432 



Special Vertebrate Organogenesis 



much earlier than they can in anuran forms 

 (Harrison, '17). 



The area for forelimb formation has 

 been located in the body wall mesoderm 

 of Amblystoma punctatum at stage 25 (Har- 

 rison, '15, '17, '18); stage 18, high nem-al 

 fold (Detwiler, '18); slit blastopore, stage 

 13 (Detwiler, '29); Vogt ('29) located it in 

 Triton at the beginning of gastrulation. 



The time at which a definite structure can 

 be located in the embryo has an important 

 bearing on its usefulness in transplantation. 

 In anuian forms the larvae are quite well 

 developed before the limb bud can be located 

 and removed. In Amblystoma punctatum. 

 the definitive tissues which later go into the 

 formation of the limb are located before 

 any visible surface indications of the bud 

 can be observed. This location of tissue, 

 consisting of a portion of the somatopleuric 

 mesoderm covered by overlying ectoderm, 

 was originally found in the tail bud stage 

 (Harrison, '17) (stage 29). Since that time, 

 Detwiler ('18), by the use of Nile blue 

 sulfate transplants, was able to locate the 

 limb area in the stage of high nemal fold 

 (stage 18), and later traced the definitive 

 material back to the slit blastopore stage 

 (stage 13). 



The limb material can then be trans- 

 planted before it develops into any sort of 

 structure. Its effect upon the organism upon 

 which it is growing can be tested, as well as 

 its normal and abnormal relationships to it. 

 It affords us, then, a perfect experimental 

 tool for studying various effects and has been 

 used as such to great advantage. The limb 

 disc, at the usual operating stage (29), is 

 localized as a thickening of the somatopleure 

 centered ventral to the fom'th myotome and 

 extending ventral to the third and fifth 

 myotomes. The area involved is usually 

 described as a circular area three and one- 

 half somites in diameter (Harrison, '17, '18). 



The constant localization of the limb ma- 

 terial with reference to other structures 

 which can plainly be observed in the em- 

 bryo is a very important factor in the ease 

 with which the limb materials can be located. 

 Harrison's experiments in determining the 

 extent of the limb-forming materials furnish 

 an interesting example of the capacity of the 

 limb region to regenerate. The transplanta- 

 tion experiments test its capacity to develop 

 in new and strange surroundings. 



The experiments were performed by re- 

 moving the material (both ectoderm and 

 somatopleuric mesoderm) from a given area. 

 The loose mesoderm left behind after the 



removal of the majority of the limb-forming 

 tissue was either careiully removed or left 

 m place. VVhen the mesoderm was carefully 

 removed there was less likelihood of re- 

 generation. When the operative area was 

 large the number of regenerating limbs was 

 reduced. 



Alter the removal of tissues from this re- 

 gion, the tissues forming a ring around the 

 outside of tlie wound tend to migrate toward 

 the center and lorm a new limb-forming 

 region. This is an area of mesodermal ma- 

 terial which under normal conditions would 

 never form a limb but which under the 

 imposed operative conditions moves into 

 the region formerly occupied by limb mate- 

 rial. This region Harrison would term as 

 normally possessing a weak potency for limu 

 formation. Under normal conditions it is 

 marginal tissue. It is only under unusual 

 circumstances of limb removal that it pos- 

 sesses the capacity for limb formation. 



It is interesting that when a transplant (5 

 somites in diameter) larger than iV-i somites 

 is removed from the limb region and trans- 

 planted to the flank region, and then the 

 central portion of the graft is removed, the 

 behavior of the ring of material around the 

 limb is the same as though it were in normal 

 position. A considerable number of regen- 

 erating limbs can be secured after this pro- 

 cedure. 



When a wound of V/k somites in diameter 

 is made in the limb region and the meso- 

 derm carefully cleaned from the floor of the 

 wound, regeneration of a limb seldom occurs. 

 This Harrison ('18) has taken as the region 

 which contains the preponderance of normal 

 limb-forming material. The limb is formed 

 by the rapid multiplication of the cells con- 

 tained within the limb disc and not by the 

 migration of tissues which lie outside of 

 the disc. 



This fact again emphasizes the inde- 

 pendence of the limb. The histology of the 

 so-called limb bud shows numerous mitoses 

 while the regions surrounding it show many 

 less karyokinetic figures. This is a histologi- 

 cal indication of the limb independence 

 which is proved by transplantation and also 

 by the lack of relationships to the meso- 

 dermal somites. 



When the limb disc of diVz somites is re- 

 moved and the wound is covered by indif- 

 ferent ectoderm, no limb develops (Harrison, 

 '18). The extirpation of the limb region 

 with the cleaning of mesodermal cells is suf- 

 ficient to reduce to 14 per cent the chances 

 of regeneration of the limb. When the limb 



