The overall aim of this procedure is to graft a dorsal neural tube explan from a quail embryo into a staged matched chicken embryo, so as to trace the migration and fate of neural crest cells. This is accomplished by first tearing a small hole in the host chicken embryo's IGN membrane to access the neural tube. The donor quail embryo is then removed from its egg to prepare it for graft excision.
In the next step, the region of interest is excised from the host embryo's neural tube. A similar region is then excised from the donor neural tube for grafting. Next, the donor neural tube is transplanted into the host neural tube.
Finally, the chimeric embryo is incubated to the desired stage for detection of neural crest arrived cells in the organ of choice. Ultimately, the migration and differentiation of donor derived neural crest cells to various locations in the host embryo can be visualized through immunofluorescence microscopy. Visual demonstration of this method is critical because the micro dissection and grafting steps require very fine motor skills, making them difficult to learn.
Begin by washing any debris from the chick eggs with tepid water. Arrange the eggs horizontally on a tray and then mark the sides facing up with pencil to denote the region where the embryo will be localized. For X ovo preparation of donor embryos, incubate the quail legs blunt side.
Up next, place the eggs in a 38 degrees Celsius humidified incubator and turn on the rocking function. After the eggs have finished, incubating sterilize the tops by spraying them with 70%ethanol and then wiping it off quickly with a kim wipe To avoid any absorption through the egg, shell place each sterilized egg onto an individual egg holder, similar to the Petri dish lined with folded Kim wipes seen here. Then using sterilized AA forceps, tap a small hole into the upper surface of the egg shell at the pointed end of the egg.
Equip a five milliliter syringe with an 18 and a half gauge hypodermic needle. Then with the bevel facing the pointy end of the egg, insert the needle vertically into the hole in the egg shell. To avoid accidentally damaging the yolk via suction, clean the hole with 70%ethanol as before and seal the hole with scotch tape.
Then with the double A forceps, tap another hole into the marked upper surface of the horizontal leg, not quite at the apex. Next, insert one side of the curved iris forceps into the hole parallel to the egg. Shell then pinching down on the shell and working in a circular motion.
Break a window, two centimeter in diameter in the chicken egg, shell discard the removed egg shell. The embryo should appear as an opaque disc on top of the yolk. Discard any unfertilized eggs identified by a small white spot on the surface of the yolk or the absence of the blaster derm.
Now while wearing proper eye protection, bend a 26 and a half gauge hypodermic needle at a 45 degree angle starting at the base of the needle. After attaching the bent needle bevel side up to a one milliliter syringe, fill the needle with diluted India ink. Puncture the yolk membrane outside the perimeter of the blaster, and slide the tip of the needle beneath the embryo, close to the surface of the yolk, but not in the embryonic layers.
Then inject just enough ink to outline the embryo and avoiding injecting any bubbles. Carefully withdraw the needle. Now using a stereo microscope and a light source with a limited heat load, such as the fiber optic gooseneck lamp shown here.
Stage the embryos according to hamburger and Hamilton. Record the stage on the eggshells. Then apply two to three drops of warm sterile ringer solution to the surface of the embryos to prevent dehydration and contamination.
Then stretch para film over the surface of the eggs to temporarily seal the windows. Next, prepare the quail eggs for transplantation. Before removing the eggs from the incubator, fill a Petri dish with ringer solution.
Then open the eggs on the blunt side with curved forceps to reveal the embryo. Using dissecting scissors, make four cuts in the shape of a square through the vili membrane and blaster derm outside the embryo, making sure that all the cuts meet. Then using curved iris forceps, gently grasp one cut edge and lift the embryo from the egg and transfer it to the prepared petri dish.
Finally, gently remove the vitelline membrane with number five forceps, and then stage the embryos under a dissecting microscope. Remove the para film from the host chick embryo. Then using a sharpened tungsten needle, tear a small hole in the VII membrane at the desired region of the neural tube.
Add a drop of ringer solution to the embryo. Next, for unilateral grafts, like the one being performed in this video, use a poured glass needle to carefully make transverse rostral and cordal incisions to the midbrain region of the neural tube, not extending beyond the lumen. Then cut bilaterally between the dorsal neural tube and the parial mesoderm.
Finally, carefully separate the excised ex explan from the neural tube, and then remove it from the embryo by aspirating it into a glass micro pipette. First, use a stage matched quail embryo. Then excise a similar sized region of neural tube as just shown and aspirate the donor X explan into a glass micro pipette containing ringers solution.
Transfer the X explan adjacent to the excised region of the chick host. Then using a pulled glass needle, orient the ex explan gently guiding it into the ablated region. Carefully add a few drops of ringer solution to the embryo to prevent dehydration.
Finally, seal the window with packing tape, making sure that the tape seals the entire region of the windowed egg, return the egg to the incubator until the desired stage, turning the rocking function off while incubating the kyira, the X may be gently turned by hand twice a day to increase their viability. If later stages of development are targeted, this representative image of the grafted region of the neural tube after six hours of re incubation to HH 11, shows the expected incorporation of the grafted quail donor tissue into the host chick neural tube. This cross section through the grafted region at HH 11 shows neuro crest cells labeled with hnk, one migrating laterally away from the neural tube.
Quail cells contributing to the neural crest cell migratory stream, and to the neural tube are clearly labeled with QCPN. At later stages, quail neural crest cell derived cells can be traced to their final target tissue. As can be seen in this figure, QCPN labeled cells are interspersed within the chick embryo meen chi of the maxillary process At E five, the QCPN antibody can easily be combined with other antibodies to examine the differentiation of quail derived neural crest cells.
In the host environment. As seen here, quail neural crest cell derived trigeminal sensory neurons are labeled by QCPN and T UJ one antibodies. Following this procedure, other methods like immunohistochemistry and in situ hybridization can be performed in order to answer additional questions like what tissues do neuro crest cells give rise to, and what genes do neuro crest cells express as they differentiate into various tissues.
