^^^ LIMB FIELD OPERATIONS 



REGENERATION FOLLOWING EXTIRPATION OF THE ANLAGE* 



Secure embryos at stage #29 and remove all membranes. Place the embryos in a Petri 

 dish with an agar base. Select those which are exactly at stage #29 and compare with the 

 accompanying diagram on following page. 



Place the embryo (stage #29) on its left side in a Permoplast or paraffin depression in 

 an operating (Syracuse) dish. With a double-edged lancet or glass needles make cuts as 

 indicated below and in the accompanying diagram. The circular cut can be made most 

 easily by rotating the operating dish while cutting with iridectomy scissors in an immobile 

 hand. The ectoderm is pigmented, the underlying mesoderm is white and the deeper endo- 

 derm is grayish-tan (having slight pigmentation). The purpose of the following procedures 

 is to determine the areal extent and the depth of the limb field as of stage #29. The limb 

 area is ventral to myotomes #3-#5, just posterior to the gill anlagen. 



a. Remove ectoderm alone from area marked "A" (see acconnpanying diagram). 



b. Remove ectoderm and all underlying mesoderm from area "A". Be certain to 

 clean out all the white mesodermal cells beneath the excised ectoderm. 



c. Remove ectoderm from the larger area "B", leave the mesoderm undisturbed. 



d. Remove the ectoderm and niesoderm from the larger area "B". Again scoop 

 out all of the white mesoderm cells by means of a hair loop. 



e. Make a semi-circular cut (as indicated by arrows) peripheral to the margin of 

 area "B", deflect this flap of ectoderm anteriorly, and clean off and scoop out 

 all mesoderm cells from beneath the flap of ectoderm. Should the excavation be 

 so extensive that the flap of ectoderm does not cover, fill the cavity with yolk 

 cells from another embryo of the same age. Apply a bridge (Brlicke) for 30 

 minutes to hold the ectodermal flap in place until it heals. 



The unoperated side of the embryo will function as the normal or control for comparison 

 with the operated side in respect to limb development. Maintain in the same environ- 

 ment, however, some other unoperated embryos of the same age and stage as supple- 

 mentary controls. The best controls would be operated embryos but with the tissue left 

 in place, particularly since this is an attempt to determine the effect of extirpations. 



HETEROTOPIC TRANSPLANTS 



The orthotopic (homotopic) position is the normal position, the heterotopic one being a 

 different or strange position for the anlage. It is the purpose of this experiment to de- 

 termine the differentiating capacity of the limb anlage' at other than the normal regions. 

 If several donors are available, supernumerary limbs may be provided. Removing the 

 ectoderm and/or the mesoderm from the donor, make the following transplantations: 



a. Ectoderm alone from the area "A" of donor to region "X" of host. 



b. Ectoderm and mesoderm from area "A" to region "X" of host. 



c. Ectoderm and mesoderm from area "B" to region "X" of host. 



d. Ectoderm and mesoderm from area "A" to region "Y" of host (on hand). 



In all cases it is well to place the donor and the host side by side and to locate the re- 

 gions concerned in both embryos before the operation is begun. Use MS 222 (1/3, 000 

 cone. ) if necessary, to quiet any muscular activity. The transplant must be made 

 quickly and the transfer may be made on the point of a needle or on a small hair loop. 

 With a ball tip gently pat the transplant into place and hold it there by means of a chipped 

 coverslip (Brlicke) for at least 30 ininutes. If the glass cover tends to adhere to the 

 ectodernn, try a small piece of lens paper whose ends may be fastened to the P.ermoplast 

 base. In the case of the transplant to region "Y" on the host it will be necessary to place 

 the host in a Permoplast depression with the head protruding. Provide an adequate 

 wound area in the host, for healing is rapid. 



* Limb extirpation is included in the exercise on "Extirpations" but the procedure here is considerably more detailed. 



