axon guidance

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 decade 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.