This method allows investigators to assess tissue interactions between the basal forebrain and the face. The main advantage of this technique is that it isolates the basal forebrain for assessment and prevents contamination with neural crest cells, which would confound the analysis. The difficult part of this technique is the removal of the forebrain from the host and replacement with the donor tissue.
Demonstrating the procedure will be Diane Hu, a research scientist from Dr.Ralph Marcucio's laboratory. To begin, prepare DMEM media with neutral red, a glass transfer pipette, and sharpened tungsten. Using a 10 milliliter syringe and an 18 gauge needle, remove 0.5 milliliters of albumin from the pointed end of the eggshell.
Make a small hole on top of the shell using the point of scissors. Then place a piece of tape over the hole and cut a circular opening to expose the embryo. Stain the embryo with neutral red.
Harvest tissue grafts from the left side of the basal forebrain of stage seven or eight embryos. Use curved sharpened tungsten needles to gently incise a piece of the forebrain. In order to not include the underlying endoderm, slide the needle beneath the forebrain and parallel to the axis of the neural tube.
Pick the graft up from the donor embryo using the glass transfer pipette, and transfer it into DMEM containing neutral red for two minutes to stain it. Then place the stained graft into DMEM that does not contain neutral red until ready for engraftment. Incubate fertilized eggs from white Leghorn chicken at 37 degrees Celsius in a humidified chamber until Hamburger-Hamilton stage seven to eight.
Expose embryos as previously demonstrated. Using sharpened tungsten needles, prepare the graft site by cutting and then removing a 0.3 by 0.2 millimeter piece of basal forebrain to accommodate the graft. Avoid excessive disruption of the underlying endoderm, which will be evident as yolk granules will begin to leak through any tear that is made.
After transferring the graft to the host, position the graft to replace the extirpated basal forebrain of the host. Place tape tightly over the hole, and return the embryos to the 37 degree Celsius incubator until they are ready for analysis. Initially, chimeras were created by transplanting quail tissues into chick embryos.
Using the QCPN antibody, quail cells were visualized and distinguished from the host tissues. The quail-chick system confirmed that all of the graft was comprised only of neural tissue and was not contaminated with other cell types. Transplantation of quail tissue into duck embryos led to severely deformed chimeras due to a faster developing quail brain.
Therefore, transplantation of duck tissues into chicken embryos was performed. The resulting duck-chick chimeras suggest that the brain participates in regulating morphology. Whole mountain C2 hybridization was used to assess Sonic hedgehog expression in chimeras.
Similar to morphology, Sonic hedgehog expression on the duck side of the chimera appeared more duck-like. Preparing the host site takes practice. A large number of embryos do not survive, so creating a large number of chimeras can help ensure proper samples for analysis.
This method made it possible to assess how signals from the brain shape the Sonic hedgehog expression domain in the frontonasal ectodermal zone.
