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Genetic Analysis of Neural Circuit Assembly
Summary: Liqun Luo seeks to understand how neural circuits assemble during development and contribute to sensory perception. He has developed the MARCM technique to track neurons in the fruit fly brain and is perfecting a similar system for studying mice.
We use molecular genetics to study the logic of neural circuit organization and assembly. The human brain is made of hundreds of billions of neurons. Most individual neurons have complex dendrites and axons that allow them to receive and send information to thousands of other neurons. Specific neurons participate in specialized neural circuits and perform dedicated functions. To comprehend this bewildering complexity, we use simpler brains of model organisms to uncover fundamental principles that are likely to be used in our own brain.
Much of our current knowledge of neuronal structure, connectivity, and development derives from Santiago Ramón y Cajal's classic studies a century ago. He used the Golgi staining method, which randomly labels a small number of individual neurons in their entirety—unobscured by the mass of other brain structures and connections. We developed a genetic analog of Golgi staining, mosaic analysis with a repressible cell marker (MARCM), that has allowed us to label small groups or isolated single neurons in the brain of the fruit fly Drosophila (which is made of hundreds of thousands of neurons). With MARCM we can also genetically manipulate only these labeled neurons by, for example, deleting a gene of interest to assess its function in the assembly of neuronal circuits. We have used MARCM to study the morphological development of individual neurons and how individual neurons assemble into functional circuits. We have also recently developed an analogous genetic method in mice to address similar questions in the mammalian brain.
Neuronal Morphogenesis and Axon Pruning
The first steps in the assembly of functional neural circuits are for individual neurons to send out axons and dendrites, which eventually form specific connections with their targets. Our previous studies have shown that Rho GTPases, acting as intracellular molecular switches that transduce extracellular signals to regulate the actin cytoskeleton, play important roles in many aspects of morphological development of neurons and in structural plasticity in adults. We now focus on studying axon and dendrite pruning during development. Using candidate gene and MARCM-based forward genetic screening, we have identified genes that are required for the axon-pruning process, and we are studying the cell biological machinery that executes the pruning process and its regulation. We have also found common mechanisms of axon degeneration during developmental pruning and after injury.
Organization and Assembly of Olfactory Circuits
A central focus in our lab is the logic that governs the assembly of individual neurons into functional circuits. We use the Drosophila olfactory system as a model. As in mammals, Drosophila olfactory receptor neurons (ORNs) that express a given receptor converge their axons onto a specific glomerulus in the antennal lobe, creating an odor map. This information is transferred and transformed by olfactory projection neurons (PNs), whose dendrites innervate single glomeruli and whose axons project stereotypically to higher brain centers. We have recently combined MARCM-based single-cell labeling of PNs with state-of-the-art image methods to create high-resolution quantitative maps of high olfactory centers. Among other findings, these maps reveal spatially segregated representations of fruit odors and pheromones. Thus, the olfactory circuit could allow specific odorants to activate stereotyped sets of third-order neurons, contributing to different innate olfactory-driven behaviors. We are using a combination of molecular genetic, imaging, and behavioral methods to characterize higher-order olfactory processing circuits and to uncover the underlying principles of information processing.
During the assembly of the fly olfactory system, a given ORN must target its axons to 1 of ~50 glomeruli, while a given PN must also target its dendrites to 1 of ~50 glomeruli. MARCM-based lineage analysis revealed that PNs are prespecified by lineage and birth order to send dendrites to specific glomeruli. This circuit thus provides an excellent system to understand how wiring specificity is achieved through genetic instructions. We found that the antennal lobe is first patterned by PN dendrites, which target to their appropriate positions and form a coarse prototypic map before ORN axons arrive. We have recently determined that the molecular gradient of semaphorin is used in the formation of this coarse dendritic map. At a later stage, ORN axons project to their appropriate target by using a variety of strategies, including axon-axon interactions among ORN axons and specific matching of ORN axon to PN dendrite at the final stage. We found, for example, that semaphorin-plexin–mediated axon-axon repulsion allows axons that arrive early to confine the targeting area of axons that arrive late. We are continuing to study genes and mechanisms that instruct the establishment of the wiring specificity of the olfactory circuit.
Mouse MADM
For labeling and genetic manipulation of individual cells, we recently developed MADM (mosaic analysis with double markers) in mice, analogous to MARCM in Drosophila. We are using MADM to explore the organization of neuronal circuits and the rules of their assembly in the mammalian brain. MADM also allows conditional knockout of a candidate gene in specifically labeled cells to analyze gene function during development and to create mouse models of human diseases. For example, to model cancer in mice, we have recently used MADM to create loss of heterozygosity of tumor-suppressor genes.
These studies have also been supported by grants from the National Institute of Neurological Disorders and Stroke and the National Institute on Deafness and Other Communication Disorders.
Last updated: September 11, 2007
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