W3005
1/29/02
Axonal navigation
Prof. Kelley
Motility is an important part of the developmental program for neurons.
The cells move themselves, both inside and outside the nervous system. Cell
migration makes important contributions to morphogenesis of nervous systems
as we saw in the previous lecture.
We can see the importance of axonal navigation very clearly in the
disordered state of the cerebellum from mice with the weaver mutation, named
after the disordered locomotion they display. Normally, cells within the
cerebellum migrate on the processes of a special kind of glial cell, the radial glial
cell (in the cerebellum these are called Bergman glia). In weaver mice, the
migration fails to take place and the orderly structure of the cerebellum is
disordered. Studies in tissue culture show that the problem with the mutant is not
in the glial cells but instead with the migrating granule cells. Study question: In
which direction are the granule cells migrating?: Pia to ventricle or ventricle to
pia?
In addition, parts of developing neurons move. A specialized structure,
the axonal growth cone, is responsible for the navigation of developing axons to
their final targets. Cajal described the growth cone from Golgi impregnations of
developing neurons. Somehow he was able to infer that growth cones were
mobile; he described them as “sniffing” their way through the nervous system, a
description that emphasizes the receptive capabilities of these structures. Ross
Harrison, the inventor of tissue culture, was the first to describe growth cones in
living tissues: neurons of developing frogs. When we examine a young neuron in
tissue culture we can see the emergence of various processes, usually called
neurites. Eventually one of these exhiibits the morphological features of the
growth cone and we call the process that bears the growth cone an axon. If,
however, we amputate the young axon, another neurite can differentiate into an
axon.
Why is axonal navigation important? This process is essential for the
formation of specific connections between nerve cells, the basic “wiring diagram”
of the brain. Navigation results in an initial mapping of neurons to their targets;
synaptic connections are further refined by experience (activity). Mutants that
are deficient axon migration are neurologically abnormal.
This is the wiring diagram of axons from identified neurons in embryonic
grasshoppers. Corey Goodman and his laboratory used the grasshopper for
these studies because each neuron is unique and can be identified by location
and characteristics of the cell body from animal to animal (this is true of many
invertebrates but is rare in vertebrates). They have not only described the
choices made by growth cones but have discovered many of the molecular cues
that guide fasiculation. Drosophila melanogaster (the fruit fly) has a very similar
wiring diagram. In this genetic organism, mutants can be used to identify
molecules involved in axon guidance because the mutants are miswired (we will
consider the case of the robo mutant later in this lecture). In Drosophila, the
entire genome is sequenced and powerful methods are available to determine
which gene has been mutated. Many of our insights into the rules that govern
axonal navigation come from studies in Drosophila and other invertebrates.
Cajal in Golgi stained sections and Ross Harrison in one of the earliest
uses of tissue culture observed axons emerging from developing neurons. When
the axon emerges it often has an enlarged, club-shaped end. This protruberance
is called the growth cone. David Bentley at Berkeley observed axons emerging
from sensory neurons in the developing limbs of embryonic grasshoppers.
These axons traveled in predictable ways making brief contacts with certain other
cells along their way into the brain (recall that the cell bodies of primary sensory
neurons are inside the CNS). Often only the earliest arriving growth cones
actually have to make the hard choices, later arriving growth cones just fasiculate
on older actions (no brainer). The early emerging axons are called pioneer
axons and the specific cells that they encounter along the way are called
guidepost cells. When the growth cone is traveling between guidepost cells is
has a simple shape. When it encounters these cells, however, many spikes
called philipodiae emerge from the growth cone itself. When growth cones reach
decision points, their morphology becomes more complex. We can see this
change in behavior in the pioneer axons on the previous slide. One of the
clearest examples of choice behavior is at the optic chiasm, the point at which
nerve fibers (axons) decide whether to remain on the same side of the brain
(ipsilateral) or cross (contralateral).
The growth cone is capable of behaving like an autonomous organelle.
This attribute is clearly shown when it is cut off from the rest of neuron, as in this
slide. The stump can continue to move and extend philipodia. Eventually the
growth cone will run out of steam (ATP) and be unable to move. However, it is
clear that because the growth cone doesn’t need its parent cell body, it has all
the machinery it needs locally to sense the environment and to grow towards or
away from local cues.
