Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using Chick^GFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

The overall goal of this procedure is to cos stain and visualize neural crust derived nervous tissue and the associated vascular system during embryonic development. This is accomplished by first removing the neural tube from a wild type host embryo. An equivalent neural tube region is X excised from a stage matched transgenic, GFP chicken embryo, enzymatically, digested and grafted into the wild type host.

This results in a chimeric embryo. The second step is to inject fluorescent dye into the blood system of the chimeric embryos to visualize the developing vascular system. Next, the embryos are harvested and the tissues of interest are dissected.

Finally, the samples are imaged using confocal microscopy to visualize both the GFP labeled enteric nervous system and the DAI labeled vascular networks. The main advantages of this technique over existing methods, such as the classic quail chick grafting technique, are that the GFP chicken and tissues are very bright and that the GFP of cytoplasmic allowing visualization of cell projections within neural networks. Also, the GFP and DAI labeling methods are compatible with live imaging.

To begin prepare the work area and all the tools and reagents needed for the procedure, the first step is to create a window in the eggshell to permit access to the embryo. After removing two to three milliliters of albumin, the embryo should be visible through the window. India ink is then injected to help visualize the embryo.

To do this, insert a micropipet through the yolk membrane outside of the perimeter of the blaster. Carefully angle its tip directly beneath the embryo. Once in place, deliver the ink underneath the embryo by blowing on a mouth tube.

Take care not to introduce any air bubbles which could lead to contamination. Carefully remove the glass micro pipette. Next, use a custom-made micro scalpel mounted on a needle holder to make a very small tear in the IGN membrane next to the area where the microsurgery will be performed.

To remove the neural tube region of interest, use a micro scalpel to incise across the entire neural tube, starting from so mite one or two and ending at six or seven. Next cut bilaterally between the neural tube and the somites to separate the neural tube from the surrounding tissues. Take care not to damage the somites.

When ready, very gently separate the neural tube from the underlying not cord, which should remain intact. A successful neural tube excision will leave all surrounding tissues perfectly intact. Remove and discard the excise neural tube.

The host embryo is now ready to receive the donor neural tube. After identifying a stage matched GFP embryo, remove it from the egg by making four incisions with spring scissors, create a rectangle shape around the embryo and then gently pick it up using dumont. Number five, tweezers.

Place the embryo in a square. Watch glass with a sogar polymer base. Gently shake the embryo to remove any attached yolk.

Remove the VII membrane and pin the embryo onto the polymer base using stainless steel minutia pins. Next, make four incisions in a rectangular shape around the neural tube and surrounding somites in the same region that has been removed from the host embryo. Using a plastic transfer pipette transfer the neural tube and so mite tissues into a watch glass containing 2%pancreatin in PBS for enzymatic digestion.

After 10 minutes of room temperature, use stainless minutiae pins mounted on a handle to manually separate the neural tube from all adjacent tissues. Once isolated, transfer the associated neural tube to another watch glass on ice to rinse the excess pancreatin and stop enzymatic digestion. After five minutes, the dissected neural tube is ready to be grafted.

Ortho topically into the chick host. When ready to begin the grafting procedure, carefully transfer the dissected neural tube from the watch glass to the host embryo. Position the neural tube in the correct orientation and gently push the ex explan adjacent into the excised region.

If necessary, use the micro scalpel to trim the ex explan to the exact size of the excised region. Once in the correct position, remove PBS and other fluid to help the donor and host tissues a adhere and establish the graft. The successful integration of the grafted GFP positive neural tube into the wild type chicken host can be assessed under a fluorescence microscope.

Seal the entire window with 24 millimeter wide clear tape to prevent dehydration and contamination. Label the chimeric embryo and return the egg to the incubator for further development at the desired experimental time point, retrieve the egg from the incubator and remove the clear tape to gain access to the embryo. Once visible, locate an accessible vein on the yolk, making sure the blood flow is directed towards the embryo.

Choose a branching point on one of the IGN veins for the injection location. Use tweezers to remove the viti membrane above the chosen injection point by tearing in opposite directions. Next, adjust the diameter of a pulled glass needle to the size of the vein.

Loathe the needle with five to 10 microliters of Dai solution. Swiftly insert the needle into the vein and blow steadily with the mouth tube to allow the dai to join the blood flow slowly without forming a clot. The success of the Dai injection can be assessed by briefly viewing the embryo under a fluorescence microscope, obvious the embryo immediately after injection.

To retain as much dii as possible, scoop the embryo onto a perforated spoon and cut the blood vessels and connective tissues to free the embryo from the yolk. Remove any loose membranes and dissect out the organs of interest. Take great care not to compress the tissue, which can lead to diffusion of the dry eye.

Immediately fix the tissues in 4%paraldehyde for one to two hours at room temperature and proceed with standard procedures for whole mount examination or for cryosectioning. These three dimensional reconstructions of a confocal image stack in the region of the stomach show GFP positive enteric neural crest cells, the di stained vascular system and emerged image of both networks. Representative histological sections in the stomach region also show GFP positive enteric neural crest cells, the DII stained vascular system and emerged image.

Three dimensional reconstructions can be used to produce movies with 360 degree rotations here. Three dimensional reconstructions in the cecum region show the GFP positive enteric neural crest front in green and the dye stained vascular system in red. This panel shows a merge of both networks here.

The confocal image stack was used to produce a movie of the enteric neural crest cell migration front and the associated vasculature. This technique allows us to study the mechanisms underlying the innervation and vascularization of organs during development by enabling strong fluorescent labeling of neur crass cells and blood vessels. While attempting this procedure, it's important to remember that it requires excellent end coordination under the microscope.

Overall, both the neural tube transplantation and the dye injection require lots of practice to be successful.