-air incubator. Chick embryos were developed until stage HH10 according to Hamburger and Hamilton (1951).
  2. Glass micropipets were prepared using a Bunsen burner. One side of the micropipet was introduced into a silicon tube that was used to aspirate solution while picking up each bead (Figure 1A-D). Tungsten wire was joined to a Pasteur pipet to handle easily (Figure 1E).


    Figure 1. Micropipets preparation. C-D. Tight part of Pasteur pipets was used to prepare micropipets. E. Wide part was used as a handle for the tungsten wire.

  3. Use forceps to selective heparin acrylic beads based on their size. A grid inserted in one ocular of the operating microscope was used to select beads of ~40 μm. Afterwards, beads were rinsed in PBS and then soaked in a solution of 25 μg/ml of Dkk-1 protein in PBS/0.1% BSA or in PBS overnight (o/n) at 4 °C.
  4. An opening in the shell was made with scissors. To counterstained the embryos in ovo, inject Indian ink (diluted 1:1 in Tyrode supplemented) under the blastoderm by a glass micropipet made with a Bunsen burner. The vitelline membrane that covers the embryo is slipped out with tungsten wire on the spot where microsurgery was to be performed (see Video 1).

    Video 1. Counterstaining of chicken embryos in ovo

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

  5. A grid with concentric circles was inserted in one ocular of the operating microscope. The working magnification used during microsurgery was 40x. This grid was positioned in relation to the embryo: the vertical axis followed the embryo ventral midline; the transversal axis was positioned by crossing the optic-diencephalic angle at both sides (Figure 2A) (Garcia-Lopez et al., 2004).
  6. Afterwards, the soaked-beads were cut to obtain half-beads using forceps. Then they were rinsed in PBS several times and one soaked half-bead implanted in the right side of the neural tube of embryos (Figure 2B). For control experiments, beads were soaked in PBS in the same manner.


    Figure 2. Bead-implanting procedure. A. Schematic representation of the grid positioned in relation to the embryo. B. Dkk-1 (black asterisk) or PBS-soaked beads were implanted into the prospective zona limitans intrathalamica of chick embryos at HH10. Abbreviations: Di, diencephalon; Mes, mesencephalon; Rh, rhombencephalon; Tel, telencephalon.

  7. When the operation was completed, the opening in the eggshell was sealed with a piece of tape and incubated in horizontal position until the stage selected for fixation.

Recipes

  1. 10x PBS– phosphate-buffer saline
    Mix 80 g of NaCl with 2 g of KCl, 14.4 g of Na2HPO4, 2.4 g of KH2PO4
    Adjust pH to 7.4 with NaOH
    Add dH2O to 1,000 ml
    Filter sterilize (0.2 μm)
    Stored at RT
  2. 20% PFA- paraformaldehyde
    Mix 600 ml of preheated 1x PBS at 65 °C with 200 g of PFA
    Add PBS to 1,000 ml
    Adjust pH to 7.4 with NaOH
    Filter sterilize (0.2 μm)
    Stored at -20 °C
  3. PBS/0.1% BSA
    Mix 0.1 g of BSA with 100 ml of 1x PBS
  4. Tyrodes buffer
    4 g NaCl
    0.1 g KCl
    0.13 g CaCl2.2H2O
    0.028 g NaH2PO4.2H2O
    0.022 g MgCl2.6H2O
    Add dH2O to 300 ml, sterilize and stored at 4 °C
  5. 0.5% Bicarbonate
    0.5 g sodium hydrogen carbonate NaHCO3
    Add dH2O to 100 ml, sterilize and stored at 4 °C
  6. 0.5% Glucose
    0.5 g D(+) glucose anhydre
    Add dH2O to 100 ml, sterilize and stored at 4 °C
  7. Tyrodes supplemented
    30 ml Tyrodes buffer
    10 ml 0.5% Glucose
    10 ml 0.5% Bicarbonate
    500 μl 10,000 U/ml penicillin-streptomycin

Acknowledgments

This protocol was adapted from the previously published studies, Crossley et al. (1996) and Vieira and Martinez, (2005), and it was performed by Martinez-Ferre et al. (2013). This work was supported by EUCOMMTOOLS Contract 261492, Spanish Ministry of Science and Innovation Grant BFU-2008-00588, Spanish Ministry of Education and Science-Universitary Professor Formation Grant AP2009-3644, Consolider Grant CSD2007-00023, Institute of Health Carlos III, Spanish Cell Therapy Network and Research Center of Mental Health, General Council of Valencia (Prometeo 2009/028 and 11/2011/042), and the Alicia Koplowith Fondation. We thank M. Ródenas for technical assistance.

