276 LIMB FIELD OPERATIONS 



ROTATION OF THE LIMB AXES 



In these experiments it is necessary to mark the donor material in some way so that the 

 axes may be identified when the transplant is oriented in the host site. Generally this 

 may be accomplished in the normal course of excision where identifying marginal nicks 

 may be used as identifying markers. Both anterior-posterior (A-P) and dor so-ventral 

 (D-V) axes are concerned. Use Urodele embryos of stage #29 or younger. 



a. Carefully cut out the ectoderm and the mesoderm of area "B" so that the bulk of 

 the limb area remains intact. Clean out the wound with a small hair loop, re- 

 moving all loose (white) mesodermal cells, and replace the excised mass of tis- 

 sue in the same wound area after making a 180° rotation of the graft, as indi- 

 cated by arrows in the accompanying diagrams (Fig. F). Both axes will, in this 

 instance, be reversed. 



b. Remove ectoderm and mesoderm of area "B" from the right side of the donor and 

 orient it with the D-V axis still dor so-ventral but in the host wound area at the 

 region of the left limb anlage'. In this instance the A-P axis will be reversed. 

 {Fig. A) 



c. Without disturbing the normal field of the host, transplant from other embryos 

 both areas listed above, similarly rotated (i. e. , as in "a" and "b") but to the 

 locality of "X" on the hosts. In this manner a direct comparison may be made 

 with the hosts normal limb field. (See diagrams for various other possible ro- 

 tations of the two primary axes. ) 



d. Repeat the above rotational transplantations with older embryos, using Urodele 

 stages #35-#36, in order to determine whether there has been any further deter- 

 mination of axes within the limb field. 



These transplantations should be followed as long as the larvae can be maintained so that 

 the axial relations of the well developed limbs may be determined, and also to study the 

 degree of innervation of the transplanted limb. The control for these experiments con- 

 sists of excision and replacennent without rotation of the limb field. 



EFFECT OF SPLITTING THE LIMB FIELD 



Surgical interference with the limb field will often result in duplication of limbs, the 

 frequency varying with the species. Amblystoma punctatum is much more favorable than 

 is A. tigrinum. Transplants often result in duplications, due to disturbances to the limb 

 field. 



Locate the limits of the limb field on Amblystoma stage #29 and then cut away a thin strip 

 of ectoderm along the dorso-ventral axis of the field, splitting the field into two semi- 

 circles (see figure p. 275). With a small hair loop and needles, clean out the mesoderm 

 lying in the exposed region (beneath the removed strip of ectoderm). 



From another embryo of the same age or slightly older, remove a strip of mid-dorsal 

 ectoderm with underlying neural tube material, trimming the strip down until it will fit 

 into the excavated region of the limb field. See that all loose mesoderm is removed, and 

 then place this strip of ectoderm (epithelial and neural) into the excavated region, holding 

 it in place with a coverslip chip until it "takes" hold. Such a foreign strip of tissue will 

 block the integration of the anterior and the posterior halves of the limb field. 



Similarly split the limb field into dorsal and ventral halves. Notochord with overlying 

 ectoderm provides an efficient block, but the notochord should be taken from later stages. 



