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Materials and Methods
The surgery involved in this procedure is delicate and requires a large measure of manual dexterity. These skills should be honed before relevant data is collected. In this experiment, surgery was practiced on embryos of appropriate age a week before data was attained.

All eggs were sterilized with 70% ethanol and allowed to dry before opening. Blastoderm stage chicks were harvested from 1-day old eggs using two techniques. In the first, bent forceps were used to tap a whole in the shell. Pieces of the shell were removed until the hole was large enough to allow a filter paper ring to be placed onto the yolk so that the ring framed the embryo (Figure 1). Alternately, the egg was swirled to dislodge the embryo and allowed to sit on its side so that the yolk would rotate and place the embryo on the upper side of the egg. The egg was cracked on the side opposite the embryo and the yolk and albumen were carefully placed in a large petri dish (figure 2). Filter paper was again used to frame the embryo. Many embryos were lost at the point when the yolk was broken and the embryo sank into the yolk, out of view. The procedure was the same for both techniques after this point. Sterile albumen from the egg was pipetted onto the albumen agar of the culture dish to form a thin nutrient layer for the embryo. Sterile Howard’s Ringers solution was pipetted into the moat of the culture dish. The amniotic membrane covering the embryo was allowed to adhere to the ring for one minute. The yolk was then punctured with scissors and the ring surrounding the embryo held with forceps while the chorion and other connective membranes were cut. The membrane around the embryo was cut and the ring was placed on the albumen agar such that the embryo was dorsal side down, exposing the heart on the ventral side. Drops of sterile Ringers solution were used to clear off any stray yolk that had accidentally been transferred with the ring and embryo onto the albumen agar. All embryos were staged and the culture dishes covered, labelled, and placed in a 37°C incubator for half an hour or until the embryo reached stage eight of development (Figure 3).

Glass pipette tips were flamed and pulled until they broke and formed extremely thin operating tools. Multiple attempts were required to make a straight taper because hot glass tends to curl after it breaks. Embryos at the Hamburger and Hamilton stage eight of development were operated under an Olympus DP12 microscope camera. The embryo was carefully moistened before the procedure with drops of Ringer’s solution. The drops were placed beside the embryo and not directly on or above it, in order to prevent damage to the embryo. The straightest, most thin glass taper was used to cut a slit down the midline of the embryo from the midway point of the embryo to the bottom half of the hood of the gut portal (Figure 5). Care was taken to avoid completely severing the upper half of the embryo (Figure 6). Development continues more normally if the brain is not completely bisected (J. Cebra-Thomas, personal communication, April 2004). Control embryos were not operated.
All embryos were returned to the 37°C incubator between observations. Photographs were taken daily. Video footage was recorded once the hearts formed and started to beat.


©Cebra-Thomas, 2000

Last Modified: May 2nd 2004

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Incision should be made through the anterior intestinal portal down the midline to the first somites, along the white line above.