Deconstructing Olfaction with Transcriptomics: From Whole Tissue to Single-Cells, and from Zebrafish to Humans
Authors: Saraiva Luis , Ibarra-Soria Ximena , Aguilera Fernando R.M. , Marioni John C. , Logan Darren W. 
Published: July 2019

Mammals can perceive myriad odorous molecules based on their perceived smell. It is estimated that humans can discriminate ∼10,000–1 trillion different odours. In animals, the olfactory system can also detect specific odorants that can elicit changes in behaviour and/or physiology. Thus, from identifying kin, food sources and sexually receptive mates to avoiding predation and disease, appropriate perception of environmental olfactory sensory cues is critical for survival and reproduction. The importance of sensing the molecular environment is reflected in the genetic investment in encoding olfactory receptors (ORs), which constitute the largest gene family in mammals. The OR gene repertoire is largely species-specific, and is shaped by the nature and necessity of chemosensory information for survival in each species' niche. As well as receptor differences, the morphology, size, neural projections and organization of chemosensory epithelia vary remarkably across mammals, suggesting differences in wider gene expression networks.

Odorant reception occurs primarily in the olfactory mucosa (OM), and is mediated by the ORs located in the cilia of olfactory sensory neurons (OSNs). ORs are then used in a combinatorial fashion to maximize odorant detection and discrimination. OSNs expressing the same OR are dispersed within a distinct area of the OM, and their axons coalesce into a few glomeruli in the main olfactory bulb where they synapse to second-order neurons in the olfactory pathway. It turn these neurons transmit signals to the olfactory cortex and other regions of the brain. Thus, a population of OSNs expressing a given OR constitutes an elementary unit of olfactory sensory input to the brain. However, twenty-four years after the initial discovery of the ORs, the molecular heterogeneity of the olfactory system at the intra- and inter-specific level still remains largely unknown. Our recent studies aimed to answer these questions.

Firstly, to identify single neuron-specific molecular barcodes, and to understand how the molecular heterogeneity at the single OSN level contributes to odor perception, we combined RNAseq with Fluorescent Assisted Cell Sorting (FACS) in a hierarchical fashion – from the whole tissue to single cells. Our analysis allowed us to identify hundreds of OSN-specific genes and thousands of other cell type specific genes in the OM. We were able to identify a previously uncharacterized sub-division of mature OSNs, which differentially express hundreds of genes. By sequencing single mature OSNs, we found that OSNs are extremely homogenous at the molecular level and can be subdivided in two classes based on their OR abundance levels. Notably, one of these classes is a novel class of chemosensory neurons which lack ORs, and express a unique molecular barcode. The high-sensitivity and hierarchical nature – from whole-tissue to single-cell – of our approach has the potential to unravel more novel pathways underlying olfactory neuronal diversity and function. This method can also be extended to any other cell types in the nervous system in order to discover novel genes associated with specific circuits or functions.

Lastly, to study the natural variation of the olfactory system between species with different chemosensory niches, we performed RNA-seq of the OM of zebrafish, mouse, rat, marmoset, macaque and human. Then, to better understand the evolutionary dynamics of gene expression in the olfactory system of these species, we conducted a comparative analysis of their olfactory transcriptomes. We found that ORs are expressed across a large dynamic range in all the species analysed, and that the RNA abundances correlate positively with the number of cells expressing a single receptor. The comparative transcriptomic analysis revealed a high degree of molecular conservation from zebrafish to humans. In addition, we developed a strategy that combined phylogenetics with the transcriptional profiles of OR [and other] genes to identify which receptors and gene networks may have been selected for different niches, and to better understand the evolution of olfaction.