References

  1. Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380(6569): 66-68.
  2. Garcia-Lopez, R., Vieira, C., Echevarria, D. and Martinez, S. (2004). Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos. Dev Biol 268(2): 514-530.
  3. Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J Morphol 88(1): 49-92.
  4. Martinez-Ferre, A., Navarro-Garberi, M., Bueno, C. and Martinez, S. (2013). Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate. J Neurosci 33(9): 3967-3980.    
  5. Vieira, C. and Martinez, S. (2005). Experimental study of MAP kinase phosphatase-3 (Mkp3) expression in the chick neural tube in relation to Fgf8 activity. Brain Res Brain Res Rev 49(2): 158-166.       

材料和试剂

  1. 受精鸡(Gallus gallus)
  2. 重组小鼠Dkk-1(R& D Systems,目录号:1765-DK)
  3. 肝素丙烯酸珠(Sigma-Aldrich,目录号:H5263)
  4. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  5. Pelikan绘画墨水黑色
  6. Silikon Peroxid(IDEX Health& Science,目录号:SC0083ST)
  7. 钨丝(0.380mm,10FT)(World Precision Instruments,目录号:TGW1510)
  8. 具有同心圆的网格(NE42-21mm)(Graticules LTD,Tonbridge Kent)
  9. 青霉素 - 链霉素(10,000U/ml)(Gibco,目录号:15140-122)
  10. NaCl(Sigma-Aldrich,目录号:S-3014)
  11. KCl(Sigma-Aldrich,目录号:P-9541)
  12. (Prolabo,目录号:22317297)
  13. NaH 2 PO 4 sub 2 O 2(Scharlab,目录号:SO0334)
  14. (Prolabo,目录号:27778293)
  15. D(+)葡萄糖酐(Prolabo,目录号:24370294)
  16. 10x磷酸盐缓冲盐水(PBS)(见配方)
  17. 4%来自20%PFA的多聚甲醛(PFA)(参见配方)
  18. PBS/0.1%BSA(参见配方)
  19. Tyrodes缓冲区(参见配方)
  20. 0.5%碳酸氢盐(见配方)
  21. 0.5%葡萄糖(见配方)
  22. Tyrodes补充(见配方)

设备

  1. 37℃,5%CO 2强制空气培养箱(Novital,型号:Covatutto 120-4V)
  2. 解剖显微镜(Leica Microsystems,型号:MS5)
  3. 丁烷燃烧器(Butsir)
  4. 玻璃微吸盘(自制)
  5. 巴斯德吸管(Deltalab,目录号:200001)
  6. 硅管(IDEX Health& Science,目录号:SC0083ST)

程序

  1. 将受精的小鸡(Gallus gallus)在37℃下在强制空气培养箱中温育。根据Hamburger和Hamilton(1951),将小鸡胚胎发育至HH10阶段
  2. 使用本生灯制备玻璃微量移液管。将微量移液管的一侧引入硅管中,用于吸取溶液,同时拾取每个珠(图1A-D)。将钨丝连接到巴斯德吸管以轻松处理(图1E)。


    图1.微滴制备。 C-D。使用巴氏吸管的紧密部分来制备微量移液管。 E.宽部件用作钨丝的手柄。

  3. 使用镊子选择性肝素丙烯酸珠基于其大小。在操作显微镜的一个目镜中插入的网格用于选择〜40μm的珠。然后,将珠在PBS中漂洗,然后在4℃下浸泡在25μg/ml的Dkk-1蛋白在PBS/0.1%BSA或PBS中的溶液中过夜(o/n)。
  4. 用剪刀制成壳体中的开口。为了在卵内复染色胚胎,通过用本生灯制造的玻璃微量移液器将印度油墨(在添加的Tyrode中以1:1稀释)注射在胚盘下。覆盖胚胎的卵黄膜在进行显微手术的地方用钨丝滑出(见视频1)。

    视频1. 在卵内对鸡胚进行反纹理
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    获取Adobe Flash Player

