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Transcriptional Profiling of Individual Airway Projecting Vagal Sensory Neurons

  • Stuart B. Mazzone
  • Luyi Tian
  • Aung Aung Kywe Moe
  • Matthew W. Trewella
  • Matthew E. Ritchie
  • Alice E. McGovernEmail author
Article

Abstract

Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs. Subtypes are heterogenous in their responsiveness to stimuli, phenotype, and developmental origin, but they collectively serve to regulate normal respiratory and pulmonary processes and elicit a diverse range of defensive physiological responses that protect against noxious stimuli. In this study, we aimed to investigate the transcriptional features of vagal bronchopulmonary sensory neurons using single-cell RNA sequencing (scRNA-seq) to provide a deeper insight into their molecular profiles. Retrogradely labeled vagal sensory neurons projecting to the airways and lungs were hierarchically clustered into five types reflecting their developmental lineage (neural crest versus placodal) and putative function (nociceptors versus mechanoreceptors). The purinergic receptor subunit P2rx2 is known to display restricted expression in placodal-derived nodose neurons, and we demonstrate that the gene profiles defining cells high and low in expression of P2rx2 include G protein coupled receptors and ion channels, indicative of preferential expression in nodose or jugular neurons. Our results provide valuable insight into the transcriptional characteristics of bronchopulmonary sensory neurons and provide rational targets for future physiological investigations.

Keywords

P2X2 Cough Nodose Jugular Bronchopulmonary Single-cell RNA-sequencing Vagus nerve Sensory neurons Adenosine triphosphate 

Notes

Acknowledgments

The authors acknowledge Ms. Jennifer Keller for expert technical assistance in aspects of this study.

Funding information

Funded by grants to S.B.M [1078943] and A.E.M [1121376] from the National Health and Medical Research Council of Australia.

Compliance with Ethical Standards

Experiments using pathogen-free C57BL/6 mice (8–10 weeks, male, n = 30) were approved by the University of Melbourne, Parkville, Australia, accredited institutional animal ethics committee in accordance with the Australian code for the care and use of animals for scientific purposes.

Supplementary material

12035_2019_1782_MOESM1_ESM.xlsx (15 kb)
Online resource 1. Mapping and gene count metrics for the single cell RNAseq samples, related to Figure 1. (XLSX 14 kb)
12035_2019_1782_MOESM2_ESM.xlsx (17 kb)
Online resource 2. Differentially expressed genes between the five clusters, related to Figure 1. (XLSX 16 kb)
12035_2019_1782_MOESM3_ESM.pdf (3.4 mb)
Online resource 3. Violin plots showing mean expression values of markers of bronchopulmonary sensory neuron subtypes previously reported in literature within the 5 clusters defined in Figure 1. In each graph the highlighted cluster denotes significantly greater expression of that cluster to another (e.g. Cx > Cy), suggestive of enrichment. Significance determined by multi comparison Tukey’s one-way ANOVA, confidence interval set at 90%. A) Ion channels: Htr3a, C1 > C2-5; P2rx2, C1 > C3-5; P2rx3, C1 > C2; P2ry1, C5 > C1-4; Piezo1, C5 > C2, C4; Piezo2, C4 > C1-3; Scn1a, C4 > C1-3, C5; Scn10a, C4, C5 > C2, C3; Trpa1, C1 > C2-5; Trpv1, C1 > C2-3, C5. B) Transcription factors: Phox2b, C1 > C4; Prdm12, C4 > C1-3, C5. C) G-protein coupled receptors: Npyr2, C4 > C2; Par1, C1 > C2, C3, C5; Ptgdr, C5 > C2; S1pr3, C1 > C2, C3 and C5 > C2, C3. D) Neurotrophic factors: Ntrk1, C4 > C1-3, C5. E) Neurofilaments: Nefh, C4 > C1-3. F) Other: Slc17a7, C4 > C1-3, C5. (PDF 3508 kb)
12035_2019_1782_MOESM4_ESM.xlsx (19 kb)
Online resource 4. Differentially expressed genes between the P2rx2Low and P2rx2High clusters, related to Figure 2. (XLSX 18 kb)
12035_2019_1782_MOESM5_ESM.xlsx (68 kb)
Online resource 5. Genes correlated to P2rx2 expression, related to Figure 3. (XLSX 68 kb)
12035_2019_1782_MOESM6_ESM.xlsx (12 kb)
Online resource 6. Genes for Ion channel and G-protein coupled receptors correlated to P2rx2 expression, related to Figure 3. (XLSX 12 kb)
12035_2019_1782_MOESM7_ESM.xlsx (6 mb)
Online resource 7. Expression of all detected genes in every single cell RNAseq sample (values in CPM). (XLSX 6124 kb)

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Anatomy and NeuroscienceThe University of MelbourneParkvilleAustralia
  2. 2.Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical ResearchParkvilleAustralia

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