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.
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References
Mazzone SB, Undem BJ (2016) Vagal afferent innervation of the airways in health and disease. Physiol Rev 96:975–1024
Kummer W, Fischer A, Kurkowski R, Heym C (1992) The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry. Neuroscience. 49(3):715–737
Brouns I, Pintelon I, Timmermans JP, Adriaensen D (2012) Novel insights in the neurochemistry and function of pulmonary sensory receptors. Adv Anat Embryol Cell Biol 211:1–115 vii
Mazzone SB (2005) An overview of the sensory receptors regulating cough. Cough. 1:2
Canning BJ, Spina D (2009) Sensory nerves and airway irritability. Handb Exp Pharmacol 194:139–183
Widdicombe J, Lee LY (2001) Airway reflexes, autonomic function, and cardiovascular responses. Environ Health Perspect 109(Suppl 4):579–584
Mazzone SB, Reynolds SM, Mori N, Kollarik M, Farmer DG, Myers AC, Canning BJ (2009) Selective expression of a sodium pump isozyme by cough receptors and evidence for its essential role in regulating cough. J Neurosci 29(43):13662–13671
Canning BJ, Mazzone SB, Meeker SN, Mori N, Reynolds SM, Undem BJ (2004) Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea-pigs. J Physiol 557:543–558
Kollarik M, Undem BJ (2002) Mechanisms of acid-induced activation of airway afferent nerve fibres in guinea-pig. J Physiol 543:591–600
Baluk P, Nadel JA, McDonald DMJ (1992) Substance P-immunoreactive sensory axons in the rat respiratory tract: a quantitative study of their distribution and role in neurogenic inflammation. Comp Neurol 319(4):586–598
Coleridge JC, Coleridge HM (1984) Afferent vagal C fibre innervation of the lungs and airways and its functional significance. Rev Physiol Biochem Pharmacol 99:1–110
Hunter DD, Undem BJ (1999) Identification and substance P content of vagal afferent neurons innervating the epithelium of the guinea pig trachea. Am J Respir Crit Care Med 159:1943–1948
Lundberg JM, Hokfelt T, Martling CR, Saria A, Cuello C (1984) Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res 235:251–261
Baker CV, Schlosser G (2005) The evolutionary origin of neural crest and placodes. J Exp Zool B Mol Dev Evol 304:269–273
Baker CV (2005) The embryology of vagal sensory neurons. In: Undem BJ, Weinreich D (eds) Advances in vagal afferent neurobiology. CRC, Boca Raton
Baker CV, Bronner-Fraser M (2001) Vertebrate cranial placodes. I. Embryonic induction. Dev Biol 232:1–61
D'Amico-Martel A, Noden D (1983) Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am J Anat 166:445–468
Narayanan CH, Narayanan Y (1980) Neural crest and placodal contributions in the development of the glossopharyngeal-vagal complex in the chick. Anat Rec 196:71–82
McGovern AE, Davis-Poynter N, Yang SK, Simmons DG, Farrell MJ, Mazzone SB (2015) Evidence for multiple sensory circuits in the brain arising from the respiratory system: an anterograde viral tract tracing study in rodents. Brain Struct Funct 220(6):3683–3699
Undem BJ, Chuaychoo B, Lee MG, Weinreich D, Myers AC, Kollarik M (2004) Subtypes of vagal afferent C-fibres in guinea-pig lungs. J Physiol 556(3):905–917
Kwong K, Kollarik M, Nassenstein C, Ru F, Undem BJ (2008) P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus. Am J Physiol Lung Cell Mol Physiol 295(5):L858–L865
Nassenstein C, Taylor-Clark TE, Myers AC, Ru F, Nandigama R, Bettner W, Undem BJ (2010) Phenotypic distinctions between neural crest and placodal derived vagal C-fibres in mouse lungs. J Physiol 588(Pt 23):4769–4783
