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Molecular Mechanisms That Generate Neuronal Diversity
Summary: Oliver Hobert studies molecular mechanisms that control cellular diversity in the nervous system.
A striking feature of nervous systems is their cellular complexity. My laboratory uses Caenorhabditis elegans as a model system to gain molecular and mechanistic insights into the creation of neuronal diversity. Our main concept is that the generation of neuronal diversity is a problem of establishing and interpreting "codes" that translate one-dimensional genomic sequence information into four-dimensional (i.e., temporally and spatially controlled) cellular diversification programs.
Cellular Diversity Generated by Gene Regulatory Events
We use several different neuron types as models to understand the generation of neuronal diversity: these include the two ASE gustatory neurons, ASE left (ASEL) and ASE right (ASER) (Figure 1); their two postsynaptic partners, the AIY interneurons (AIYL and AIYR) (Figure 2); and several other neuron classes defined by their use of common neurotransmitters (dopaminergic neurons and GABAergic neurons).
ASE sensory neurons and left/right asymmetry. The ASE gustatory neurons enable us to combine our interest in neuronal fate determination with our interest in the poorly understood generation of neuronal diversity along the left/right (L/R) axis. The two neurons that constitute the ASE class, ASEL and ASER, are bilaterally symmetric in most regards (cell position, axon/dendrite morphology, synaptic connectivity) yet express a different spectrum of putative chemoreceptors, thus allowing them to broaden their chemosensory capacities. The L/R asymmetry of the ASE neurons is stereotyped and developmentally programmed, mirroring the directional L/R asymmetries of many other vertebrate and invertebrate nervous systems.
The ASE neuron class therefore poses two intriguing questions: First, how are these two neurons made to be different from other classes of neurons (e.g., other sensory neurons)? Second, how are the left and the right ASE neurons made different from one another? We have used mutant screening approaches to identify genes that disrupt the asymmetry of the ASE neurons (lsy genes, for lateral symmetry). In most isolated lsy mutants, the two ASE neurons revert to a bilaterally symmetric ground state. We found that lsy genes define a genetic regulatory cascade composed of several transcription factors (TFs) and microRNAs (miRNAs). Together, lsy genes diversify a bilaterally symmetric ground state and ultimately generate behavioral laterality.
Exploiting the unique genetic amenability of C. elegans, we have recently expanded our genetic screens for lsy mutants to investigate the central question of how the L/R asymmetric activity of the regulatory factors is brought about. We have retrieved at least 20 genes that define the L/R asymmetric state of the ASE neurons, and we are characterizing these genes on a molecular level.
Our retrieval of mutant alleles of a general ASE cell fate determinant indicates that our genetic screening for lsy mutants will also reveal the mechanisms underlying bilaterally symmetric fate specification, that is, the events that induce the bilaterally symmetric ground state of ASEL/R and therefore make ASEL/R different from other neuron types. Since the ASE neurons (like any other cell in C. elegans) are generated by a stereotyped lineage program, which itself is defined by a series of binary decisions (divisions along the L/R and anterior/posterior axis), we expect to identify the mechanisms that translate lineage programs into specific "TF codes" (and/or some parallel codes, such as miRNA codes), which then determine the set of terminal differentiation markers of the ASE neuron type. Although the concept of TF codes is not novel, the current understanding of these codes is largely correlative and descriptive and often only describes a specific state in the lineage history of a cell. We expect that our unbiased genetic analysis of ASE neuron development will reveal distinct TF codes that act hierarchically at different points along the lineage history of the ASE neurons, thus providing a comprehensive picture of neuronal lineage commitment and differentiation.
