Axon Guidance: Determining the intracellular signals that allow the nerve growth cone to read " road signs" and sense direction
(This article was published in UMDNJ Research 2000, v.2, No. 3.)
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One of
the most fascinating and critical events in brain development
is precise wiring of distinct neuronal circuits that underlie
diverse functions of the mature nervous system, ranging from sensorimotor
responses to cognition. Such precise wiring of neuronal connection
is also required in adult brain for functional recovery after
brain injury and diseases. However, the magnitude and complexity
of the task involved in wiring the complex nervous system is staggering.
In the human brain, there are more than a billion neurons, including
hundreds of different types, to assemble into highly complex neuronal
networks with over 109 connections. An ultimate challenge
in developmental neurobiology is to understand how this intricate
pattern of neuronal connections is achieved with high precision
during development. Over a century ago, Ramón y Cajal made
his landmark observations on the patterns of nerve process outgrowth
and connectivity in developing mammalian brains and proposed that
each elongating process of developing neurons, the axon, is lured
to the target cells by diffusible molecules secreted from the
target cells. However, direct evidence to support the guidance
mechanism has only started to emerge recently. An increasing
number of families of guidance molecules has now been identified.
It is well established that developing axons are guided to their
targets by a variety of environmental cues, including long-range
diffusible and short-range surface-bound molecules that can either
attract or repel the axon. The presence of these guidance cues
in a temporal and spatial pattern enables the axon to navigate
through the complex environment of the developing embryo to reach
its correct target.
In
1880, Ramón y Cajal observed and named the growth
cone ("cono de crecimiento") as the motile structure
leading the axonal extension. During development, the growth
cone leads the elongating axon navigating through the complex
environment of developing tissues, senses and responds to a variety
of attractive or repulsive cues by turning towards or away from
the source, respectively, and after it has reached the target
region, recognizes and makes the synaptic connection with the
target cell. It is believed that an axon encounters a combination
of guidance cues during its journey to the target. A fundamental
question yet to be addressed is how the growth cone reads these
"road signs" and accurately senses the direction for
its directed extension. One of my lab's major research focuses
is on the molecular and cellular mechanisms underlying axon guidance
by long-range diffusible cues. Specifically, we are to dissect
the intracellular signaling evens that allow the growth cone to
process and transduce the directional information from extracellular
space to intracellular motility apparatus for directed growth
cone movement. Using state-of-art cellular imaging techniques,
we have been able to examine intracellular signals involved in
growth cone guidance in high temporal and spatial resolutions.
Our recent studies have established calcium ions as the second
messenger to mediate growth cone guidance by a number of diffusible
guidance cues. We have shown that attractive turning of nerve
growth cones in response to a number of guidance molecules, including
neurotramitters, neurotrophins, and netrin-1, involves localized
elevation of intracellular Ca2+ concentration at the
growth cone. These findings suggest that the directional information
for growth cone extension is encoded in the local Ca2+
signal intracellularly, and the subsequent local activation of
downstream effectors in the growth cone relays the directional
cue to the cytoskeleton for localized modification of its dynamics
to result in the turning response. To further test this hypothesis
and to understand the precise role of local Ca2+ signals
in growth cone guidance, we have developed a sophisticated intracellular
manipulation technique to directly elevate the intracellular Ca2+
concentration in a spatially-restricted subcellular region. We have now
provided the direct evidence, for the first
time, that a localized Ca2+ signal in the growth cone provides
the directional cue intracellularly for axonal extension
and is sufficient to instruct both attractive and repulsive turning
responses of the growth cone 
(Nature
403:89-93, 2000). We
have further shown that the growth cone is capable of integrating
local and global cytosolic Ca2+ signals for specific
turning behavior. Such integration could provide the flexibility
for the growth cone to generate distinct responses required for
specific and accurate wiring of millions of axons through a limited
number of guidance cues available during development.
Different extracellular guidance cues are likely to initiate different intracellular signaling cascades. We are actively investigating the involvement of other second messengers, for example, cAMP, in growth cone guidance. Furthermore, different signaling pathways are likely to interact and eventually converge to give rise to the common cytoskeletal activity for directed growth cone movement. We are currently examining the cellular events downstream of second messengers in growth cone turning induced by a number of guidance cues. Our goal is to determine the common sets of cellular events utilized by the growth cone to respond to different extracellular cues to directional steering. The results from these studies are likely to provide the "missing link" between extracellular cues and directed motility of the nerve growth cone.
The cell's ability to sense the environment and to determine the direction and proximity of an extracellular stimulus is critical not only for the development of nervous systems but also for immunity, angiogenesis, wound healing, and embryogenesis. Understanding how direction is accurately read and processed from extracellular diffusible cues by nerve growth cones is fundamental to our understanding of how the functional brain is constructed, which in turn provides the groundwork for potential development of strategies for repairing damaged neuronal connections after brain injuries and diseases. Using a well-defined neuronal culture system for growth cone guidance assays, sophisticated high-resolution imaging, and direct manipulation of intracellular signals, we are at a unique position to dissect the intracellular mechanism underlying accurate direction sensing by nerve growth cones during axon guidance. Results from this study would not only advance our knowledge of molecular mechanisms underlying precise neuronal wiring during development but also provide important insights into cellular mechanisms underlying directional sensing of migrating cells during important biological responses such as chemotaxis of leukocytes during inflammatory response.