Abstract
Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.
Highlights
Taste buds are composed of 50–100 specialized neuro-epithelial cells that have classically been divided into type I, II, and III taste cells based originally on morphology and more recently on gene expression patterns[1, 2]
Using dissociated taste cell preparations made from the circumvallate (CV) papillae of Tas1r3-green fluorescent protein (GFP) transgenic mice, Tas1r3GFP cells (n = 9) were identified by their intrinsic fluorescence and collected manually (Supplementary Fig. S1)
To gain further insights into the nature of type III taste cells, we identified these cells by physiological methods
Summary
Taste buds are composed of 50–100 specialized neuro-epithelial cells that have classically been divided into type I, II, and III taste cells based originally on morphology and more recently on gene expression patterns[1, 2]. Molecular genetic studies have led to the discovery of multiple genes encoding the receptors and downstream transduction molecules for a few of the basic tastes They include the GPCRs responsible for sweet (Tas1r2 + Tas1r3), umami (Tas1r1 + Tas1r3), and bitter (Tas2rs) tastes, and an ion channel, the epithelial sodium channel (ENaC), that mediates one of the two salty taste transduction pathways in mammals[3,4,5,6,7,8]. Genes encoding key components of the downstream signalling pathway shared by sweet, bitter, and umami tastes have been identified, including Gnat[3], Gnat[1], Gnat[2], Gna[14], Gng[13], Plcb[2], Itpr[3], and Trpm[] Many of these discoveries were driven by methods such as differential display PCR and genetic mapping, which are limited in throughput and sensitivity compared with newer techniques such as microarrays and next-generation RNA sequencing (RNA-Seq). The methodology described in this article could be used for single-cell RNA-Seq analysis of any cell type
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