254 



EXTIRPATION OF ORGAN ANLAGEN 



or the Urodele embryos at stage #25 or #26 may be used. Remove the membranes and 

 place the embryo on its left side in a Permoplast or agar depression in a Syracuse dish. 

 Locate the optic bulge and with a glass needle and hair loop cut a rectangular piece of 

 epidermis directly over the eye, leaving the upper margin of the rectangle uncut (a hinge). 

 Lift the flap of epidermis and locate the underlying optic vesicle. 



1. In some embryos, take the hair loop and cut off the most lateral (outer portion) of the 

 optic vesicle, that portion known to give rise to the retina. 



2. In other embryos, excavate the entire optic vesicle, clipping it off at its base. 



If the flap of epidermis is still intact, replace it over the wound. If it has been damaged, 

 simply leave the embryo in 10% Standard Solution until the wound has closed completely 

 by the spreading of adjacent epidermis, and then transfer to full strength Standard Solu- 

 tion. The extirpation of the optic vesicle does not do away with the eye muscles entirely 

 but they are reduced in size and are atypical. 



THE HEART ANLAGE 



The partial or even the complete extirpation of the heart forming mesoderm of Ambly- 

 stoma is generally followed by regeneration, which may mean the entire heart. The 

 heart field extends well beyond the limits of the ultimate heart forming area so that even 

 the extirpation of an area equivalent only to the ultimate heart may result in the formation 

 of a small heart or hearts. With these facts in mind, derived from the work of Copen- 

 haver, attempt to extirpate parts of the heart as follows: 



Amblystoma stage #15 or Anuran stage #14 should be used, for the heart mesoderm is 

 derived from two separate lateral primordia representing the free ventral margins of the 



two hypomeres. These fuse in the mid-line, ventral to the 

 pharynx and just posterior to the thyroid anlage to give rise to a 

 single tubular heart. The two primordia meet ventrally at about 

 stage #27 in the Urodeles and about stage #17 in the Anura. 



Remove the membranes and orient the medullary plate stage 



with the right ventro-lateral side uppermost, in a depression in 



agar or Permoplast. The exci^'ion and healing should be in 10% 



Standard Solution. With sharp needles outline the heart area on 



the right side of the mid-ventral line and cut out a rectangular 



area deep enough to include all three germ layers (see figure). 



It may be necessary to excavate cells with a hair loop, and this 

 Medullary plate stace of an 11111 ^ • 1 ^1 • 1 ^ ■ 1 t^ ..1 l 



' should be done extensively on the right side. Keep the embryo 



amphibian embryo showing .,-,„ ,,_,. .,, ,.,i, ,, 



^, .. u ^ in 1 Ovo Standard Solution until the wound is healed over and then 



the presumptive heart area 



to be extirpated (stippled). gradually return to full strength Standard Solution. 



Urodele embryos may be allowed to develop as long as possible because the heart devel- 

 opment can be viewed through the thin covering ventral epidermis. The Anuran embryos 

 should be dissected, or fixed, stained, and sectioned within about a week following the 

 operation to determine the extent of damage or recovery of the above extirpation. (See 

 section on Heart Field Operations. ) 



THE BRAIN 



Amblystoma punctatum (or tigrinum) embryos should be freed from their jelly membranes 

 at stage #14 to #16 and allowed to grow at about 18°C. in Urodele Growing Medium until 

 they reach stage #32. At this time the primary brain vesicles have been developed and 

 can be outlined from the exterior of the embryo. 