How do growth cones move? One molecular motor of the growth cone is
actin. We can see the distribution of molecules such as actin very clearly in
growth cones grown on a flat surface, such as a tissue culture dish. The growth
cone has a central club-shaped region from which emerges a veil of ruffling,
membrane-bound cytoplasm. The filopodia emerge from this veil. The filopodial
domain of the growth cone is filled with microfilaments made up of polymerized
actin,, here revealed by staining to its binding partner fluorescent phalloidin. The
central club shaped region contains numerous microtubules (revealed by staining
with an antibody to tubulin, a major component of microtubules.
The current model for how the growth cone moves is that the filopodia
treadmill. The filopodium elongates by addition of actin monomers at the leading
edge. The filopodium moves via breaking and attaching connections of actin
filaments to myosin strands, in an energy dependent process that resembles that
used in muscle contraction and relaxation. Growth of the axon behind the growth
cone requires addition of microtubules.
Growth cones move towards some targets and away from others. The
recognition of repulsive cues by a growth cone is often followed by growth cone
collapse, illustrated in this slide. The growth cones grows actively up to the
boundary. When the philopodia sense the repulsive molecule they retract and
the entire growth cone becomes smaller or collapses. The growth cone then
retracts from the boundary. Growth cone collapse is often used to distinguish
repulsive cues from the absence of attractive cues.
The importance of local cues in establishing axon trajectories is shown
very clearly in this experiment of Bill Harris (then at UCSD now at Cambridge).
Harris labeled axons emerging from retinal ganglion cells with the fluoescent dye,
diI, and the followed the routes they took to their major target in this case, the
optic tectum. The experiments were carried out in embryos of the South African
clawed frog, Xenopus laevis, as they are transparent and the axons all very
superficial. The upper right hand panel shows the normal pathway of the axons
as they head across the forebrain to the tectum. What happens when a small
square through which the axons would pass is rotated? How did Harris interpret
his results? What is the point of the tissue rotation shown in panel e?
Where did the ides of growth cone guidance come from? One very
important contribution to understanding the molecular basis of directed growth
was made by Paul LeTourneau in his PhD thesis at Stamford. LeTourneau, now
at Minnesota, plated individual neurons onto tissue culture plates that contained
a grid-like pattern of the positively charged amino acid, polyornithine (porn) over
a background of palladium (Pd). When the axons extended they grew along the
porn highways and avoided the Pd squares. This result could have been due
either to avoidance of Pd or preference for porn. How would you distinguish
these possibilities experimentally? What accounts for the preference for porn?
LeTourneau went on to show that the effects of the various substrates that
he used could be accounted for by the ability of the growth cone to stick or
adhere to the substrate. This observation lead to a search for adhesive
molecules some of which are shown in the next slide.
In the periphery, adhesion to the substrate is often mediated by the
extracellular matrix (ECM) molecule laminin. The laminin interacts with integrin
receptors on the surface of emerging axons. Other molecules are associated
with adherence of one axon to another (fasiculation). These include the fasiclins
discovered by Goodman and colleagues and the molecule NCatenin (panel D).
Adhesion can de calcium dependent mediated, for example, by the neural cell
adhesion molecule NCAM (panel A). Adhesion can be calcium dependent,
mediated, for example, by the molecule Ncadherin. Fibronectin can mediate
substrate adhesivity in the central nervous system. Shown here are the
structures of laminin, fibronectin and the integrin receptor.
Axons like to grow on other axons. This phenomenon is known as
fasiculation (or bundling). Fasiculation can be promoted by mutual attraction of
axons for each other or for their laminin or fibronectin highways. Cell Adhesion
Molecules (CAMs, especially fasiclin) pull axons together. Alternatively the
surrounding areas of axons can be repulsive and axons are then pushed
together. Semaphorins, for example, push axons together. To actually make
connections with a target, axons need to leave the bundle (defasiculate).:
Polysialic acid residues added to CAMs help axons defasiculate Semaphorins
come in several forms, some attached to the membrane and others which can
diffuse. Semaphorins can be either attractive or repulsive, as can other guidance
molecules depending on the biochemical state of the cell.The ability of
developing axons to grow towards some brain regions and away from others was
recognized very early in experiments on the retinotectal system of frogs. As we
saw in Bill Harris’ experiment, the optic tectum is a major target for axons of
retinal ganglion cells. Cells from the nasal or anterior part of the eye (the part
nearest the nose) grow into posterior tectum while axons from retinal ganglion
cells in the posterior part of the eye (temporal near the temple) grow into the
anterior part of the tectum.
