The overall goal of this procedure is to use quail duct chimeras as a means to investigate mechanisms through which neural crust cells generate species specific patterns during cranial facial development. This is accomplished by first dissecting and removing a strip of the neural fold from the mid and anterior hind brain region of a stage 9.5 quail embryo. The second step involves transferring the quail donor neural fold to the duck host embryo.
The third step involves dissecting and removing the equivalent region of a stage 9.5 duck host embryo. The final step is to place the coil neural fold into the duck, mid and anterior hindbrain region. Ultimately, the resulting chimeric embryos can pinpoint neural crest mediated changes in gene expression through in C two hybridization, and can also reveal morphological changes through histological analyses.
I first had the idea for this method when I was watching Brown pelicans fly over Ocean Beach in San Francisco. I realized that neural crest cells must be the source of species specific pattern since they give rise to the bones and cartilages of their long beak skeleton. Although I was unable to track down brown pelican eggs, I was able to find white Peking duck.
Given that San Francisco has the largest Chinatown outside of Asia By exploiting the differences in the rates of maturation and morphology between quails and ducks. This method can help us answer key questions in developmental biology such as understanding the origins of species specific pattern, Performing the surgery will be Dr.Jennifer Fish, who's a postdoctoral scholar In my laboratory, assisting will be Kate Ovitz, who is a graduate student Using a buns and burner heat the narrow end of a paste pipette until the glass along the taper begins to melt. Remove it from the flame and pull the tip until the pipette becomes sealed.
Be sure that a portion of the tapered part has an outer diameter between 0.7 and one millimeter. Then break off the sealed end about four centimeters from the taper. Next, attach about two feet of rubber tubing to the open end of the pipette blow on the other end.
There should be no air passing through the connection or other end of the pipette while providing gentle pressure by blowing on the tube. Use a propane pencil, flame torch to heat an oval area on one side of the pipette just below the start of the taper. As soon as the glass begins to buckle outwards, remove the pipette from the flame and blow gently through the rubber tubing.
This will cause the heated part of the glass to form a sausage shaped bubble. Too much pressure will cause the glass bubble to pop, and this should be avoided. The resultant window in the pipette should be about one centimeter wide and one and a half to two centimeters long.
Now point the tapered end upwards and scrape the bubble into a glass waste container while gently blowing through the tubing to prevent glass fragments from entering the pipette. Then using the propane flame, carefully burn off the remaining edges of the bubble and fire polish the sides of the open window. Next, hold the diamond tip pencil in a vertical position, use a diamond tip pencil to score the tapered end of the pipette one centimeter from the beginning of the taper.
Using forceps, gently break the pipette at the scored line. To remove the tip, check under a dissecting microscope to make sure the break is clean and not jagged. Hold the pipette as one would hold a pencil with an index finger over the open window at approximately 9.5 millimeters from the tip.
Soften the glass on the pipette by holding it adjacent to the edge of the flame of an alcohol burner. Apply pressure to the tip with forceps to bend the narrow end of the pipette down at a 60 degree angle under a dissecting scope. Quickly and carefully, fire polished the tip with the alcohol burner.
The finished opening should be rounded and unobstructed. Note that the tip will become constricted and unusable if held in the flame too long. Now lubricate a 2.5 centimeter length of rubber tube with 70%ethanol.
Slide it over the shaft of the pipette from the bottom up to cover the open window. Then place a two milliliter latex bulb onto the open end of the pipette. To maintain internal air pressure, first, prepare the host egg.
Cut the Vitale membrane using a flame sharpened tungsten needle, and pull the membrane over the embryo. Then reseal the egg with tape as done for the donor egg. Remove the tape from the window on the donor egg to cut the neural fold from the donor embryo.
Make slits on both sides of the neural tube using a flame sharpened tungsten needle. Then cut across the neural tube at the desired anterior and posterior levels of the graft and tease the graft apart from the rest of the neural tube. Pay attention to any particular anatomical details like the anterior versus posterior shape of the graft, which will the donor tissue to be oriented properly.
When placed in the host, Be sure the tungsten needle is extremely sharp. Use quick and deliberate strokes in order to remove the tissue completely and in one piece, Remove the neural fold from the donor embryo using a shaman pipette. Draw up as little liquid as possible or the tissue could float away on transfer.
Now remove the tape from the window on the host egg and use the shaman pipette to place the donor neural fold alongside the host embryo adjacent to the region of the neural tube that will receive the transplant. Now separate the neural fold from the neural tube of the host embryo as done with the donor. Take care to remove a graft of equal size as the donor tissue.
Gently push this host tissue far away from the embryo. Then with a blunt tungsten needle or the rounded tip of a micro pipette, gently move the donor neural fold into the host neural tube, maintaining the proper anter posterior and dorsal ventral orientations. Make sure the graft is tucked in along the sides, but be sure not to poke or damage the underlying tissues.
Sterile saline should be carefully and gently applied over the host embryo. If there are any signs of desiccation, now carefully reseal and label the egg and gently return it to a high humidity incubator where the chimeric embryo can develop to the desired stage. For analysis.
For analysis, collect the chimeric embryos in freshly made cold Sarah's fixative. Then immediately place them on a rocker at four degrees Celsius for an overnight fixation. This will allow for more sensitive detection of quail eggs with the anti quail antibody.
Another important step for gene expression analyses by real-time quantitative PCR is to freeze the embryos and liquid nitrogen prior to RNA. Extraction coils and ducks show considerable differences in size and shape, and thus their embryos are ideally suited for making a chimeric system. To study C craniofacial development.
Specifically, the system is useful for investigating the role of neural crest cells. In patterning the facial skeleton in the chimeric qua embryo donor quail neural crest cells migrate into the host duck mandibular arch. In a duck like pattern, quail donor cells in green are viewed with an anti quail antibody from the ventral view.
In a stage 12 chimeric embryo, the donor neural crest generates a mandibular skeleton that is quail like in size and in shape. Once mastered, each transplant can be done in approximately five minutes. If done properly any longer, puts the embryo at risk for desiccation and survival rate will decrease.
While performing this procedure, it's important to be aware of your posture, relax your shoulders, and maintain good hand position. Also, develop a routine and place your surgical tools and reagents in the same place every time so that you can keep your eyes in the microscope and move your hands in an automated manner.
