The overall goal of this procedure is to show how to determine the effects of growth factors and inhibitors, in the gene expression of developing chicken embryo limbs. This method can help answer key questions in development biology, such as how signalling molecules influence the growth and differentiations of cells in vivo. The main advantage of this technique is that it provides a method to test these factors directly in the embryo.
Though this method can provide insight into the limb development, it can also be applied to other systems, such as neural tissue, somites or gastrulation. Demonstrating the procedure will be Rebeea Mohammed, a graduate student from my laboratory. To begin, transfer one milliliter of the beads from the original stock solution into a fresh tube.
Allow the beads to settle, then remove the liquid, and replace it with one milliliter of PBS. Repeat this step five times and store the washed beads at four degrees Celsius, as a slurry in PBS. Select the beads for grafting, by removing them from the washed stock solution using a 20 microliter pipette and place them into one milliliter of PBS.
Using a stereoscope, choose beads that are around 100 microns in diameter. Transfer the selected beads in two micro-liters of liquid into a new Petri dish containing a 20 milliliter drop of PBS Next, remove the PBS from the beads. First, with 20 micro-liter pipette, and then, remove the residual liquid by capillary action using a fine watch-maker's forceps.
It's important to remove as much liquid as possible so that the growth factor is not diluted when it's added to the beads. Pipette the growth factor directly onto the beads. For FGF18, add 0.5 micro-liters of recombinant FGF18, reconstituted add 0.5 milligrams per milliliter in PBS with 0.1 percent BSA.
Then, place several drops of water around the edges of the dish to prevent the liquid from evaporating. When ready, incubate the beads for one hour at room temperature. Following incubation, remove the drops of water from the dish with a Pasteur pipette and place the dish on ice.
Derivatize the beads, by first using a spatula to transfer the beads to a 1.5 milliliter micro-centrifuge tube. Then, add one milliliter of 0.2 normal formic acid and incubate the beads in the acid on a shaking platform for one hour. Next, allow the beads to settle, remove the formic acid and replace it with one milliliter of water.
Wash the beads for one hour on the shaking platform and repeat the wash six times to remove any residual formic acid. Following the last wash, remove the remaining liquid and store the beads at four degrees Celsius. Use a spatula to remove a small palette of beads approximately five to ten micro-liters in volume from the stock, and place the beads into a 30 milliliter Petri dish.
Then, add one milliliter of DMSO. Next, use a P2 pipette set to two micro-liters, to transfer beads of approximately 100 microns in diameter, to a 20 micro-liter drop of solvent in another 30 milliliter Petri dish. Remove the bulk of the solvent with a 20 micro-liter pipette and any residual solvent with a two micro-liter pipette.
Then, cover the beads with 20 micro-liters of the drug to be applied, dissolved in DMSO. Incubate the beads in the drug solution for one hour. Protect the beads from light, as many drug molecules are light sensitive.
To help visualize the beads, transfer four to five beads in two micro-liters of liquid, into 20 micro-liters of two percent phenol red dissolved in water. Remove a single bead with fine watch-maker's forceps and rinse the bead in PBS. Do not leave beads in the phenol red solution for more than 15 minutes before rinsing them, as this can cause lost of activity.
Cut fine tungsten wire into three to four centimeter lengths Then, insert one piece of the wire, into the end of a glass Pasteur pipette and melt in a Bunsen burner to fix it in position. Prepare up to 10 needles to ensure that there are spares if they are damaged. Next, place the end of the wire into a blow-torch flame and hold it there until the tungsten glows white and tip is sharp.
This takes around two to three minutes. Add five to six drops of artist India ink to 15 milliliters of PBS containing 100 units of Penicillin and 0.1 milligrams of Streptomycin per milliliter. To prepare the chicken embryos, incubate eggs with the blunt end up until they reach the desired stage for most limb bud manipulation, which is typically three to five days.
When the desired time point is reached, use blunt forceps to tap on the blunt end of the egg and break the shell. Then, use the forceps to remove the shell and expose the embryo. Ensure the egg cell membrane is also removed.
To visualize the embryo, inject PBS supplemented with a few drops of artist India ink, under the embryo with a one milliliter syringe and a 21 gauge needle. The vitelline membrane overlying the embryo, can then be removed with fine forceps. Next, add two to three milliliters of a supplemented PBS with one percent fetal calf serum, 100 units per milliliter of Penicillin, and 0.1 milligrams per milliliter of Streptomycin, into the egg to ensure that embryo does not dehydrate.
Using sharpened watch-maker's forceps, open amnion over the developing limb buds. At these stages, the embryo will typically turn, so that the right side is upper most. Ensure the embryo does not dry out by adding more of the supplemented PBS solution when it is needed.
The amnion surrounding the embryo can be opened using a fine tungsten needle to reveal the developing limb. Then, use the sharpened tungsten wire to make an incision into the limb bud where the bead will be implanted. Avoid cutting all the way through the limb bud, where possible.
Although at younger stages, this is difficult. Pick up a bead, coated with either the growth factor or drug using extra fine, watch-maker's forceps. If the bead has been soaked in either DMSO or phenol red, be sure it gets rinsed with PBS before applying it to the embryo.
Transfer the bead to the embryo using the forceps, and then, use the sharpened tungsten wire to insert the bead into the incision location. Add an additional one to two milliliters of the supplemented PBS solution to the egg to ensure that the embryo is kept hydrated. Avoid applying the solution directly onto the embryo itself, as this can damage the embryo and cause the bead to be dislodged.
Next, use a ten milliliter syringe, equipped with a 19 gauge needle to remove only as much albumin as is needed to ensure that the embryo does not make contact with the tape used to seal the egg. Ensure that the needle does not damage the yolk, by sliding it down the inside of the shell. Finally, seal the egg with tape and return it to the incubator.
In order to confirm that FGF18 is able to act through MEK to phosphorylate ERK, the embryos were harvested one hour after bead grafting and immunostained for the presence of phosphorylated ERK, which can be seen surround the grafted bead in the image on the right. At HH stage 21, MyoD is not expressed in developing limb myoblasts. Although staining can be seen in the myotome of the developing somites.
Six hours following an FGF18 bead graft, MyoD can be seen induced in myoblast close to the bead. While there is no expression in the contralateral limb. This expression can be inhibited by co-grafting a bead, coated in a MEK inhibitor such as U0126.
Similarly, grafting a U0126 bead at HH Stage 21 reduces the endogenous expression of MyoD after 24 hours, compared with the untreated contralateral limb. In limb buds, at HH stage 19, FGF18 does not induce MyoD. However, co-grafting of beads soaked in FGF18 and a retinoic acid antagonist can overcome this, and ectopic MyoD is detected.
Once mastered, this technique can be done in three to four minutes, if it's performed properly. The development of this technique, allowed researchers in the field of development biology to explore growth factors signalling in embryo development. While attempting this technique, it's important to remember not to let the embryo dry out.
After watching this video, you should have a good understanding of how to graft beads into developing embryos to determine how growth factors signalling affects development.
