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spinal cord relied on the use of many molecular
markers of cell identity that were obviously not around
at the time Holtfreter was doing his experiments. These
genes include the pax class of transcription factors dis-
cussed in the previous section, as well as a variety of
other genes that are restricted to particular populations
of both differentiated and/or undifferentiated cells
within the spinal cord. The expression of some of the
critical genes that define the dorsal-ventral polarity of
the spinal cord are summarized in Figure 2.24. To track
down the polarity signal released by mesoderm, a cell
culture system was devised in which the notochord
and the neural tube were co-cultured in collagen
gels. The signal was first shown to be diffusible, since
pieces of notochord could induce floorplate without
touching the neural tube. In addition, the expression
of motoneuron-specific genes, such as choline acetyl-
transferase, was also shown to depend on the noto-
chord. A clue to the identity of the factor was
uncovered in a rather roundabout manner. A crucial
clue about the identity of the notochord signal would,
again, come from Drosophila. During a large screen for
developmental mutants in the fruit fly (Nusslein-
Volhard and Wieschaus, 1980), a severely deformed
mutant was found, named hedgehog for its truncated
appearance. Subsequent cloning of the gene showed
that this molecule resembled a secreted protein.
The link between hedgehog and the notochord-
signaling molecule began with the identification of the
mammalian homolog, called Sonic hedgehog ( Shh ). Shh
is expressed initially in the notochord at the time when
the dorsal-ventral axis of the neural tube is being spec-
ified (Roelink et al., 1994). Shortly after this time, the
expression of sonic hedgehog begins in the differenti-
ating ventral neural tube, leading to floorplate. This
expression pattern is consistent with the transplanta-
tion experiments of Harrison and more recently of Tom
Jessell and co-workers; both found that initially the
ventralizing signal arises from the notochord, but soon
after it is also found in the floorplate. To determine
whether Shh was indeed the inducer of dorso-ventral
polarity in the spinal cord, a small aggregate of Shh
expressing Cos cells was placed next to the neural
tube. The Shh released from these cells was sufficient
to induce a second floorplate, as well as other genes
normally expressed in the ventral neural tube. In
further experiments, simply adding recombinant Shh
protein to explants of neural tube was sufficient to
induce them to differentiate as ventral neural tissues,
including floorplate and motor neurons (Figure 2.25).
These experiments thus show that Shh is sufficient to
ventralize the neural tube during development. Two
additional results show that Shh is required during
normal development to specify the dorsal-ventral axis
Neural crest
Roof plate
Neural crest
Sulcus limitans
Floor plate
FIGURE 2.22 The overall organization of the neural tube
emerges soon after closure. The most ventral part of the neural tube
becomes flattened into a distinct “floorplate.” The most dorsal aspect
of the neural tube develops into a tissue known as the roof plate. A
distinct fissure, the sulcus limitans , forms between the dorsal and
ventral parts of the neural tube along most of its length.
along the dorsal-ventral axis. Later, the neural tube
will become even more polarized along this axis; in the
ventral part of the tube, motor neurons will begin to
arise, while in the dorsal part, the sensory neurons
form. Experiments over many years have led to the
conclusion that the distinct polarity of the neural tube
arises largely because of the interaction between the
surrounding nonneural tissue and the neural tube.
Experiments during the early part of the twentieth
century by Holtfreter demonstrated that the basic
dorsal-ventral polarity of the neural tube was depend-
ent on an adjacent, nonneural structure, called the
notochord. Isolation of the neural tube from the sur-
rounding tissues resulted in an undifferentiated tube,
without obvious motoneuronal differentiation in the
ventral tube. However, when he transplanted a new
notochord to a more dorsal location, this induced a
second floorplate (Figure 2.23) and motoneuron dif-
ferentiation in the dorsal neural tube. Thus, the noto-
chord is both necessary and sufficient for the
development of the dorsal-ventral axis of the spinal
The studies that led to identification of the signals
that control dorsal-ventral polarity in the developing
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