Lieu T, Kollarik M, Myers AC, Undem BJ (2011) Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract. Am J Phys Lung Cell Mol Phys 300(5):L790–L798
D’Autréaux F, Coppola E, Hirsch MR, Birchmeier C, Brunet JF (2011) Homeoprotein Phox2b commands a somatic-to-visceral switch in cranial sensory pathways. Proc Natl Acad Sci U S A 108(50):20018–20023
McGovern AE, Driessen AK, Simmons DG, Powell J, Davis-Poynter N, Farrell MJ, Mazzone SB (2015) Distinct brainstem and forebrain circuits receiving tracheal sensory neuron inputs revealed using a novel conditional anterograde transsynaptic viral tracing system. J Neurosci 35:7041–7055
Kollarik M, Ru F, Undem BJ (2019) Phenotypic distinctions between the nodose and jugular TRPV1-positive vagal sensory neurons in the cynomolgus monkey. Neuroreport. 30(8):533–537
Trancikova A, Kovacova E, Ru F, Varga K, Brozmanova M, Tatar M, Kollarik M (2018) Distinct expression of phenotypic markers in placodes- and neural crest-derived afferent neurons innervating the rat stomach. Dig Dis Sci 63(2):383–394
Surdenikova L, Ru F, Nassenstein C, Tatar M, Kollarik M (2012) The neural crest- and placodes-derived afferent innervation of the mouse esophagus. Neurogastroenterol Motil 24(10):e517–e525
Hu G, Huang K, Hu Y, Du G, Xue Z, Zhu X, Fan G (2016) Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons. Sci Rep 6:31851
Li C, Wang S, Chen Y, Zhang X (2018) Somatosensory neuron typing with high-coverage single-cell RNA sequencing and functional analysis. Neurosci Bull 34(1):200–207
Li CL, Li KC, Wu D, Chen Y, Luo H, Zhao JR, Wang SS, Sun MM et al (2016) Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity. Cell Res 26(1):83–102
Usoskin D, Furlan A, Islam S, Abdo H, Lönnerberg P, Lou D, Hjerling-Leffler J, Haeggström J et al (2015) Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat Neurosci 18(1):145–153
Hockley JRF, Taylor TS, Callejo G, Wilbrey AL, Gutteridge A, Bach K, Winchester WJ, Bulmer DC et al (2019) Single-cell RNAseq reveals seven classes of colonic sensory neuron. Gut. 68:633–644
Kupari J, Häring M, Agirre E, Castelo-Branco G, Ernfors P (2019) An atlas of vagal sensory neurons and their molecular specialization. Cell Rep 27(8):2508–2523.e4
McGovern AE, Mazzone SB (2010) Characterization of the vagal motor neurons projecting to the guinea pig airways and esophagus. Front Neurol 1:153
Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finnell R, Zettel M, Coleman P (1992) Analysis of gene expression in single live neurons. Proc Natl Acad Sci U S A 89:3010–3014
Hashimshony T, Wagner F, Sher N, Yanai I (2012) CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell Rep 2:666–673
Liao Y, Smyth GK, Shi W (2013) The subread aligner: Fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res 41(10):e108
Liao Y, Smyth GK, Shi W (2014) FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 30(7):923–930
Lun ATL, Bach K, Marioni JC (2016) Pooling across cells to normalize single-cell RNA sequencing data with many zero counts. Genome Biol 17(1):75 http://genomebiology.biomedcentral.com/articles/10.1186/s13059-016-0947-7
Kiselev VY, Kirschner K, Schaub MT, Andrews T, Yiu A, Chandra T, Natarajan KN, Reik W et al (2017) SC3: Consensus clustering of single-cell RNA-seq data. Nat Methods 14(5):483–486 http://www.nature.com/doifinder/10.1038/nmeth.4236
Robinson MD, McCarthy DJ, Smyth GK (2009) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 26(1):139–140 http://bioinformatics.oxfordjournals.org/cgi/doi/10.1093/bioinformatics/btp616
Wickham H (2009) ggplot2: elegant graphics for data analysis. Applied Spatial Data Analysis with R 2009. 21–54 p. http://link.springer.com/10.1007/978-0-387-98141-3.
