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ity is high enough, the axon would attach rather than
be repelled. Two mechanisms have been discovered
that appear to resolve this problem. The first is extra-
cellular protein clipping in which the ectodomains of
activated receptors are attacked by metalloproteases
and cleaved, breaking the attachment between the
growth cone and its substrate (Hattori et al., 2000). The
second is endocytosis in which the entire receptor-
ligand complex is internalized into the growth cone
(Zimmer et al., 2003). In each case, the molecular bonds
holding the growth cone to the repellent surface are
neutralized allowing the growth cone to retract.
The repulsive factor responsible for the guidance of
olfactory tract axons away from the septum has been
identified as the vertebrate homolog of a Drosophila
protein called Slit, which is the ligand for Slit receptor,
Robo (Li et al., 1999). The olfactory bulb axons express
Robo, which enables them to sense the Slit. Motor
neurons of the vertebrate spinal cord also express Robo
and grow away from the ventral midline, which
expresses Slit (Brose et al., 1999). The axons of motor
neurons and olfactory bulb neurons also grow away
from cells transfected with Slit in culture. Slit and
Robo will become more important to us later in the
chapter when we delve further into the issue of
midline crossing.
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NGF
NGF
22 min
90 min
NGF
NGF
FIGURE 5.29 Growth cones can rely on chemotaxis to orient
their growth. A sensory neuron turns toward a pipette that is eject-
ing nerve growth factor (NGF) and thus producing a diffusible gra-
dient. Each time the pipette is moved, the axon reorients its growth.
(After Gundersen and Barrett, 1979)
centration of one molecule in a thousand from one side
of the growth cone to the other, making the growth
cone the most sensitive reader of chemical gradients
known in biology. In the case of pipette-based turning
assays, growth cones grow forward at a reasonable
pace and turn toward or away from the pipette without
speeding up, suggesting that these guidance factors
are not simply acting as a general growth-promoting
or inhibiting substances but as a directional cues.
Growth cones that turn toward attractants also send
out more filopodia on the side where the concentration
is higher and fewer filopodia on the other side, and the
opposite is true for diffusible chemorepellents. This
presumably creates differential traction forces toward
the attractant or away from the repellent.
Do axons use similar gradient-based mechanisms of
growth cone guidance in vivo? One of the first exam-
ples of an in vivo gradient attracting axons by chemo-
taxis was that of trigeminal ganglion sensory axons in
the mouse growing to the maxillary pad epithelium at
base of the whiskers. This is the most heavily inner-
vated skin in the entire body. When the maxillary
pad and trigeminal ganglion are removed and placed
near each other in a three-dimensional collagen gel,
the trigeminal axons preferentially grow toward the
explant of whisker pad, even when there are compet-
ing target explants of neighboring pieces of epidermis
even closer (Lumsden and Davies, 1986) (Figure 5.30).
Thus, it was hypothesized that the maxillary pad emits
a tropic agent for axon growth. This factor was nick-
named “max factor,” which was later identified as the
combination of BDNF and NT-3 (O'Connor and
Tessier-Lavigne, 1999), two neurotrophins that will be
discussed further in Chapter 7.
CHEMOTAXIS, GRADIENTS,
AND LOCAL INFORMATION
In a process termed chemotaxis , growth cones claw
their way up concentration gradients of diffusible
attractants to their source, or in the case of negative
chemotaxis turn down concentration gradients of
repellents. For chemotaxis to occur, the growth cone
must be positioned in the gradient such that one side
is exposed to a higher level of the factor than the other.
Gradients of different molecules can be experimentally
produced by ejecting solution from the tip of an elec-
trode and allowing the concentration to dissipate as it
spreads out into the tissue culture media. By using this
method, it was demonstrated that chick dorsal root
axons turn toward a source of Nerve Growth Factor
(Figure 5.29) (Gundersen and Barrett, 1979; Zheng
et al., 1994), although since then a large number of
molecules have been shown to have activity in such
pipette-based turning assays. These experiments
indicate that growing processes have a mechanism for
recognizing small concentration differences across a
relatively small distance. Using a controlled gradient
in a collagen gel, Rosoff et al. (2004) have been able to
show that growth cones can sense a difference in con-
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