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, 7 (1)

Identification of a Sacral, Visceral Sensory Transcriptome in Embryonic and Adult Mice

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Identification of a Sacral, Visceral Sensory Transcriptome in Embryonic and Adult Mice

C J A Smith-Anttila et al. eNeuro.

Abstract

Visceral sensory neurons encode distinct sensations from healthy organs and initiate pain states that are resistant to common analgesics. Transcriptome analysis is transforming our understanding of sensory neuron subtypes but has generally focused on somatic sensory neurons or the total population of neurons in which visceral neurons form the minority. Our aim was to define transcripts specifically expressed by sacral visceral sensory neurons, as a step towards understanding the unique biology of these neurons and potentially leading to identification of new analgesic targets for pelvic visceral pain. Our strategy was to identify genes differentially expressed between sacral dorsal root ganglia (DRG) that include somatic neurons and sacral visceral neurons, and adjacent lumbar DRG that comprise exclusively of somatic sensory neurons. This was performed in adult and E18.5 male and female mice. By developing a method to restrict analyses to nociceptive Trpv1 neurons, a larger group of genes were detected as differentially expressed between spinal levels. We identified many novel genes that had not previously been associated with pelvic visceral sensation or nociception. Limited sex differences were detected across the transcriptome of sensory ganglia, but more were revealed in sacral levels and especially in Trpv1 nociceptive neurons. These data will facilitate development of new tools to modify mature and developing sensory neurons and nociceptive pathways.

Keywords: autonomic regulation; nociceptor; pelvic pain; sexual dimorphism; urogenital; visceral pain.

