The Olfactory System of the Mouse
The sense of smell (olfaction) is designed to detect a very wide variety of chemical structures in the external environment. In the mouse, chemosensory detection at the molecular receptor level is mediated primarily by a repertoire of ~1,100 odorant receptor (OR) genes expressed in olfactory sensory neurons (OSNs) in the main olfactory epithelium. These ORs have the seven-transmembrane structure that is typical for G-protein coupled receptors. Each mature OSN is thought to express just one OR gene, and only one of the two alleles of that OR gene. Axons of OSNs that express the same OR gene terminate and coalesce into a few glomeruli, which are well-delinead anatomical structures in the olfactory bulb numbering ~3,600 in an adult mouse. What are the molecular mechanisms that enable an OSN to restrict expression of the immense OR gene repertoire to just one gene and one allele of it? What are the molecular and cellular mechanisms that enable axons of OSNs that express the same OR gene to coalesce with high precision into glomeruli? We are approaching these fundamental biological questions by genetic manipulation of the mouse.
Relationship between number OSNs and volume of glomeruli
How many OSNs per mouse express a given OR gene? A traditional approach for visualizing OSNs and their axons consists of tagging an OR gene genetically with an axonal marker that is cotranslated with the OR by virtue of an internal ribosome entry site (IRES). We have now generated full cell counts for gene-targeted strains of the OR-IRES-marker design coexpressing a fluorescent protein . These strains represent 11 OR genes, a 1% sample of the OR gene repertoire. We found a 17-fold range in the OSN number per mouse across these OR genes. We discovered a strong linear correlation between OSN number and the volume of the corresponding glomeruli. A practical outcome of our findings is that total glomerular volume can be used as a surrogate measurement for estimating OSN numbers in these gene-targeted strains.
A mouse OR with an extremely broad odorant response profile
How selective or how broad are OSNs that express a given OR gene with regard to their odorant responsiveness, that is the profile of chemical structures (odorants) that active these cells physiologically? We have previously documented the broadest odorant response profile in native mouse OSNs, for OSNs expressing OR gene SR1. We have now found that that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256-17 is even broader than that of OSNs expressing SR1. Of 35 single chemical compounds belonging to distinct structural classes, OSNs expressing MOR256-17 responded to 31 chemicals, compared to 10 for OSNs expressing SR1. The 10 compounds that activated OSNs expressing SR1 also activated OSNs expressing MOR256-17. We conclude that OSNs expressing MOR256-17 reside on one extreme of odorant responsiveness among the ~1,100 populations of OSNs expressing distinct OR genes in the mouse. The biological function and evolutionary emergence of OSNs with such extremely broadly responsiveness remains unclear.
RNA-seq of whole olfactory mucosa and single OSNs
The main olfactory epithelium is a component of the main olfactory mucosa, a complex tissue that is composed of multiple cell types, neuronal and non-neuronal. RNA-seq, also called deep sequencing, has emerged as a powerful approach to quantify gene expression in tissue samples and very recently also in single cells. We have now applied RNA-seq hierarchically in three steps of decreasing cellular heterogeneity: starting with crude tissue samples dissected from the nose, proceeding to flow-cytometrically sorted pools of mature OSNs, and finally arriving at single mature OSNs. We showed that 1,087 of 1,099 (=98.9%) of intact olfactory receptor (OR) genes are expressed in mature OSNs, indicating that essentially every member of the OR gene repertoire is a candidate receptor for odorants based on the minimal criterion of expression in mature OSNs. We found that single mature OSNs typically express a single intact OR gene abundantly, consistent with the one neuron-one receptor rule. For the single OSNs where the two alleles of the abundantly expressed OR gene exhibit single-nucleotide polymorphisms, we demonstrate that monoallelic expression of the abundantly expressed OR gene is extremely tight. We also found that two single cells lack OR gene expression but express Trpc2 and Gucy1b2, representing the type B cells that we had discovered earlier with another single-cell based method for gene expression. We discovered that these cells can be distinguished from canonical OSNs by the differential, higher expression of at least 55 genes. We can thus firmly establish these type B cells as a novel type of chemosensory neuron that is fundamentally distinct from canonical, OR-expressing OSNs.