- Olfactory Development
How does the olfactory apparatus of vertebrates detect and discriminate thousands of odors? Our approach to elucidating the mechanisms of olfactory discrimination involves the characterization of odorant receptors and the neural pathways that they activate. We are also interested in the developmental mechanisms responsible for specifying odorant receptor expression in olfactory neurons and the pathfinding of these cells' axons to their appropriate targets. Finally, our lab is developing DNA microarray technologies to elucidate genome-wide patterns of gene expression in the nervous system.
Patterning in the olfactory bulb. Olfactory neurons expressing the same odorant receptor converge with great precision to a small number of glomeruli in the olfactory bulb. This suggests that spatial patterns of afferent innervation in the bulb are used to encode olfactory information. What are the mechanisms for specifying the pattern of olfactory neuron projections in the olfactory bulb? In one model, each olfactory neuron type recognizes and extends toward aparticular glomerulus by responding to specific guidance cues presented by the target. We are pursuing several complementary approaches to identify the molecules involved in olfactory axon pathfinding. Transgenic manipulations in the mouse and zebrafish are being used to assess the potential role of candidate genes in the formation of the olfactory sensory map. We are also utilizing DNA microarrays to search for molecules expressed in spatially-restricted patterns in the olfactory bulb; such molecules would be good candidates as guidance cues for ingrowing olfactory axons.
The zebrafish olfactory system. The numerical and anatomical simplicity of the zebrafish olfactory system facilitates an analysis of the molecular and cellular basis of olfactory coding in a vertebrate species. In one line of investigation, we are defining the odorant-binding properties of cloned fish odorant receptors. This study is facilitated by the observation that fish respond to water soluble cues that are more amenable as probes for biochemical analyses. The zebrafish also offers advantages for studying development, and continuing improvements in methods for the generation and screening of mutant zebrafish may permit genetic approaches for studying odorant receptor gene expression and olfactory neurogenesis. To gain insight into the mechanisms governing odorant receptor gene expression, we have carried out genomic mapping of odorant receptor genes and have found that, as in other vertebrate species, odorant receptor genes are clustered in the zebrafish genome. However, genes tightly linked within a cluster are not coordinately regulated, suggesting that the regulation ofindividual receptor genes requires the interaction of specific trans-acting factors with proximal cis-regulatory sequences. We are pursuing a variety of approaches, including transgenic manipulations, to define the promoter sequences responsible for directing the developmentally-regulated expression of the odorant receptor genes.
DNA microarrays. World-wide consortia are rapidly generating the complete nucleotide sequences of the genomes (entire DNA content) of a wide variety of organisms, including a large number of eubacteria and archaebacteria, budding yeast (S. cerevisiae), several plant species, nematodes (C. elegans), fruit flies (D. melanogaster), mice, and humans. Concomitant with these genome sequencing efforts, investigators are also attempting to determine which sets of genes, out of the entire repertoire in an organism, are expressed under what conditions, in which tissues, at what stage in development, and in response to what internal or external cues. This latter information the "expressed genome" of an organism has opened up entirely new ways of addressing and understanding basic biological processes. The methodology for assessing the expressed genes in an organism or cell is based on using glass slides onto which DNAs corresponding to all of the genes in a organism are arrayed in an addressable pattern. These DNA microarrays Ð also referred to as "gene chips" are then probed with mRNA isolated from the cells or tissues of interest. In this way, a global picture of which genes are expressed, and which are not, under any circumstance can be obtained. With currently available technology, it is now possible to monitor simultaneously the expression of all of the recognizable genes in yeast (6,307), fruit flies (~14,200), worms (~19,100) and even mice and humans (~35-50,000). The ability to perform such analyses en masse provides the ability to characterize biological and pathological processes at unprecedented levels of detail. At the same time, this field is still in its infancy with regard to optimizing and refining both the technology for producing and querying microarrays and the computer-based methods for analyzing and extracting biological meaning from such massive amounts of data.
We have established in our laboratory the full capabilities for carrying out DNA microarray analysis of gene expression. These techniques allow the analysis of mRNA expression from tens of thousands of genes at a time. To date, we have created high density cDNA microarrays from the mouse and the zebrafish. We are using these microarrays as tools to investigate patterns of developmentally-regulated and spatially-restricted patterns of gene expression in the vertebrate central nervous system, as well as more generally during development. We have also generated DNA microarrays containing all identified open reading frames in the worm and are using these microarrays as tools for monitoring gene expression in this model genetic organism. In collaboration with Terry Speed's group (UC Berkeley Department of Statistics, we are also working to develop improved methods for DNA microarray experimental design and statistical analysis.