Materials and Reagents

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

Equipment

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

Procedure

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).
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.
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.
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
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).
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.
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

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
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
PBS/0.1% BSA
Mix 0.1 g of BSA with 100 ml of 1x PBS
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
0.5% Bicarbonate
0.5 g sodium hydrogen carbonate NaHCO₃
Add dH₂O to 100 ml, sterilize and stored at 4 °C
0.5% Glucose
0.5 g D(+) glucose anhydre
Add dH₂O to 100 ml, sterilize and stored at 4 °C
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

Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380(6569): 66-68.
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.
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.
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.
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.