278 LIMB FIELD OPERATIONS 



RELATION OF ECTODERM AND MESODERM IN LIMB FORMATION 



The experiments on regeneration (above) throw some light on the relationship of limb bud 

 ectoderm and mesoderm to normal limb development. Since mesoderm alone cannot be 

 grafted, it is necessary first to transplant ectoderm to which the foreign mesoderm at- 

 taches, and then to make a second transplantation, carrying along together the normally 

 unrelated ectoderm and mesoderm. 



a. Transplant the ectoderm alone from area "B" (see diagram) to the region "X" on 

 a second embryo. The transplant should be exclusively ectoderm, i. e. , all 

 original mesoderm must be cleaned away with a hair loop. Within 24 hours the 

 graft should be completely healed. Prepare another host by removing the entire 

 limb aniage, ecto- and mesoderm of area "X" of the previously operated embryo. 

 This consists of original limb ectoderm plus flank mesoderm. Note carefully 

 the exact stage of development of all embryos used; the size of the donor tissue; 

 size of the host area; and the extent of the excision of mesoderm. Should this 

 operation produce a perfectly normal limb it would indicate that the limb disc 

 ectoderm is of prime and exclusive importance, in the development of the limb. 



b. Place flank ectoderm over limb-disc mesoderm, making certain that all flank 

 mesoderm has been cleaned off of the transplant. Should a normal limb develop 

 from this operation, it would suggest the importance of the limb disc mesoderm 

 and the lability of the flank ectoderm. 



c. Transplant flank ectoderm only over limb disc mesoderm for 24 hours, until 

 thoroughly healed. Then excise and transplant the entire disc material (ecto- 

 and mesoderm) to the flank region of another host. In this case the limb disc 

 mesoderm alone is being transplanted to a foreign region. Should a normal limb 

 develop, this would confirm the importance of the mesoderm in limb development. 



These heterotopic transplants are more graphic if they are made between A. punctatum 

 and A. tigrinum (Harrison, 1933) and at stage #29 and #35. 



GENETIC FACTORS RELATING TO LIMB FORMATION 



RATES OF GROWTH - HETEROGONIC: 



The following experiments are devised to demonstrate the growth rate differences in- 

 herent in transplants, especially when made between fast and slow growing species. Up 

 to the beginning of the larval period, when the yolk has been absorbed, the more slowly 

 developing Amblystoma punctatum has a considerable forearm with two digits while at 

 the corresponding stage of development of Amblystoma tigrinum (fast developer) and A. 

 mexicanum larvae have only mesenchymatous nodules. Shortly after this stage, however, 

 the A. tigrinum limbs reach and surpass the size of those found in A. punctatum. To 

 demonstrate that the transplanted limb generally maintains its inherent (genetic) rate of 

 growth (Harrison, 1924) the following experiments are designed: 



a. Amblystoma tigrinum limb-bud is completely replaced by A. punctatum limb bud 

 at stage #35. 



b. A. punctatum limb bud completely replaced by A. tigrinum limb bud at stage #3 5. 



c. Heterotopic transplants made between A. tigrinum and A. punctatum, with host 

 limb fields untouched. This gives an excellent basis for comparisons. 



Such transplantations as these, involving mesoderm alone, would demonstrate most con- 

 clusively the relative functions of ectoderm and mesoderin in limb formation, outlined 

 in the previous set of experiments. The mesoderm of the limb bud seems to control the 

 fornn, the rate of growth, and the ultimate size of the urodele limb. And in turn, the 

 limb affects the number of ganglion cells in the adult spinal ganglia (Schwind, 1932). 



