Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The goal of this surgical intervention is to elucidate mechanisms of limb induction in the chick embryo by preventing signaling molecules from diffusing from medial tissues to cells in the lateral plate mesoderm and studying its effect on gene expression. This method can help answer key questions in developmental biology such as, what are the inductive signals to control limb bud initiation. The advantage of this technique is that the chick embryo is accessible to surgical manipulation and very robust, in addition, the unoperated contralateral control limb bud serves as a very useful internal control.

Susan Wilde will demonstrate the procedure. To begin, cut a 1 cm square piece of 0.013 mm thick aluminum foil and sterilize it by wiping it with 70%ethanol. Then, place the square onto a 6 cm sterile plastic petri dish.

To prevent static cling discard the lid and make a new one for the dish from the same foil. Next, set the petri dish on top of a stage graticule under a binocular microscope set at 1.6x magnification. Use a number 15 blade small scalpel to cut 1.3 mm wide strips of the foil that are 2 mm long.

Separate the foil strips using two pairs of number four watch makers forceps. Then, make a right angled bend in the center of the foil barrier such that it resembles a hinge. Line up the barriers in the center of the petri dish with one side flat against the dish and the other pointing up out of the dish, prepare 20 or more barriers before operation day.

Place the foil lid over the dish and store the barriers in a place where they will not be disturbed. With the egg lying on its side, grasp a pair of type AA strong forceps firmly and use a controlled stabbing motion to pierce the blunt end of the egg containing the air sac while holding the egg with the free hand. Make sure that the inner shell membrane has been broken.

Next, take the egg in both hands and turn it 180 degrees along its horizontal axis. Next, take a piece of clear tape, approximately 5 cm square and stick this closely over the uppermost surface of the egg to stop the eggshell from crumbling onto the embryo when the egg is opened. Use a pair of curved scissors to gently break through the shell and its associated inner cell membranes in the center of the uppermost part of the egg.

Make a very small hole with one blade of the scissors so that air can enter the egg over the embryo. Use the scissors to enlarge the hole in the shell to a circle with a diameter of approximately 1 cm. Remove this piece of egg size shell from the egg.

Cover the hole on the egg with a piece of clear tape approximately 5 cm square. Press the tape to stick it only at the lip of the hole leaving the remaining tape free. The free tape can then be grasped to easily unseal the egg.

Take a stage 15 embryo from the incubator and place the egg on an egg rest under a binocular microscope set to a 3.2x magnification or higher. Then, remove the upper layer of clear tape. Using a steel micro knife, make an incision through the vitelline membrane and lateral place mesoderm adjacent to somites 26-32 on the right side of the embryo.

Pick up the barrier with number 5 micro forceps at one end and twist the barrier to insert the free end into the incision. As soon as possible, seal up the egg with clear tape and return it to the incubator. If a bead is to be inserted in conjunction with a barrier, firstly, prepare retinoic acid soaked beads as described in the accompanying text protocol.

Next, take the bead and a pair of number 5 micro forceps and insert it into the cut face of the lateral place mesoderm on the distal side of the incision. Then, insert the barrier into the incision proximal to the bead. Seal the egg and return it promptly to the incubator.

If a bead or beads are to be inserted without a barrier, do not make the long incision. Instead, make a small incision through the vitelline membrane and into the lateral plate mesoderm at the position the bead is to be placed. Then, take the bead and number 5 micro forceps and insert it into the hole in the lateral plate mesoderm.

Push it in a little further with the closed ends of the forceps. Take a windowed stage ten embryo from the incubator and place the egg on an egg rest under binocular microscope set to 3.2x magnification or higher if preferred. Then, using a steel micro knife, make an incision through the vitelline membrane and lateral plate mesoderm, lateral to the primitive streak at the caudal end of the embryo in line with the somites, approximately six somite lengths long.

Next, pick up the barrier with a pair of number 5 micro forceps, at the end protruding from the petri dish and twist the hand to insert the free end of the barrier into the incision. As soon as possible, seal up the egg with clear tape and return it to the incubator. If a bead is to be inserted without a barrier, make a small incision through the vitelline membrane and into the lateral plate mesoderm at the position the bead is to be placed.

Then, take the bead and a pair of number 5 micro forceps and insert it into the hole in the lateral plate mesoderm. Finally, push it in a little further with the closed ends of the forceps. Shown here is the barrier position at the presumptive leg level of somites 26-32 in a stage 15 embryo.

This barrier leads to both Fhf10 and Fhf8 expression being absent in the leg bud region in the operated side and present in the unoperated left side. However, Tbx4 expression is observed at both stage 19 and stage 23 on the operated side. This implies that Tbx4 expression alone is not sufficient to initiate hind limb outgrowth from the lpm.

When rhetinoic acid soaked beads are placed distal to the barrier at stage 15, Tbx4, Fgf10 and Fgf8 are all expressed on the operated side as they're on the left control side. Thus, in normal development, retinoic acid from the somites is essential before limb bud outgrowth can be initiated. After watching this video, you should have a good understanding of how to use impermeable barriers and candidate soaked factored beads to study how inductive signals regulate limb bud initiation in the chick embryo.