276 LIMB F lELO OPERATIONS 



Place the emtryo (stage #29) on its left aide In a Permoplast or paraffin depression 

 In an operating (Syracuse) dlah. With a douhle-edged lancet or glass needles make cuts as 

 Indicated below and In the acconipanying diagram. The circular cut can be made most easily 

 by rotating the operating dish while cutting with iridectony scissors in an immobile hand. 

 The ectoderm is pigmented, the underlying mesoderm is white and the deeper endoderm is 

 grayish-tan (having alight 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 ncrotomes #5-#5, Just posterior to the gill anlagen. 



a. Eemove ectoderm alone from area marked "A" (see accompanying 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. Bemove ectoderm from the larger area "B", leave the mesoderm undisturbed. 



d. Hemove the ectoderm and mesoderm 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 (Briicke) for 



50 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 compari- 

 son 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 supplementary 

 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. 



KECOHD OF LIMB EXTIRPATION EXPERIMENTS 