AIY interneurons: from gene expression diversity to phenotypic diversity. In the past, we have gained significant insights into the development of the AIY interneurons. Besides diving deeper into earlier aspects of AIY development, we are using the AIY interneurons as a paradigm to correlate specific anatomical and functional features of AIY with the defining molecular features of AIY, a gene expression battery that we have recently defined. The starting point for these studies was our use of a combination of forward and reverse genetic approaches to identify cis- and trans-regulatory aspects of AIY differentiation. We have shown that two homeobox transcription factors (ttx-3 and ceh-10) are combinatorially required for AIY development and therefore define a TF code that determines AIY differentiation. We have defined the cis-regulatory elements through which these homeobox genes act, allowing us to identify—through the use of computational tools that we developed—a large battery of AIY-expressed genes. This gene battery provides a potential molecular correlate to the functional diversity of AIY. For example, we found that one-quarter of all predicted neuropeptide-receptor genes are expressed in AIY. These and other AIY-expressed genes will help us understand on a molecular level how the AIY interneurons control specific C. elegans behaviors that we have defined and how these neurons are linked into a functional circuit with ASE and other neurons. In a specific application of the rationale, we have recently shown that an AIY-expressed gene, sra-11, is required for the correct execution of olfactory imprinting, a novel memory paradigm that we found to be controlled by AIY.
Recently, we have begun to work our way "upstream" from the ttx-3/ceh-10–mediated control of AIY differentiation. Following the rationale of TF codes, we have screened for mutants in which the ttx-3/ceh-10–induced AIY differentiation is defective and have identified upstream regulatory mechanisms.
Other neuron classes that we study are more diverse. For example, dopaminergic neurons are a class of neurons originating from different neuronal lineages. Surprisingly, we found that even though diverse, all dopaminergic neurons use a common regulatory logic to turn on their terminal differentiation gene. We found this phylogenetic logic to be phylogenetically conserved.
Taken together, our work on gene regulatory control in the nervous system has led to our proposal of the concept of "terminal selector genes," transcription factors that coregulate batteries of genes that determine the terminal phenotype of a neuron. We are currently testing how widely applicable this principle is.
Maintaining Nervous System Architecture
The complexity of nervous system architecture is an obvious indicator of neuronal diversity. Our genetic approaches to mechanisms that control this architectural complexity have revealed a novel concept in axon patterning. Specifically, we discovered that the relative position of axons in defined axon fascicles must be actively maintained, a previously unknown phenomenon we have termed "axon maintenance." An intriguing aspect of this phenomenon is its cell-type specificity: different sets of maintenance factors appear to be required for different sets of neurons to maintain their position. Thus, axons do not require unselective "glues" to maintain their relative position but sample cell-specific information from their environment.
The axon maintenance phenomenon emerged from our studies of six zig genes, which we had identified in a genome-wide analysis of expression patterns of predicted proteins containing immunoglobulin (Ig) domains. We focused on zig genes because of their temporally strictly controlled coexpression in a known guidepost neuron long after development of the nervous system has terminated. Postdevelopmental removal of this guidepost neuron or loss of zig gene function has no impact on axon development but causes a failure of the axons to be maintained in their correct position in defined fascicles. More recently, we have identified additional factors involved in axon maintenance, including a specific splice-form of the fibroblast growth factor receptor (FGFR), egl-15(5A), and a novel, giant extracellular protein, DIG-1. Our studies not only implicated the FGFR in a new biological process but also, even more surprisingly, revealed a non-kinase-dependent role of the protein. Its extracellular domain is sufficient to provide axon maintenance function. This observation should prompt a reinvestigation of long-known, but functionally uncharacterized, extracellular domain-only forms of various FGFRs.
We are currently attempting to understand the molecular mechanism of action of the ZIG, DIG-1, and EGL-15(5A) maintenance factors by genetic analysis and by direct in vitro interaction tests using standard tissue culture binding assays. The working model is that these factors, all bearing Ig domains, may interact in different, cell-type-specific combinations to build adhesive complexes that anchor axons in their appropriate environment.
From Molecules to Behavior
Elucidating the molecular machinery that generates and defines the identity of the individual neuron types not only improves our understanding of cell fate specification but also provides a platform to determine how simple neuronal circuits are assembled and how they generate behavioral patterns.
For example, we have described a novel behavioral paradigm, olfactory imprinting, controlled by the AIY neuron classes. We are studying molecules that are required for the gustatory function of the ASE neuron class and, most recently, have begun to investigate how previous experience modulates gustatory behavior.
This work was supported in part by grants from the National Institutes of Health.
Last updated November 16, 2009
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