Olfactory Sensory Neuron Diversity Measured By RNA-Seq

The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes.

Smells are simply chemicals in the air that are recognized by nerves in our nose. Each nerve has a receptor that can identify a limited number of chemicals, and the nerve then relays this information to the brain. Animals have hundreds to thousands of different types of these nerves meaning that they can detect a wide array of smells.

Smell receptors are proteins, and the genes that encode these proteins can be very different in two unrelated people. This could partly explain, for example, why some people find certain odors intense and unpleasant while others do not. However, having different genes for smell receptors does not by itself completely explain why some people are more sensitive than others to particular smells. The amounts of each nerve type in the nose might also differ between people and have an effect, but to date it has not been possible to accurately count them all.

A team led by researchers at the Wellcome Trust Sanger Institute have now devised a new method to essentially count the number of each nerve type in the noses of mice from different breeds. The method makes use of a technique called RNA-sequencing, which can reveal which genes are active at any one time, and thus show how many nerves are producing each type of smell receptor. The researchers learned that different breeds of mice had remarkably different compositions of nerves in their noses. Further analysis revealed that this was due to changes to the DNA code near to the genes that encode the smell receptor.

RNA-seq is highly sensitive for OR mRNA detection and provides a measurement of OSN diversity

rna-seq

(A) Barplot of the mean normalized expression of 1249 OR genes from six biological replicates, accounting for gene length. Genes are ordered by decreasing abundance. The horizontal line is the median expression (32.06) and all the genes below it are shown in the inset. (B) Mean normalized mRNA expression values for the OR genes in chromosome 9 of the Olfr7 cluster deletion mouse line (green; n = 3). The corresponding abundances in wild-type animals (orange) are shown as a mirror image (n = 3). The break on the x-axis separates the two OR clusters. The dotted box encloses the deleted ORs. (C) Unequal RNAseq expression levels for different OR genes can be explained by two scenarios: (left) an OR gene with high RNAseq levels is expressed by a larger number of OSNs than a gene with low RNAseq abundance; and/or (right) an OR with high RNAseq values is expressed in the same number of OSNs as one with low RNAseq values, but at higher levels per OSN. (D) Comparison of the number of OSNs that express nine OR genes assessed by in situ hybridization (ISH; x-axis) to the corresponding RNAseq values (y-axis). Error bars are the standard error of the mean (ISH n = 4, RNAseq n = 6). The line is the linear regression and the Spearman’s correlation coefficient (rho) indicates a very strong correlation. Representative ISH images of two OR genes (in red) are shown. (E) In single-cell RNAseq experiments, 63 OSNs were randomly collected from the MOE. The distribution of OR mRNA expression in WOM samples is plotted (left), alongside the equivalent values for the ORs that were present in the picked single-OSNs (right). There is a significant enrichment (p<6.44 × 10−9) toward collecting OSNs that express OR genes with high RNAseq counts in WOM. (F) Comparison of the normalized expression value for the highest OR detected in each of the 63 single-OSNs (y-axis) to the corresponding mean value in WOM (x-axis, n = 3). The line is the linear regression and the Spearman’s correlation coefficient (rho) indicates there is no correlation.

Next, the research team sought to find out how the amount of each nerve type is controlled by giving mice water with different smells for weeks and looking how this affected their noses. These experiments revealed that a small number of the nerve types became more or less common after exposure to a smell. The altered nerves were directly involved in recognizing the smells, proving that the very act of smelling can change the make-up of nerves in a mouse’s nose.

These results confirm that the diversity in the nose of each individual is not only dictated by the types of receptors found in there, but also by the number of each nerve type. The next challenge is to understand better how these differences change the way people perceive smells.

Dr Darren Logan, the lead author on the study from the Wellcome Trust Sanger Institute, said: “The neurons in the olfactory system are highly connected to the neurons in the brain and studying these can help us understand neuronal development. We have shown that each individual has a very different combination of possible olfactory neurons, driven by genetics. In this study we also show that, with experience of different smells, these combinations of neurons change, so both genetics and environment interplay to give every individual a unique sense of smell.”

Ibarra-Soria X, Nakahara TS, Lilue J, Jiang Y, Trimmer C, Souza MAA, Netto PHM, Ikegami K, Murphy NR, Kusma M, Kirton A, Saraiva LR, Keane TM, Matsunami H Mainland J, Papes F, Logan DW. (2017) Variation in olfactory neuron repertoires is genetically controlled and environmentally modulated. eLife 6:e21476. [article]

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