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FIGURE 5.8 Time-lapse images of two GFP-expressing retinal axons growing in the optic tract of a
Xenopus brain. The images on the left are successive time points spaced about 20 minutes apart showing the
two elongating axons, tipped with growth cones, rearranging their relative positions in the optic tract. The
image in the top right shows the initial branching of the same two axons in the tectum, the target structure
for these axons. The image at the bottom right is a low-power view of the preparation at the beginning and
at the end of the time-lapse. (Courtesy of Sonia Witte and Christine Holt)
engineering chimeric genes, combining GFP with a
protein potentially involved in axon growth, one can
test the effect of misexpressing the protein of interest
in live axons. By putting the GFP gene under the
control of a promoter that is active in a subset of devel-
oping nerve cells in a transgenic animal, it is possible
to monitor a whole class of growing axons. One
problem, often encountered in thick specimens, is the
excessive level of out-of-focus fluorescence. Confocal
microscopes overcome this obstacle and allow one to
observe sharp fluorescent images in a limited depth
of field (0.5-1.5 mm), called optical sectioning . This is
accomplished by scanning a focused laser beam across
the specimen in a point-by-point fashion. The emitted
light from each “point” is acquired and used to recon-
struct an image of the specimen. Using a combination
of these techniques, it has become possible to watch
axons grow out and innervate their target in the CNS
of live embryos (Figure 5.8).
As growth cones crawl forward, they leave axons
behind. This means that new material must be continu-
ally incorporated into the axon. In culture, when a par-
ticle is attached to a growing axon, it remains relatively
stationary compared to the distal tip of the axon. This
suggests that this new material is assembled distally, at
the growth cone. Indeed, new glycoproteins are added
preferentially at the distal tips of axons in the growth
cone region (Hollenbeck and Bray, 1987). The incorpo-
ration of this new material is calcium dependent,
suggesting that membrane is added by the calcium-
mediated fusion of internal vesicles to the growth
cone's surface. The addition of cytoskeletal compo-
nents also takes place primarily at the tip of the growing
process. This was shown by labeling neurons with fluo-
rescent tubulin and actin, and then illuminating part of
the axon with a bright spot to bleach the fluorescence at
a particular location (Figure 5.9). The result is that the
bleached spot stays relatively still as the growth cone
continues to advance, suggesting that these compo-
nents are assembled distally. If one looks carefully at the
middle of the axon, however, it is possible to see that
there is also forward transport of some assembled
microtubule fragments as well as cargo moving up and
down the axon. In addition, if two beads are attached
along the shaft of the axon, one notices a growing sepa-
ration between these two markers, suggesting that
some membrane is also added along the axon. Such,
transport and interstitial growth is crucial as the brain
or body enlarges after initial connections are made.
THE DYNAMIC CYTOSKELETON
If we want to know how a growth cones navigates,
we have to understand how it moves, and this means
looking into its dynamic cytoskeleton. The cytoskele-
ton of a growth cone is filled with molecules that
are involved in cell movements (Heidemann, 1996;
Letourneau, 1996; Dent and Gertler, 2003; Gordon-
Weeks, 2004). The two most important elements are the
microtubules that extend along the axon and splay out
in the central domain, C-domain, of the growth cone,
and the actin fibers, which are more prominent in the
peripheral, P-domain (Figure 5.10). In the P-domain,
actin forms the basic cytoskeleton of the lamellipodia,
the veils between filopodia, and the filopodia them-
selves, which are filled with thick bundles of actin.
Many other cytoskeletal-associated proteins in the
growth cones do a variety of jobs such as anchoring
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