3.4.1. Improving Visibility of the Embryo
Have the syringe filled with contrast medium before starting. Push the needle into the yolk outside the area pellucida, and move the tip, as horizontally as possible, into the subblastodermal space. Be careful not to scrape the underside of the embryo. Expel 0.l mL, taking care not to introduce air bubbles (see Note 4).
3.4.2. Grafting the Donor Notochord into the Host
1. Illuminate the embryo with a single fiber optic lens on an oblique path. Use around 50x magnification, hold egg with one hand. Gently press down on the compressible egg stool to keep the object plane in focus. Support wrists on hand-rests and steady the third to fifth fingers of the operating hand on the side of the egg—this will enable you to stabilize the needle holder better. Avoid large movements.
2. Under the dissecting microscope, place one drop of Howard's Ringer over the host embryo. Nick the vitelline membrane with a fine needle, grasp the edge of the membrane with no. 5 forceps and reflect a flap above the embryo; be sure to pull back both layers of the vitelline. The hole in the vitelline should be just large enough to expose the operation site. As the vitelline ruptures, the drop of Ringer will flood in and prevent albumen from covering the embryo (if this should happen, both host and donor tissues become very sticky, and grafting becomes difficult).
1. The manipulation described below will result in ectopic induction of floor plate, since the graft will face the dorsoventrally uncommitted neural plate (Fig. 1, see Note 5 for sclerotome induction). Using a very sharp tungsten needle, make a longitudinal slit through the ectoderm at its junction with the neural plate in the region of the open posterior neuropore: push the point through and cut by pulling sharply upward (50x magnification). The slit should be 500-600 |im long. Periodically clean the needle of tissue or albumen by flaming (see Note 6).
2. Using the mouth pipet, and suck a small drop of Dispase solution into the fine capillary. Apply to the opened ectoderm of the host embryo. Use the tungsten needle to gently separate the neural plate from the adjacent paraxial mesoderm: roll off or push away the neural plate as it comes loose. Rinse the treated side with Howard's Ringer immediately to wash off the Dispase. The aim of this procedure is to make a pocket beneath the lateral part of the neural plate to house a notochord implant in close apposition with the basal surface of the neuroepithe-lium. Therefore, do not fully separate neural plate and paraxial mesoderm, as the graft will then settle down too deeply next to the host notochord.
3. Cut donor notochords into 300-400 |im lengths, transfer one piece in minimum fluid using a serum-coated 20-|L tip.
4. Deposit the notochord piece over the posterior end of the embryo, avoiding the albumen.
5. Maneuver the piece over the prepared slit. Open the slit by pulling on the lateral ectoderm with a needle. The notochord should drop into the hole. Push it in more deeply with the point of the needle.
1. After operations, place another drop of Howard's Ringer over the embryo, check that a graft has not been dislodged, return the shell flap, and reseal egg with canvas sticky tape.
2. Stretch the tape before applying, and smooth it down carefully onto the egg. Make sure the tape adheres smoothly to the shell without creases that would admit air. High humidity inside the egg is essential after operations—drying, even slightly, is the major cause of mortality. Mark the shell with name and number/stage of operation using a pencil and place in the recovery incubator (38-39°C, >90% humidity). Do not open the egg until you want to harvest the embryo—checking up on progress causes drying and reduces survival.
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