Figures

Figure 1.
Figure 1.
Analysis of DRG from two different spinal levels (lumbar: L4-5; sacral: L6-S1) taken from five male and seven female adult C57Bl/6 mice. A, Principal component analysis (N = 23 168; all detected genes) indicates spinal region as the major source of variance in the data. B, Volcano plot illustrates differential expression between lumbar and sacral DRG. Light gray points are genes not differentially expressed, dark gray points are differentially expressed at adjusted p <0.05; examples of specific differentially expressed genes are highlighted in red. A negative value for the FC indicates an upregulation in sacral DRG, whereas a positive FC indicates upregulation in lumbar DRG. Full dataset are provided in Extended Data Figure 1-1. C, Heatmap with hierarchical cluster for all differentially expressed genes (N = 466; adjusted p <0.05) for adult lumbar and sacral samples (N = 12). Both samples and rows are clustered using Pearson correlation. Heat color reflects row-wise z score, and samples are colored according to spinal level and sex. Ranked gene lists for heat map are provided in Extended Data Figure 1-2.
Figure 2.
Figure 2.
Gene set enrichment analysis of DRG neurons taken from five male and seven female adult C57Bl/6 mice, comparing expression at lumbar (L4-5) and sacral (L6-S1) levels. Seven of 10 gene sets (grouped according to shared function; see Extended Data Fig. 2-1) were enriched (adjusted p <0.05): LGICs, TFs, GPCRs, NTFRs, Ca, and K channels, and TRP channels. A, B, Five gene classes showed upregulation in sacral DRG (blue) and two showed upregulation in lumbar DRG (red). C, D, Genes differentially expressed between lumbar and sacral spinal levels from the LGIC and GPCR classes. Blue indicates upregulation in sacral DRG and red upregulation in lumbar DRG. Full list of differentially genes identified by this analysis is provided in Extended Data Figure 2-2, and complete set of heatmaps is provided in Extended Data Figure 2-3.
Figure 3.
Figure 3.
Strategy for isolation of Trpv1-expressing neurons in mouse DRG. AF, Neurons isolated from lumbosacral adult DRG of TrpV1PLAP-nLacZ mice were incubated with the fluorescent galactosidase substrate, DDAOG (excitation/emission maxima ∼460/610 nm). A, Five neurons that have taken up DDAOG. B, Three neurons (arrows) are visible by their fluorescent hydrolysis product (excitation/emission maxima ∼645/660), also evident in the nucleus, so deduced to express galactosidase, i.e., Trpv1 neurons. C, Merge of panels A, B. D, Neurons expressing β-galactosidase visualized using immunohistochemistry; expression is restricted to the nucleus. E, Each neuron in the field has converted DDAOG to DDAO. F, Merge of panels D, E. Bar in A applies to all micrographs and represents 50 μm. GI, Representative outputs from flow cytometry of mouse lumbosacral DRG. G, Neurons isolated from wild-type mice were not treated with DDAOG. H, Neurons isolated from wild-type mice were treated with DDAOG, but without expressing β-galactosidase cannot hydrolyze this to form DDAO. I, Neurons isolated from of TrpV1PLAP-nLacZ mice were treated with DDAOG; many cells have hydrolyzed DDAOG to DDAO (i.e., Trpv1 neurons) and others have taken up DDAOG but not hydrolyzed this to DDAO (i.e., Trpv1-negative neurons).
Figure 4.
Figure 4.
Analysis of neurons isolated by flow cytometry to enrich the Trpv1 population from adult mice (five male, five female). A, Box plot of key markers associated with unmyelinated or myelinated DRG neurons; x-axis reflects gene of interest, y-axis reflects Log2(RPKM) gene expression. B, Volcano plot illustrating genes differentially expressed between Trpv1 lumbar and sacral DRG neurons. Light gray points are genes not differentially expressed, dark gray points are differentially expressed at adjusted p <0.05; examples of specific differentially expressed genes are highlighted in red. A negative value for the FC indicates an upregulation in sacral DRG, whereas a positive FC indicates upregulation in lumbar DRG. Full dataset provided in Extended Data Figure 4-1. C, Proportional Venn diagram showing the number of genes differentially expressed between spinal levels, in the total adult DRG population and adult Trpv1 neurons. A total of 466 genes were differentially expressed (adjusted p <0.05) between lumbar and sacral levels in the total population of adult DRG neurons, 327 of which were also detected as differentially expressed between lumbar and sacral levels in adult Trpv1 neurons. An additional 6511 genes were detected as differentially expressed (adjusted p <0.05) between lumbar and sacral levels when only the Trpv1 neurons were included. Gene lists summarized in Venn diagram are provided in Extended Data Figure 4-2. D, Heatmap with hierarchical cluster for all differentially expressed genes (N = 6838; adjusted p <0.05) for TRPV1 lumbar and sacral samples (N = 10). Both samples and rows are clustered using Pearson correlation. Heat color reflects row-wise z score, and samples are colored according to spinal level and sex. Ranked gene lists for heat map are provided in Extended Data Figure 1-2.
Figure 5.
Figure 5.
Gene set enrichment analysis of Trpv1-sorted DRG neurons taken from five male and five female adult mice, comparing expression at lumbar (L4-5) and sacral (L6-S1) levels. Nine of 10 gene sets (grouped according to shared function) were enriched (adjusted p <0.05): LGICs, TFs, GPCRs, NTFRs, K, Ca, and Cl channels, and TRP channels. A, B, All nine gene classes showed upregulation in sacral DRG (blue). C–F, Genes differentially expressed between lumbar and sacral spinal levels from the LGIC, GPCR, K, and Ca channel classes. Blue indicates upregulation in sacral DRG and red upregulation in lumbar DRG. Full list of genes identified by this analysis is provided in Extended Data Figure 2-2, and a complete set of heatmaps is provided in Extended Data Figure 5-1.
Figure 6.
Figure 6.
Analysis of DRG from two different spinal levels (lumbar: L4-5; sacral: L6-S1) taken from five male and five female E18.5 mouse embryos. A, Volcano plot illustrating genes differentially expressed between lumbar and sacral DRG neurons. Light gray points are genes not differentially expressed, dark gray points are differentially expressed at adjusted p <0.05 (N = 466); examples of specific differentially expressed genes are highlighted in red. A negative value for the FC indicates an upregulation in sacral DRG, whereas a positive FC indicates upregulation in lumbar DRG. Full dataset provided in Extended Data Figure 6-1. B, Proportional Venn diagram showing the number of genes differentially expressed between spinal levels (pooled male and female data), in the total E18.5 and total adult DRG population. A total of 466 genes were differentially expressed between lumbar and sacral levels in the total population of adult DRG neurons (adjusted p <0.05), 111 of which were also detected as differentially expressed between lumbar and sacral levels in E18.5 DRG neurons. An additional 1109 genes were detected as differentially expressed between lumbar and sacral levels only in the E18.5 DRG population. Gene lists summarized in Venn diagram are provided in Extended Data Figure 6-2. C, Heatmap with hierarchical cluster for all differentially expressed genes (N = 1220; adjusted p <0.05) for E18.5 lumbar and sacral samples (N = 10). Both samples and rows are clustered using Pearson correlation. Heat color reflects row-wise z score, and samples are colored according to spinal level and sex. Ranked gene lists for heat map (clusters 1 and 2) are provided in Extended Data Figure 1-2.
Figure 7.
Figure 7.
Sex differences in sensory neurons. Proportional Venn diagrams indicate the number of genes differentially expressed (adjusted p <0.05) between male and female groups in each spinal region of adult (A), Trpv1 (B), and E18.5 (C) populations. A, In adult DRG, 17 genes were detected as differentially expressed between males and females; of these, 10 were differentially expressed only in sacral DRG, one only in lumbar DRG and six were differentially expressed between males and female DRG of both lumbar and sacral spinal levels. B, In the Trpv1 adult DRG neurons, 33 genes were detected as differentially expressed between males and females; of these, 26 were differentially expressed between sex only in sacral DRG, seven were differentially expressed between sex in DRG of both spinal levels and no genes were differentially expressed between sexes only at the lumbar level. C, In E18.5 DRG neurons, 233 genes were detected as differentially expressed between males and females; of these, 153 were differentially expressed between sex only in sacral DRG, seven only in lumbar DRG and 73 were differentially expressed between sex at both spinal levels. Where <30 genes were identified as differentially expressed between males and females, the specific genes are listed in the table below the relevant Venn diagram. These lists distinguish genes differentially expressed only in the lumbar or sacral levels, or differentially expressed in both levels. Genes listed in blue were upregulated in males, whereas genes listed in pink were upregulated in females. Asterisks indicate genes located on sex chromosomes. Full lists of differentially expressed genes for A–C are found in E. Full gene lists for A–C are provided in Extended Data Figures 7-1, 7-2, 7-3.
Figure 8.
Figure 8.
Summary of experimental outcomes in adult mice. Sensory neurons from DRG at different spinal levels contain either entirely somatic sensory neurons (S) or a mixture of somatic and visceral (V) neurons. Genes differentially expressed between DRG at these spinal levels indicate features of pelvic visceral sensory neurons and are represented here by volcano plots (all genes) and heat maps (GPCRs). Very few genes are differentially expressed between males and females. Isolation of Trpv1-expressing nociceptors provides a more sensitive assay for revealing genes differentially expressed between spinal levels (all genes and GPCRs shown as per upper panel) and sex differences.

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