The overall goal of the following procedure is to track opus Lavis neural crest cell migration. This is accomplished by first dissecting the neural crests from embryos at the neural stage. In the second step, the explants are plated in a fibronectin coated dish to study their in vitro cell migration.
Alternatively, after removal of the neural crest from a host embryo, explants can be grafted back to track their migration in vivo. Ultimately, the contribution of embryonic neural crest cell migration in opus lavis development can be investigated. Generally, people who are new to this method may find it difficult because early embryos are fragile.
The neural crest is a rather small cell population, and you have to make your own dissection tools. The visual demonstration of this protocol is critical. Each element of the dissection process has been optimized to ensure a clean removal of the neuro quest.
On the day of the experiment, take a 60 millimeter dissection dish coated with about 10 milliliters of 2%aros, and fill it with three quarters, normal amphibian medium, or nam almost to the top. Then use a plastic pasture pipette to transfer stage 16 XUS lavis embryos into the dish and place the dish under a binocular microscope using two sets of forceps. Remove the Vitale membrane from the embryos, taking care not to damage the dorsal part of the embryos.
Then incubate the embryos at room temperature until they reach stage 17 when the embryos have reached the proper stage of development. Dig a small hole in the aros at the bottom of the dish to make a niche for the embryos during the dissection, and then place an embryo inside the hole dorsal side. Up.Next, use an eyebrow knife and an insect pin to make an incision through two to three cell layers in the lateral part of the neural fold.
Then make a second medial parallel incision in the more dorsal part of the neural fold. After turning the dish 90 degrees if needed, make a third perpendicular incision at the posterior side of the first two incisions. The neural crest is a thick gray mass of cells underneath the pigmented outer layer of the ectoderm lateral to the neural tube and supra to the cephalic mesoderm, which is white.
After identifying neural crest, use the tip of the eyebrow knife to detach the pigmented ectoderm layer from the underlying neural crest starting from the third incision edge. Next, starting from the third incision, again, carefully separate the tissue from the mesoderm. Then detach the neural crest tissue anteriorly from the optic vesicle and free the explan.
When first, learning the procedure using C two hybridization to check that at least some of the explan express snail two, a canonical neural crest marker to confirm that the correct cell population is being dissected to plate the X explan for in vitro migration imaging First, fill a fibronectin coated dish with modified danal chicks medium. Then to prevent the X explan from coming into contact with the liquid air interface, aspirate some liquid into the tip of a P 1000 pipette. Next, draw the X explan into the pipette tip, and then more liquid.
Once the tip is inside the medium in the fibronectin coated dish, allow the X explants to slowly dispense by gravity, pushing very gently on the pipette plunger as necessary. Now, use the eyebrow knife to cut and space a few x explan throughout the dish. Avoiding the sides, allow the dish to rest without moving for 15 to 30 minutes until the X explants have attached to the fibronectin.
Then place the dish in a refrigerated incubator between 15 to 20 degrees Celsius until imaging before grafting the donor neural crest eggplants into the host embryos. Use coarse forceps to cut a fine glass cover slip into 1.5 square millimeter pieces. Next, dissect out the host neural crest in three quarters.
Nam as just demonstrated, taking special care not to damage the host Embryo. The single most difficult aspect of this experiment is to remove the host neuro crest without damaging the embryo. Some people prefer to use plasticine to hold better the embryo.
You can also either transfer the graft from another dish or make the two dissections in the same dish as you feel more comfortable with. Immediately place the neural crest explan onto the host ablated area. Then put a piece of cover slip bigger than the embryo onto the grafted embryo to maintain the graft in position.
After 15 to 20 minutes, gently remove the cover slip. Then after letting the embryo recover for another 10 minutes, carefully transfer the embryo to a new dish filled with three quarters Nam. When plated on fibronectin, the neural crest explants attach rapidly within 15 to 30 minutes, and the explan spreads flat within two hours.
After three to six hours, the cells begin to scatter by 24 hours at 15 degrees Celsius. Many cells have started to migrate away from the explan yolk makes the cells appear very bright under phase contrast, and filippo and la mepo cell protrusions are clearly visible. After Phin staining of the act in cytoskeleton.
In this representative Orthotopic grafting experiment, donor embryos were injected with GFP mRNA for following the grafted cells in an unlabeled host embryo. The cranial neural crest was then ablated on one side of the host, 30 to 60 minutes after the surgery, the graft healed replacing the ablated host's cranial neural crest. A few hours later, the GFP positive cells migrated out of the explan following the typical migration roots of the host neural crest cells towards the ventral cranial facial locations.
In these tadpoles neural crest derived cranial facial structures have even developed. When the cranial neural crest was ablated, the cranial facial structures disappeared, resulting in a shortened face with eyes adjacent to the cement gland. In grafted tadpoles, the donor neural crest cells contributed to the cranial facial structures resulting in a normal face morphology.
This dissection technique can be used to study many aspects of neural crest development. For example, for in vitro studies, you can treat your cells with many cues or drugs and then study the migration and differentiation properties. For in vivo studies of grafted embryos, you can manipulate the gene expression of both host and donor embryos by gaining loss of function approaches, and this way you will be able to study cell autonomous and non-cell autonomous aspects of the neuro cross development.