Core Team, R. C. T. R. R: a language and environment for statistical computing. R Foundation for statistical computing, Vienna (2013).
Wang J, Kollarik M, Ru F, Sun H, McNeil B, Dong X, Stephens G, Korolevich S et al (2017) Distinct and common expression of receptors for inflammatory mediators in vagal nodose versus jugular capsaicin-sensitive/TRPV1-positive neurons detected by low input RNA sequencing. PLoS. 12(10):e0185985
Weigand LA, Ford AP, Undem BJ (2012) A role for ATP in bronchoconstriction-induced activation of guinea pig vagal intrapulmonary C-fibres. J Physiol 590(16):4109–4120
Riccio MM, Kummer W, Biglari B, Myers AC, Undem BJ (1996) Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways. J Physiol 496(Pt 2):521–530
Driessen AK, Farrell MJ, Mazzone SB, McGovern AE (2015) The role of the Paratrigeminal nucleus in vagal afferent evoked respiratory reflexes: a neuroanatomical and functional study in guinea pigs. Front Physiol 6:378
Driessen AK, Farrell MJ, Dutschmann M, Stanic D, McGovern AE, Mazzone SB (2018) Reflex regulation of breathing by the paratrigeminal nucleus via multiple bulbar circuits. Brain Struct Funct 223(9):4005–4022
Patthey C, Clifford H, Haerty W, Ponting CP, Shimeld SM, Begbie J (2016) Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick. Neural Dev 11:3
Desiderio S, Vermeiren S, Van Campenhout C, Kricha S, Malki E, Richts S, Fletcher EV, Vanwelden T et al (2019) Prdm12 directs nociceptive sensory neuron development by regulating the expression of the NGF receptor TrkA. Cell Rep 26(13):3522–3536.e5
Bartesaghi L, Wang Y, Fontanet P, Wanderoy S, Berger F, Wu H, Akkuratova N, Bouçanova F et al (2019) PRDM12 is required for initiation of the nociceptive neuron lineage during neurogenesis. Cell Rep 26(13):3484–3492.e4
Goswami SC, Mishra SK, Maric D, Kaszas K, Gonnella GL, Clokie SJ, Kominsky HD, Gross JR et al (2014) Molecular signatures of mouse TRPV1-lineage neurons revealed by RNA-Seq transcriptome analysis. J Pain 15(12):1338–1359
Acknowledgments
The authors acknowledge Ms. Jennifer Keller for expert technical assistance in aspects of this study.
Funding
Funded by grants to S.B.M [1078943] and A.E.M [1121376] from the National Health and Medical Research Council of Australia.
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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.
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Electronic Supplementary Material
Online resource 1.
Mapping and gene count metrics for the single cell RNAseq samples, related to Figure 1. (XLSX 14 kb)
Online resource 2.
Differentially expressed genes between the five clusters, related to Figure 1. (XLSX 16 kb)
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)
Online resource 4.
Differentially expressed genes between the P2rx2Low and P2rx2High clusters, related to Figure 2. (XLSX 18 kb)
Online resource 5.
Genes correlated to P2rx2 expression, related to Figure 3. (XLSX 68 kb)
Online resource 6.
Genes for Ion channel and G-protein coupled receptors correlated to P2rx2 expression, related to Figure 3. (XLSX 12 kb)
Online resource 7.
Expression of all detected genes in every single cell RNAseq sample (values in CPM). (XLSX 6124 kb)
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Mazzone, S.B., Tian, L., Moe, A.A.K. et al. Transcriptional Profiling of Individual Airway Projecting Vagal Sensory Neurons. Mol Neurobiol 57, 949–963 (2020). https://doi.org/10.1007/s12035-019-01782-8
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DOI: https://doi.org/10.1007/s12035-019-01782-8