Apical Ridge Recombinations with Limb Mesenchyme Fig 3B

1. First remove the ectoderm from a limb bud that will be used to provide the mesenchyme core, and remove this ectoderm from the dish.

2. Now transfer limb bud that will provide the ectodermal jacket for the recombination into the dish containing the isolated mesenchyme core.

3. Separate ectodermal jacket, and immediately push mesenchymal core inside. Steps 1-3 are to be carried out in ice-cold medium on a cold slab.

4. Take the dish with recombined tissues off cold slab, and leave at room temperature for 10 min. Then place at 38°C for about 1 h to allow the ectodermal jacket to shrink on to the mesenchyme.

5. Graft recombined tissues to prepared site in a new embryo to continue development.

a. Pin to limb bud stump. The embryo normally lies on its left side, so that dorsal surfaces of right limbs are uppermost. It is therefore easy to manipulate the right limb bud and leave the left as a control. Cut off right wing bud to give a stump (stages 20-22 would be suitable). Pin recombined tissues to stump. It is best to push one pin through one edge of the tissue recombination in a dish so that the hooked end marks the ectoderm surface and the pointed end without a hook protrudes from the naked mesenchyme surface. Then transfer speared tissue into host egg and pin tissue loosely to stump, so that the bare mesenchyme surface abutts the mesenchyme of the stump. Then take a second pin and secure other edge of graft. (Pins can be removed the next day.)

b. Place recombination in trench on dorsal surface of wing bud. Cut a square away from the tip on dorsal surface of right wing bud (developmental stages 22-24). Lift up ectoderm and a layer of mesenchyme to create a shallow trench, and remove tissue from the egg on a small spatula. (The bleeding will soon stop.) Transfer tissue recombination into host egg and stand tissue recombination in trench with mesenchyme surface downward.

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