I. PROBLEM OF THE ORGANIZER
23
small amount of RNA. Subfractions of the acid-precipitable fraction
were prepared by ultracentrifugation and electrophoresis, and tested
after dialysis and ethanol precipitation. All of them indicated strong
mesoderm inducing abilities in sandwich experiments (Yamada, 1958c).
Up to this stage of investigation the sandwich technique has been
exclusively used for testing morphogenetic effects of samples. Although
the technique is superior to the insertion technique in simplifying the
reacting system, it has the drawback of needing a solid sample and of
being unsuited for quantification. Difficulty is experienced especially
when two samples with relatively high levels of activity have to be
compared. Obviously it is necessary to work out a new technique in
which the sample can be tested in solution. Simple immersion of the
isolated ectoderm in a solution of the substance to be tested was found
to give unreliable data, because the impermeable surface coat which
occupies the original external surface of the ectodermal layer may
quickly cover the whole external surface of the explant, as the latter,
subsequent to isolation curls up into a sphere. Thus it is possible that
the inductive activity of the solution is prevented from revealing its
effects. According to an oral communication of Kawakami, the success
of mesodermal induction in an ectodermal explant of the Triturus
gastrula, kept in a suspension of chick embryo extract, depends upon
whether the explant curls up or not. A technique to prevent the curling
of the explant and to test the morphogenetic effects of a sample in
solution has recently been worked out by the present author. The
technique consists of placing a piece of the ectoderm immediately after
isolation between two sheets of nylon. This sandwich is put in a drop of
the medium containing the sample in solution, on the bottom of a watch
glass. The watch glass is kept in an air-tight Petridish. Under the surface
tension of the medium, the upper nylon sheet is gently pressed upon the
explant so that the explant is kept flat, exposing its originally internal
surface to the medium. After two days the ectoderm is released from the
nylon and transferred into a culture dish containing standard culture
medium. According to the nature and amount of the sample administered
in the medium, the explant can differentiate any of the regional types.
If the presumptive ectoderm of the early gastrula is treated similarly
with nylon but kept in the standard medium (Holtfreter's solution
containing 0-02% sulphadiazine sodium, and buffered to pH 7-4 with
Tris) it forms only a group of epidermal cells (cf. Fig. 14).
Using the nylon technique for testing the inducing ability of the
sample, a renewed attempt at isolation of the bone marrow factor is being
made in our laboratory (Yamada and K. Takata, unpublished work).
In the new series of fractionations (Fig. 6) sucrose was used instead of
sodium chloride as the medium for the tissue homogenate. Comparing
23
small amount of RNA. Subfractions of the acid-precipitable fraction
were prepared by ultracentrifugation and electrophoresis, and tested
after dialysis and ethanol precipitation. All of them indicated strong
mesoderm inducing abilities in sandwich experiments (Yamada, 1958c).
Up to this stage of investigation the sandwich technique has been
exclusively used for testing morphogenetic effects of samples. Although
the technique is superior to the insertion technique in simplifying the
reacting system, it has the drawback of needing a solid sample and of
being unsuited for quantification. Difficulty is experienced especially
when two samples with relatively high levels of activity have to be
compared. Obviously it is necessary to work out a new technique in
which the sample can be tested in solution. Simple immersion of the
isolated ectoderm in a solution of the substance to be tested was found
to give unreliable data, because the impermeable surface coat which
occupies the original external surface of the ectodermal layer may
quickly cover the whole external surface of the explant, as the latter,
subsequent to isolation curls up into a sphere. Thus it is possible that
the inductive activity of the solution is prevented from revealing its
effects. According to an oral communication of Kawakami, the success
of mesodermal induction in an ectodermal explant of the Triturus
gastrula, kept in a suspension of chick embryo extract, depends upon
whether the explant curls up or not. A technique to prevent the curling
of the explant and to test the morphogenetic effects of a sample in
solution has recently been worked out by the present author. The
technique consists of placing a piece of the ectoderm immediately after
isolation between two sheets of nylon. This sandwich is put in a drop of
the medium containing the sample in solution, on the bottom of a watch
glass. The watch glass is kept in an air-tight Petridish. Under the surface
tension of the medium, the upper nylon sheet is gently pressed upon the
explant so that the explant is kept flat, exposing its originally internal
surface to the medium. After two days the ectoderm is released from the
nylon and transferred into a culture dish containing standard culture
medium. According to the nature and amount of the sample administered
in the medium, the explant can differentiate any of the regional types.
If the presumptive ectoderm of the early gastrula is treated similarly
with nylon but kept in the standard medium (Holtfreter's solution
containing 0-02% sulphadiazine sodium, and buffered to pH 7-4 with
Tris) it forms only a group of epidermal cells (cf. Fig. 14).
Using the nylon technique for testing the inducing ability of the
sample, a renewed attempt at isolation of the bone marrow factor is being
made in our laboratory (Yamada and K. Takata, unpublished work).
In the new series of fractionations (Fig. 6) sucrose was used instead of
sodium chloride as the medium for the tissue homogenate. Comparing