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Wiring the Fly Brain
Summary: Lawrence Zipursky is interested in uncovering the mechanisms by which neurons make highly specific patterns of connections during development.
Communication between neurons relies on precise patterns of interconnections between them. We are interested in understanding the molecular mechanisms by which these connections, referred to as synapses, are specified. This is a problem of daunting complexity. In the human brain there are about 1012 neurons linked together into a communication network by some 1015 synaptic connections. Even in the fruit fly Drosophila melanogaster, which we study, there are 250,000 neurons and millions of synaptic connections. How do correct connections form during development? Presumably specific molecular labels on the surface of different neurons provide a basis for the cellular recognition that underlies this specificity. Identifying these labels and understanding how they work is the central goal of my laboratory.
The developing fly brain is well suited to the identification of mutants in which connection specificity is disrupted. We can easily visualize the patterns of connections made by different neurons and manipulate these cells genetically.
Dscam Encodes a Large Family of Cell Surface Proteins That May Specify Neuronal Identity
We have studied a number of genes encoding cell surface proteins that are required for neuronal connections in the fly. Among these genes, Dscam is the most intriguing. Through a process called alternative splicing, the Dscam gene potentially encodes more than 38,000 different proteins. All forms or isoforms of Dscam have the same overall domain structure or shape but different amino acid sequences within specific regions of the protein. Do these different isoforms have different recognition specificities, and if so, does this contribute to connection specificity?
Dscam is required for normal connections to form in many regions of the nervous system. In the olfactory system it is required for different subclasses of olfactory neurons to connect to the correct group of cells in the brain that process olfactory information. It is also required for the construction of the mushroom body (MB), a central brain structure that is important for learning and memory. During normal development, MB axons bifurcate, with each sister branch projecting to a different region of the MB. By removing Dscam from single MB neurons, we and others showed that Dscam is crucial for the two branches to segregate. We have taken a combined biochemical and genetic approach to consider the mechanism by which Dscam promotes branch segregation.
We have used a biochemical approach to show that single isoforms of Dscam interact with each other, indicating that one form of Dscam on the surface of an axon or dendrite will bind to the same isoform on an apposing cell surface. Remarkably, these interactions are very specific. Indeed, two very closely related isoforms do not interact. Dscam thus provides a family of closely related proteins with similar functions that display literally hundreds to thousands of distinct binding specificities.
Through genetic studies we concluded that interactions between identical isoforms signal contact-dependent repulsion. How does this contribute to the mechanism of MB axon branch segregation? In collaboration with Andrew Chess (Whitehead Institute for Biomedical Research at MIT), we have shown that each MB neuron expresses multiple isoforms of Dscam. Furthermore, each MB neuron expresses a combination of isoforms that is distinct from their neighbors. As such, each MB neuron may exhibit a combination of labels that specifies its unique identity. We proposed that in wild-type MB neurons, sister branches express the same isoforms of Dscam (the isoforms on branches of other MB neurons are different) and that interactions between Dscam on the two branches induce a repellent response and thus segregation of the two branches to different pathways.
It is possible that the specific combination of isoforms in a single MB neuron is not essential, it is only important for each neuron to have a cell surface identity that is different from its neighbors. Consistent with this view is the observation that we can remove all isoforms of Dscam from a single neuron, express only a single isoform in its place, and restore normal branching. As a consequence of this genetic manipulation, the two sister branches will express the same isoform, albeit only a single one, and this will be different from the isoforms expressed on neighboring axons.
Studies in other regions of the developing fly nervous system also suggest that Dscam may function more broadly to facilitate the segregation of axonal and dendritic processes of the same cell. As Chess and colleagues have shown that different populations of neurons express biased subsets of isoforms, interactions between different isoforms of Dscam may also contribute to wiring specificity. Additional genetic, cell culture, and biochemical studies are in progress to address these questions. (A grant from the National Institutes of Health provided support for the work on the role of different Dscam isoforms in MB development.)
Formation of Neuronal Connections in the Fly Visual System
We have been studying the formation of the connections between photoreceptor neurons (R cells) and their targets in the brain. The compound eye of the fly contains some 800 simple eyes, or ommatidia, and each ommatidium contains eight R cells. These cells fall into three classes based on synaptic specificity. R1–R6 neurons connect to the first optic ganglion, called the lamina, and R7 and R8 neurons extend axons through the lamina and terminate in two distinct layers in the second optic ganglion, the medulla. There are 10 distinct layers in the medulla. Other visual system neurons make connections in distinct layers. How do axons and dendrites select specific layers in which to make synaptic connections? To address this question, we have focused on a single neuron, the R7 neuron. This neuron is particularly well suited to genetic manipulation and developmental analysis.
We have identified three different classes of mutations that affect the pattern of R7 connections. One leads to R7 neurons terminating in the layer appropriate for R8 neurons. Another leads to abnormal connections within the appropriate layer. In the third class, R7 neurons appear to make connections in multiple layers in the medulla. So far, our studies have largely focused on mutations resulting in R7 connecting to the layer appropriate for R8. Mutations in N-cadherin fall into this class.
N-cadherin is a cell surface protein that promotes interactions between two closely apposed membranes. Molecular analysis revealed that 12 isoforms of N-cadherin are generated by alternative splicing. To our surprise, one mutation in N-cadherin disrupted alternative splicing such that only 6 of the 12 isoforms were generated in the eye. While R7 neurons lacking these isoforms exhibited a defect indistinguishable from R7 lacking all 12 isoforms, olfactory receptor neurons that require N-cadherin for targeting were unaffected by it. This specific isoform requirement reflects selective expression of a subclass of isoforms during a crucial stage of R7 targeting rather than differences in biochemical properties of different isoforms. In addition to N-cadherin, other cell surface proteins, including the receptor tyrosine phosphatases Lar and Ptp69D, are required for R7-targeting specificity, exhibiting phenotypes similar to those seen for N-cadherin. All three cell surface proteins are broadly expressed in the developing visual system as growth cones select their targets. Thus it is likely that they play a permissive rather than an instructive role in target selection. A central goal of our studies is to identify genes that act in an instructive fashion. To this end we have identified and are characterizing additional genes regulating R7 targeting.
Formation of Connections in the Fly Olfactory System
During the past several years we have been studying the mechanisms regulating synaptic target specificity in the developing olfactory system. Here different subpopulations of neurons express different odorant receptors. Neurons expressing the same odorant receptor then connect to the same group of neurons in the brain within an anatomical structure called a glomerulus. There are some 43 different odorant receptors and a similar number of glomeruli. Using specific genetic reagents, we can directly visualize and genetically manipulate the wiring specificity of different classes of olfactory receptor neurons. In previous studies we characterized the requirement for N-cadherin and Dscam in the elaboration of these connections. Dscam plays a key role in promoting targeting to the correct region of the antennal lobe, while N-cadherin functions at a later step to promote the formation of a protoglomerulus, a precursor structure to the glomerulus. Over the past year we have identified through forward genetic screens a panel of genes required for targeting specificity. These genes encode a wide spectrum of different proteins. Some genes are required for the connections of all olfactory receptor neurons, while others are only required for a subclass of them. Studies are in progress to understand how these proteins act in olfactory receptor neurons to regulate specificity.
Last updated: March 6, 2008
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