I. PROBLEM OF THE
ORGANIZER
29
should be remembered that when the inducing ability of the dead
organizer was discovered, it was taken as evidence for the presence in
the organizer cells of a chemical factor, the inducing substance, which
is responsible for the inductive effects of the organizer. Thus it is logical
to question the existence of the inducing substance itself on the grounds
of the above results, as was done by Okazaki in his paper. He suggested
as one of the possibilities that the normal induction may be brought
about by the propagation of some change in the state of the protoplasm
from the organizer to the adjacent ectoderm, rather than by the transfer
of a specific substance. The possibility is certainly of theoretical importance, and should not be lost sight of in our discussion. However,
another possibility left open is that in the organizer some inducing agents
unstable toward ethanol are present, and are transferred to the reacting
ectoderm cell by a process which occurs only in the living condition.
From the above discussion it is clear that subcellular components of
embryos should be tested in a fresh condition avoiding denaturation.
This excludes the use of the sandwich technique as the test method,
because it necessitates precipitation of a soluble substance before the
test. Fortunately the nylon technique introduced above (Section IV)
seems to fulfil the requirement, and was used exclusively in the present
experiments. The method of fractionation consisted in differential
centrifugation of the homogenate of neurulae of Triturus pyrrhogaster
in 8% sucrose buffered with phosphate. After centrifuging at 10,000 g
for 40 min, the microsome fraction was sedimented from the supernate
by applying a force of 100,000 g for 90 min. The sediment was suspended
in M/20 Tris buffer (pH 7-4), cleared by centrifugation, and tested in
Holtfreter solution containing 0-02% sulphadiazine sodium and buffered
to pH 7-6 with Tris. Freezing of the sample was avoided, because of possible inactivation. The supernate after ultracentrifugation was brought
to pH 5 with acetic acid. The precipitate developed was resuspended
in Tris, dialyzed, centrifuged, and tested as the pH 5 fraction. The
final supernate obtained was tested similarly as the soluble fraction after
dialysis and clearing. As the donor of the presumptive ectoderm, early
gastrulae oí Triturus pyrrhogaster were used.
The results presented in Table IV show that inducing ability is mainly
restricted to the microsome fraction. Concerning the inductive effect of
the microsome fraction there are a few points to be scrutinized. First of
all, the level of activity of the microsome fraction is relatively low as
compared with that of the bone marrow factor (Section IV). This is perhaps understandable from the fact that the homogenate of embryos and
not of the organizer area was used as the source of fractionation, hence
the microsome fraction contained, beside the material from the inducing
areas, a larger amount of material from the non-inducing areas, which
ORGANIZER
29
should be remembered that when the inducing ability of the dead
organizer was discovered, it was taken as evidence for the presence in
the organizer cells of a chemical factor, the inducing substance, which
is responsible for the inductive effects of the organizer. Thus it is logical
to question the existence of the inducing substance itself on the grounds
of the above results, as was done by Okazaki in his paper. He suggested
as one of the possibilities that the normal induction may be brought
about by the propagation of some change in the state of the protoplasm
from the organizer to the adjacent ectoderm, rather than by the transfer
of a specific substance. The possibility is certainly of theoretical importance, and should not be lost sight of in our discussion. However,
another possibility left open is that in the organizer some inducing agents
unstable toward ethanol are present, and are transferred to the reacting
ectoderm cell by a process which occurs only in the living condition.
From the above discussion it is clear that subcellular components of
embryos should be tested in a fresh condition avoiding denaturation.
This excludes the use of the sandwich technique as the test method,
because it necessitates precipitation of a soluble substance before the
test. Fortunately the nylon technique introduced above (Section IV)
seems to fulfil the requirement, and was used exclusively in the present
experiments. The method of fractionation consisted in differential
centrifugation of the homogenate of neurulae of Triturus pyrrhogaster
in 8% sucrose buffered with phosphate. After centrifuging at 10,000 g
for 40 min, the microsome fraction was sedimented from the supernate
by applying a force of 100,000 g for 90 min. The sediment was suspended
in M/20 Tris buffer (pH 7-4), cleared by centrifugation, and tested in
Holtfreter solution containing 0-02% sulphadiazine sodium and buffered
to pH 7-6 with Tris. Freezing of the sample was avoided, because of possible inactivation. The supernate after ultracentrifugation was brought
to pH 5 with acetic acid. The precipitate developed was resuspended
in Tris, dialyzed, centrifuged, and tested as the pH 5 fraction. The
final supernate obtained was tested similarly as the soluble fraction after
dialysis and clearing. As the donor of the presumptive ectoderm, early
gastrulae oí Triturus pyrrhogaster were used.
The results presented in Table IV show that inducing ability is mainly
restricted to the microsome fraction. Concerning the inductive effect of
the microsome fraction there are a few points to be scrutinized. First of
all, the level of activity of the microsome fraction is relatively low as
compared with that of the bone marrow factor (Section IV). This is perhaps understandable from the fact that the homogenate of embryos and
not of the organizer area was used as the source of fractionation, hence
the microsome fraction contained, beside the material from the inducing
areas, a larger amount of material from the non-inducing areas, which