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extracts. To obtain more tissue to work with, several
investigators screened a variety of adult tissues for
similar inducing activities. Although some found a
certain degree of specificity, liver and kidney being the
most potent neural inducers, others found that “frag-
ments from practically every organ or tissue from
various amphibians, reptiles, birds, and mammals,
including man, were inductive” (Holtfreter, 1955).
Perhaps most disconcerting to the investigators at the
time were the results from the candidate molecule
approach. Some of the factors found to have neuraliz-
ing activity made some sense: polycyclic hydrocarbon
steroids, for example. However, other putative induc-
ers, such as methylene blue and thiocyanate, most
likely had their effects through some toxicity or
In the early 1980s, a number of investigators began
to apply molecular biological techniques to study
embryonic inductions. The first of these studies
attempted to test for factors that would trigger the
process of mesoderm induction in the frog. As
described above, the frog embryo is divided into an
animal half and a vegetal half; the animal half will ulti-
mately give rise to neural tissue and ectodermal tissue,
while the vegetal half will give rise primarily to endo-
derm. The mesoderm, which will ultimately go on to
make muscle and bone and blood, arises in between
these two tissues, from the cells around the embryo's
equator (see Figure 1.16). It has been known for many
years, based on the work of Peter Nieuwkoop, that the
formation of the mesodermal cells in the equatorial
region requires some type of interaction between the
animal and vegetal halves of the embryo. If this animal
half or “cap” is isolated from the vegetal half of the
embryo, no mesodermal cells develop. However,
when Nieuwkoop (1973, 1985) recombined the animal
cap with the vegetal half, mesodermal derivatives
developed in the resulting embryos (Figure 1.16). He
postulated that a signal from the vegetal half of the
embryo induced the formation of mesoderm at the
junction with the animal half of the embryo. The iden-
tification of the molecular basis for this induction
has been the subject of intense investigation, and
the reader is referred to more general textbooks on
developmental biology for the current model of this
At the same time these studies of mesodermal
inducing factors were taking place, a number of inves-
tigators realized that the animal cap assay might also
be a very good way to identify neural inducers. Not
only do isolated animal caps fail to generate mesoder-
mal cells, but they also fail to develop into neuronal
tissue. Several factors added to animal caps caused the
cells to develop into neural cells as well as mesoder-
Neural tissue
Keller Sandwich
FIGURE 1.15 Planar neural induction can be contrasted from
vertical neural induction by Keller sandwiches. The organizer
region, including the IMZ cells, along with some of the surrounding
ectodermal tissue, can be cultured in isolation and will not involute
when two IMZ regions are placed back-to-back. The tissue under-
goes morphological changes similar to those that occur during
gastrulation, except the tissue extends rather than involutes. Never-
theless, neural tissue (red) is induced in the attached ectoderm, indi-
cating that the signals for neural induction can be passed through
the small region that connects the mesodermal cells and the ecto-
dermal cells.
Early efforts to define the chemical nature of the
neural inducer were unsuccessful. In the initial
attempts at characterization, Bautzman, Holtfreter,
Spemann, and Mangold showed that the organizer
tissue retained its inductive activity even after the cells
had been killed by heat, cold, or alcohol. Holtfreter
subsequently reported that the neuralizing activity
survived freezing, boiling, and acid treatment; how-
ever, the activity was lost at temperatures of 150°C.
Several embryologists then set out to isolate the active
principle(s) in the dorsal lip of the blastopore using the
following three approaches: (1) extracting the active
factor from the dorsal blastopore cells; (2) trying out
candidate molecules to look for similar inductive
activities; and (3) testing other tissues for inductive
The initial attempts at direct isolation of the induc-
ing activity from the blastopore lip cells resulted in
failure, largely because of the small amounts of tissue
that could be obtained and the limited types of
chemical analyses available at the time. From the initial
report in 1932 to the late 1950s, over one hundred
studies tried to characterize the neural inducing activ-
ity. The search for the neural inducer was one of the
major preoccupations of developmental biologists
in this period. Whereas one group reported that the
active principle was lipid extractable, another would
report that the residues were more active than the
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