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  5. 将具有同心圆的网格插入手术显微镜的一个目镜中。显微外科手术中使用的工作放大率为40倍。该网格相对于胚胎定位:垂直轴遵循胚胎腹侧中线;横向轴通过两侧的视 - 间脑角定位(图2A)(Garcia-Lopez等人,2004)。
  6. 然后,使用镊子切割浸泡的珠粒以获得半珠粒。然后将它们在PBS中漂洗数次,并将一个浸泡的半珠植入胚胎的神经管的右侧(图2B)。对于 对照实验中,珠子以相同的方式浸泡在PBS中

    F 图2.珠粒植入程序。A.与胚胎相关的网格的示意图。 B.将Dkk-1(黑色星号)或PBS浸泡的珠子植入HH10的雏鸡胚胎的预期的带状疱疹内。缩写:Di,hypencephalon; Mes,中脑; Rh,rhombencephalon;电话,telencephalon。

  7. 当操作完成时,蛋壳中的开口用一条胶带密封,并在水平位置孵育直到选择固定的阶段。

食谱

  1. 10x PBS-磷酸盐 - 缓冲盐水
    将80g的NaCl与2g的KCl,14.4g的Na 2 HPO 4,2.4g的KH 2 PO 4
    用NaOH调节pH至7.4 将dH <2> O添加至1,000 ml
    过滤灭菌(0.2μm)
    存储在RT
  2. 20%PFA-多聚甲醛
    将600ml预热的1x PBS在65℃下与200g PFA混合 将PBS加入到1000 ml
    用NaOH调节pH至7.4 过滤灭菌(0.2μm)
    储存于-20°C
  3. PBS/0.1%BSA 将0.1g BSA与100ml 1x PBS混合
  4. Tyrodes缓冲区
    4克NaCl
    0.1克KCl
    0.13g CaCl 2 2H O 0.028g NaH 2 PO 4 4 2 H 2 O 0.022g MgCl 2 。 6H 2 O 将dH 2 O加入到300ml中,灭菌并在4℃下保存
  5. 0.5%碳酸氢盐 0.5g碳酸氢钠NaHCO 3溶液 将dH 2 O加至100ml,灭菌并在4℃下贮存
  6. 0.5%葡萄糖
    0.5克D(+)葡萄糖酐 将dH 2 O加至100ml,灭菌并在4℃下贮存
  7. 蒂罗德补充了
    30 ml Tyrodes缓冲液
    10ml 0.5%葡萄糖 10ml 0.5%碳酸氢盐 500μl10,000U/ml青霉素 - 链霉素

致谢

该方案改编自以前发表的Crossley等人(1996)和Vieira和Martinez,(2005)的研究,并且由Martinez-Ferre等人进行。 >(2013)。这项工作得到EUCOMMOLOLS合同261492,西班牙科技创新基金BFU-2008-00588,西班牙教育和科学部教育和科学大学教授Formrant Grant AP2009-3644,Consolider Grant CSD2007-00023,健康卡洛斯III研究所,西班牙语细胞治疗网络和心理健康研究中心,瓦伦西亚总理事会(Prometeo 2009/028和11/2011/042)和Alicia Koplowith基金会。我们感谢M.Ródenas的技术援助。

参考文献

  1. Crossley,PH,Martinez,S.and Martin,GR(1996)。由FGF8诱导的中脑发育 380(6569):66- 68.
  2. Garcia-Lopez,R.,Vieira,C.,Echevarria,D。和Martinez,S。(2004)。 在雏鸡胚胎的10个体节阶段的间脑和Zona极限的命运地图。 a> Dev Biol 268(2):514-530。
  3. Hamburger,V。和Hamilton,H.L。(1951)。 鸡胚发育的一系列正常阶段。 J Morphol 88(1):49-92
  4. Martinez-Ferre,A.,Navarro-Garberi,M.,Bueno,C.and Martinez,S.(2013)。 Wnt信号通过调节Shar诱导在alar板中指定动脉内限制及其组织器性质。 a> J Neurosci 33(9):3967-3980。    
  5. Vieira,C。和Martinez,S。(2005)。 在与Fgf8相关的鸡神经管中MAP激酶磷酸酶-3(Mkp3)表达的实验研究活动。 Brain Res Brain Res Rev 49(2):158-166。       
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How to cite this protocol: Martinez-Ferre, A., Navarro-Garberí, M., Bueno, C. and Martinez, S. (2013). Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos. Bio-protocol 3(21): e963. DOI: 10.21769/BioProtoc.963; Full Text



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