246 Origin and Differentiation of the Lens 
experimental study of the question of the self-differentiation of the lens 
in Amblystoma punctatum. It seems possible that by removing the 
optic vesicle or optic cup at various stages before and during lens forma- 
tion, one would be able to determine whether the continued influence of 
the optic vesicle was necessary for the normal differentiation of the lens. 
The operation of cutting out or removing the optic vesicle without 
injury to the lens forming ectoderm or to the developing lens rudiment, 
is a simple one. The embryos were operated upon in tap water under 
the binocular microscope. They were held in position with a small pair 
of fine forceps, and a semi-circular incision was made, with a very finely 
pointed needle, through the ectoderm a little caudal to the bulge made 
on the side of the head by the developing eye. The skin flap was then 
turned forward, exposing the rounded optic vesicle. The latter was cut 
off from the side of the brain, and then when the skin flap was turned 
farther forward the optic vesicle was carefully pulled away from the 
ectoderm, sometimes as a whole, or else in small pieces. Great care was 
taken, however, not to injure the developing lens or surrounding ecto- 
derm. After removal of the optic vesicle or the optic cup, the skin flap, 
which in later stages had attached to it the lens rudiment, was turned 
back into its original position, and held there by turning the embryo over 
on its side, with the skin flap against the bottom of the dish. The mere 
weight of the body sufficed to hold the flap in place. Healing was rapid; 
one to two hours generally being sufficient for complete closure of the 
wound. The operations were all made on the right side, while the left 
remained intact for purposes of comparison. The older embryos were 
first anesthetized in acetone chloroform in order to keep them quiet dur- 
ing the operation. 
The embryos thus operated upon were allowed to live from two hours 
to thirty days, then killed in Zenker’s fiuid, thoroughly washed, embedded 
in paraffin, and cut into serial sections 10 micromillimeters in thickness. 
They were stained in hematoxylin and Congo red. 
It has already been noted that in Rana fusca as well as in Rana palus- 
tris a lens will not arise from the normal lens-forming region of the 
ectoderm, if the optic vesicle is removed about the time of, or shortly 
after, the closure of the neural folds. Likewise in Amblystoma punc- 
tatum the lens fails to arise when the optic vesicle is removed at an early 
stage. 
The optic vesicles were removed, by the operation already described, 
from embryos of Arablystoma shortly after closure of the neural folds 
(see Fig. 1). At this age there is not the slightest visible trace of lens 
formation or of any changes in the ectoderm leading to lens formation. 
