RESEARCH OVERVIEW
|
To be updated ...
Each function of the mature nervous system, from a simple reflex response to a complex behavior, depends on the actions of distinct neuronal circuits. These circuits operate correctly because their component neurons are connected appropriately to each other. The complexity of these neuronal circuits is intimidating since, for example, there are millions of neurons forming billions of connections in the human brain. A fundamental problem in neurobiology is to understand how this intricate pattern of neuronal connection is established during development. Three major processes are know to be required for the proper formation of the extremely complex neuronal circuitry during development: migration of immature nerve cells from their birthplace to their specific locations, guidance of growing nerve processes to their target cells, and formation of specific synaptic connections with the target cells. Using a variety of approaches of cell and molecular biology, high-resolution microscopy, digital imaging, intracellular manipulation, and electrophysiology, our laboratory investigates the molecular and cellular mechanisms that govern and regulate these three developmental processes. In particular, we aim to understand how each of these three developmental processes is regulated by extracellular molecules as well as to dissect the intracellular signaling cascades that transduce extracellular signals to the cellular activities leading to the precise neuronal connections.
For all those who are fascinated by the magic of the infinitely small, hidden in the bosom of the living being are millions of palpitating cells whose only demand for the surrender of their secret, and with it the halo of fame, is a lucid and tenacious intelligence to contemplate them, to admire and to understand them. -- from Cajal's autobiography, Recuerdos de mi vida: Historia de mi laborcientica, Tercera edicion, 1923.
Neuronal Migration
A characteristic feature
of many neuronal precursors (neuroblasts)and neurons is that they
migrate from the sites at which they begin to differentiate.
For example, neurons in the peripheral nervous
system are derived from neural crest cells that migrate extensively
throughout the body before completing their differentiation into
autonomic, sensory, and enteric neurons. In the developing mammalian
brain, neuronal migration establishes the laminar pattern of cortical
regions, moving young neurons from ventricular zones where they
are generated to the layers where they establish synaptic relationships.
Although considerable progress has been made in characterizing
the migration and identifying genes and molecules involved in
neuronal migration, many questions concerning the cellular mechanisms
underlying the directed migration of neurons remain to be answered.
For example, a simple yet fundamental question is how does a
neuron migrate in a particular direction. Does a neuron move
in a similar way as a fish keratocyte? Will axonal processes
interfere with the migration? How are the rate and direction
of migration regulated by cell's endogenous activity or by exogenous
molecules? What are the signals that stop the migration? Elucidation
of these fundamental issues concerning neuronal migration will
not only provide insights on how the brain is constructed but
also have potential for the development of pharmaceutical treatments
for neuronal disorders.
We are interested in neuronal migration at the single cell level. We use cerebellar cultures to study cellular mechanisms of granule cell migration and the regulation of neuronal migration by extracellular molecules. The cerebellar granule neurons have provided an experimental model for central nervous system (CNS) migration, partially due to the fact that the specialized movement of granule neurons can be reproduced in an in vitro system. Using a variety of state-of-the-art techniques such as high-resolution imaging, laser assisted microsurgery, and photoactivated release of caged second messengers, we are addressing the following questions:
- What cytoskeletal and membranous events are involved in and responsible for the migration?
- How are the direction and rate of neuronal migration regulated by extracellular guidance molecules ?
- What is the role for Ca2+ and cAMP in the migration?
- Does synaptic activity affect the migration?
- More …
Growth Cone Motility and Guidance
Once a neuron has migrated to its final position
(sometimes even before), it begins to extend an axon. The axon extends at its
growing tip by means of a specialized structure called the nerve growth
cone. During development, the growth cone leads the elongating axon
navigating through the complex environment of developing tissues,
senses and responds to a variety of environmental cues by turning
towards or away from the source, and finally, after reaching the
target region, recognizes and makes synaptic connection with the
target cell. Recently, significant progress has been made in
identifying diffusible or surface-bound molecules that guide elongating
axons in vivo such as netrin family of chemoattractants
and semaphorin family of repellents. One of my lab's major efforts
is to understand how a growth cone detects diffusible guidance
cues and alters its direction of growth accordingly. We have
recently discovered that neurotransmitters and neurotrophic factors,
in addition to their traditional effects on nervous system, exert
chemoattractive effects on the growth cone of cultured Xenopus
embryonic neurons. Our study on neurotransmitter-induced turning
has established Ca2+ as the second messenger mediating
the turning response of the growth cone. Using a high-resolution
digital imaging technique, we have further demonstrated that
transmitter-induced turning of the growth cone was preceded by an
asymmetric elevation of intracellular Ca2+ concentration
in the growth cone followed by an asymmetric filopodia protrusion.
These results suggest that the directional information for growth
cone turning is encoded in the asymmetric elevation of intracellular
Ca2+. Our current hypothesis is as follows:
gradients of diffusible chemoattractants activate their specific
cell surface receptors asymmetrically across the growth cone,
resulting in a localized (asymmetric) elevation of cytosolic second
messenger levels and subsequent localized (asymmetric) activation
of a common set of proteins in the growth cone. The activated
proteins in turn regulate asymmetric cellular activities which result
a turning response of the growth cone. This hypothesis is supported
by our recent demonstration that direct focal elevation of
[Ca2+]i at the growth cone is sufficient to
provide the directional cue intracellularly and initiate both attractive and
repulsive turning responses (
see our new Nature paper).
To further test and validate this hypothesis, we have been examining the
cellular events involved in growth cone turning induced by a variety
of guidance molecules including attractive netrins and repulsive semaphorins.
Different extracellular guidance cues are likely
to initiate different intracellular signaling cascades which will
eventually interact and converge to give rise to the final directed
growth cone movement. Our goal is to determine the common sets
of cellular events utilized by the growth cone to steer towards
or away from different signals. The results from these studies
are likely to provide the "missing link" which associates
extracellular cues to the motile activity of the growth cone.
Growth cone-target interaction and synaptogenesis
The functions of distinct
neuronal circuits depend on the correct synaptic connections between presynaptic
terminals and postsynaptic targets. After the axons reach the
target field, each growth cone of different axonal processes needs
to interact and recognize its correct target. The contact of
nerve growth cone with the correct target cell initiates a cascade
of cellular changes in the nerve and target cells, leading to
the formation of functional synapses. In Xenopus nerve-muscle
cultures, one of the initial events during growth cone-target
contact is the induction of spontaneous acetylcholine secretion
from the nerve terminal, followed by the development of specialized
synaptic structures both in neurons and muscle cells (e.g. clustering
of AChRs on muscle surface). My laboratory is interested in several
aspects of the growth cone-target interaction. In particular,
we are interested in surface molecules involved in the growth
cone-target interaction, intracellular signaling pathways mediating
the interaction, and the cellular events responsible for transforming
motile growth cones into presynaptic terminals. For example, by
expressing functional acetylcholine receptors and other muscle surface
molecules in fibroblasts, we are taking steps to reconstitute the
postsynaptic target for Xenopus motor neurons