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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, 3536 views, 0 Q&A, How to cite
DOI: http://dx.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)

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.       


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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