Visualization of Neural and Vascular Networks in a Chicken Embryo

Abstract

Source: Delalande, J., et.al. Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using Chick^GFP Neural Tube Grafting and Carbocyanine Dye DiI Injection. J. Vis. Exp. (2015)

This video demonstrates the transplantation of a GFP-labeled donor neural tube from a stage-matched transgenic chicken embryo into a recipient embryo at the level of somites one to seven, followed by vascular labeling using a lipophilic fluorescent dye. The combined approach allows for direct visualization of the embryo under a stereofluorescence microscope, enabling the study of neural graft integration and vascular network development in ovo.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Graft the Tissue

1. Using a glass micro-pipette, carefully transfer the dissected neural tube from the watch glass to the host embryo. Position the neural tube in the correct anterior-posterior orientation and gently push the explant adjacent to the excised region of the chick host using the micro-scalpel. Leave a small fragment of ectoderm attached to, or by cutting a small nick in, the dorsal surface to identify the orientation of the neural tube.
   1. If necessary, use the micro-scalpel to trim the explant to the exact size of the excised region.
2. Gently guide the neural tube into the ablated region and position it such that the dorsal side is correctly orientated. Use a glass micro-pipette, mounted to a mouth tube, to remove PBS and/or fluid surrounding the graft. This helps the donor and host tissues to adhere and the graft to become established.
3. Seal the entire window with 24 mm wide clear tape to prevent dehydration and contamination.
4. Label the chimeric embryo by marking with a pencil on the eggshell and record its number in the lab book. Return the egg to the incubator for further development.

2. Inject DiI into Blood Vessels of Host Embryo

1. At the desired experimental time point (here, 3 - 10 days later), retrieve the chimeric embryo from the incubator and remove the clear tape using straight scissors to gain access to the embryo within the egg.
2. If necessary, enlarge the window in the shell using the scissors. Be careful not to damage the chorioallantoic membrane if it is attached to the shell, which would result in hemorrhage and jeopardize the blood vessel labeling.
3. Choose an accessible vein on the yolk making sure the blood flow is directed towards the embryo. Choose a branching point of one of the vitelline veins (Figure 1B, C).
   NOTE: At E6.5 - E7.5, the chorioallantoic membrane may need to be gently moved aside with tweezers to access the yolk veins. After E8.5, the only option is to inject into one of the chorioallantoic membrane veins since, by this stage, the chorioallantoic membrane fully covers the embryo.
4. Remove the vitelline membrane above the chosen injection point using two Dumont #5 tweezers by tearing in opposite directions.
5. Break a pulled glass needle using a Dumont #5 and adjust its diameter to the approximate size of the vein prior to loading with CellTracker CM-DiI. Make the DiI stock solution at 40 µg/µl in DMSO and store at -20 °C. Prepare the working solution in 0.3 M sucrose/PBS at a concentration of 4 µg/µl.
   1. Aspirate between 5 - 10 µl of DiI in 0.3 M sucrose/PBS into the needle using suction with a mouth tube. Older embryos might require up to 25 µl or more. From E8.5, embryos have larger, more muscular veins, which may need to be held in position with a Dumont #5 before stabbing with the DiI-loaded glass needle.
6. Swiftly insert the needle into the vein and blow steadily with the mouth tube to allow the DiI to join the blood flow slowly without forming a clot. Alternatively, use a pressure injector for DiI delivery.

Results

Figure 1. Intravenous DiI injection. (A) Recommended instruments: a) CellTracker CM-DiI drop on paraf...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fertilized chick eggs	Henry Stewart and Co, Louth, UK
Fertilized GFP chick eggs	The Transgenic Chicken Facility, The Roslin Institute, The University of Edinburgh
Egg incubator (Profi-H Hatcher)	Lyon Technologies, CA, USA	910-033
14C Incubator	Precision Cooled Incubator, Leec Ltd., Nottingham, UK	Model LT2
Stereo-microscope	LEICA	Model MZ 12.5
Digital Camera	LEICA	DC500
Image acquisition software	LEICA	IM50
Goose neck halogen cold light source	Advanced Imaging Concepts, Inc	KL 1500 LCD
Mouth tube	SIGMA - ALDRICH	A5177
Transfer pipettes, polyethylene	SIGMA - ALDRICH	Z350796
Dumont #5 forceps	Fine Science Tools	11251-30
Minutien pins	Fine Science Tools	26002-15
Dumont AA forceps, Inox Epoxy- coated	Fine Science Tools	11210-10
Sellotape (clear, 24mm width)	Any Supplier
CellTracker CM-DiI	Molecular Probes	C-7001
Borosillicate glass capillaries, thinwall without filament	Harvard apparatus	PY8 30-0035