Friedrich Bonhoeffer asked whether these two tectal targets had different
properties. He prepared membranes from each tectal region, layered the
posterior membranes with fluorescein (a yellow fluorescent dye) and laid them
down as strips on a tissue culture plate. Axons from either the nasal or temporal
retina were labeled red with rhodamine and he observed their behavior on the
strips. Describe the results of this experiment. What conclusions could
Bonhoeffer draw? Could he distinguish repulsive from attractive cues? What
other methods might he have used?
The current thinking about the retinotectal system is that, at a gross level,
the behavior of axons can be understood in terms of different receptors
expressed by cells in different parts of the retina and different ligands (molecules
to which receptors bind) expressed in different locations in the tectum.
One of the major decisions that a growth cone has to make is whether to
cross to the other side of the nervous system. We have already seen that growth
cones display a complex morphology as they make this choice at the optic
chiasm. One of the interesting questions for these kinds of axons, the
commisural or crossing axons, is why axons don’t cross back again to the other
side. If for example the midline puts out an attractive cue, what makes it
unattactive after an axon crosses? Corey Goodman tackled this question using
mutations in Drosophila. He reasoned that one way things might go wrong is to
have axons cross and recross the midline endlessly leading to very fat
commisures. This is actually the phenotype observed in the robo or roundabout
mutation.
One idea is that the midline contains both potentially attractive and
repellant signals. Axons would be kept from recrossing the midline by the
repellant activity. The state of the growth cone, however, must change in this
scenario so that it no longer finds the midline attractive
Some support for this last idea comes from experiments in the developing
spinal cord. Some of the neurons in this part of the brain will extend axons to
cross at the ventral midline and then travel forward. A series of experiments by
Tom Jessell, Mark Tessier-Levigne and colleagues established that these axons
are attracted by a molecule called netrin. Netrins are short range
chemoattractants that are chemically very closely related to the ECM molecules,
laminins. Netrins act as long range signals that guide axons to the midbrain in
both vertebrates and invertebrates. Netrins act as repellants for neurons that
grow away from the midline
That axons change their state after crossing is suggested by an
experiment in which axons that have not yet crossed (green) are attracted to the
ventral midline (floorplate or FP) while those that have already crossed (red) do
not turn towards the FP. Not the obvious club-shaped growth cones at the tips of
the red axons.
The turning behavior of a growth cone towards an attractant is often
calcium dependent as in the case of this frog growth cone being led on by nerve
growth factor, a diffusible sustance which may act as a chemoattractant for
growth cones at a distance in one of Mu-Ming Poo’s experiments while at
Columbia (he is now at Berkeley).
Poo showed that the same molecule can be either an attractant or a
repulsant depending on the biochemical state of the cell. In the first three panels,
a neuron is attracted to BDNF, another growth factor. The turning response is
calcium sensitive (middle panels) and becomes an avoidance response when
cAMP is manipulated.
• Summary
The growth cone responds to cues in the immediate environment and cues that
are further away; some of the cues are attractive and some are repulsive.
Growth cones also like to migrate on other axons (fasciculation); when they
reach their destinations (targets) they peel off (defasiculate); fasciculation and
defasiculation are due to the balance of attractive and repulsive cues. As it
travels through the developing brain, an individual growth cone samples
territories that differ successively. Axons are pushed from behind by more
distant repellents, pulled from ahead by more distant attractants and hemmed in
(kept “in line” or “on the road”) by local attractant/repellent signals. Remember
the important principle of location, location and location. Many areas of the
developing brain contain topographic representations. Antagonistic (attractive vs
repellant) gradients along two axes may establish topographic connections. How
synaptic connections actually form will be the subject of our next lecture.