Hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell RNA-seq.

Ximena Ibarra-Soria,Peter Mombaerts,John C. Marioni,Antonio Scialdone,Masayo Omura,Luis R. Saraiva,Mona Khan,Darren W. Logan

doi:10.1038/srep18178

Abstract

The mouse olfactory mucosa is a complex chemosensory tissue composed of multiple cell types, neuronal and non-neuronal. We have here 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 olfactory sensory neurons (OSNs), and finally arriving at single mature OSNs. We show that 98.9% of intact olfactory receptor (OR) genes are expressed in mature OSNs. We uncover a hitherto unknown bipartition among mature OSNs. We find that 19 of 21 single mature OSNs each express a single intact OR gene abundantly, consistent with the one neuron-one receptor rule. For the 9 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. The remaining two single mature OSNs lack OR gene expression but express Trpc2 and Gucy1b2. We establish these two cells as a neuronal cell type that is fundamentally distinct from canonical, OR-expressing OSNs and that is defined by the differential, higher expression of 55 genes. We propose this tiered experimental approach as a paradigm to unravel gene expression in other cellularly heterogeneous systems.

Highlights

We have recently characterized the transcriptome of C57BL/6 mouse olfactory mucosa by deep RNA sequencing (RNA-seq), and generated a comprehensive gene expression profile for this tissue[11]
To characterize gene expression in mature olfactory sensory neurons (OSNs), we need to purify them away from the many other cell types that are present within the crude tissue samples that can be scraped from the nasal cavity and contain pure main olfactory epithelium (MOE) and submucosa and adjacent tissues
We find that genes enriched in our Fluorescence Activated Cell Sorting (FACS)-sorted OSNs are consistent with Olfactory marker protein (OMP)+ enrichment in Sammeta et al, and that genes enriched in our whole olfactory mucosa (WOM) samples are consistent with OMP– enrichment in the same study[21] (Supplementary Fig. S1C)

Summary

Introduction

We have recently characterized the transcriptome of C57BL/6 mouse olfactory mucosa by deep RNA sequencing (RNA-seq), and generated a comprehensive gene expression profile for this tissue[11] This approach averages each gene’s expression level across the many different cell types that are present in these crude tissue scrapes, obscuring the heterogeneity of cell types and subtypes. Multiple additional types of chemosensory neuron have been identified in the mouse MOE, including cells expressing the guanylyl cyclase D receptor (Gucy2d or GC-D)[12] and cells expressing trace-amine associated receptors (TAARs)[13] These smaller cell populations have been the focus of some recent functional studies[14], but the full molecular identity and the extent of heterogeneity among the vast majority of chemosensory neuronal cell types in the MOE remain unknown. We identify 55 upregulated genes that establish these cells as a novel neuronal type within the MOE, which is fundamentally distinct from canonical OSNs

Methods

Results

Conclusion