Figure 1.
hybridization slices from adult mice. A Schematic overview for the preparation of the mouse head slices (14 µm). The nose and the mandible were removed before the slices were prepared (blue line). The black arrow indicates the cutting direction, while the red line indicates the intersecting plane that was used. B In situ hybridization for Pirt mRNA (25-fold). C 100-fold. D 200-fold magnification of the TG segment. Single cells are strongly stained. Scale bar B: 560 µm, C: 140 µm D: 75 µm.
Figure 2.
Expression pattern analysis of all detected GPCRs.
A The bar diagram shows the number of detected GPCRs that had FPKM values higher than 1 for the liver (L), skeletal muscle (SM), olfactory epithelium (OE), dorsal root ganglia (DRG), brain (B), and trigeminal ganglia (TG). Black bars: expressed GPCRs, gray bars: not expressed GPCRs. The lowest number of GPCRs was detected in liver (72) and skeletal muscle (89). The highest count was detected in the TG (197), OE (163), DRG (191), and brain (193). B To investigate not only the number of expressed GPCRs but also the general cumulative expression level for each tissue sample, we summarized all FPKM values (sFPKM). The brain (5079 sFPKM), TG (2808 sFPKM) and DRG (2871 sFPKM) had the highest presence of GPCRs. The skeletal muscle had the lowest amount of sFPKM (845), which was followed by the liver (933 sFPKM). C The bar diagram shows the expression pattern for all different GPCR subfamilies (secretin (Sec), adhesion (Adh), glutamate (Glut), frizzle and taste (Fzd/Taste), rhodopsin-alpha to -delta (Rho A-D), and not yet classified GPCRs (others). The rhodopsin-delta subfamily (shown without ORs) is expressed at a higher level in the DRG and TG compared with all other tissues. D The comparison of the distribution of GPCR subfamilies between the DRG and TG. Most members belong to the rhodopsin-alpha and rhodopsin-delta subfamiles.
Figure 3.
The 30 most highly expressed GPCRs in trigeminal
ganglia. GPCRs are listed depending on their expression level in the TG and DRG in comparison with the brain (B), liver (L), olfactory epithelium (OE), and skeletal muscle (SM). The FPKM value, which is an indicator of the expression strength, is represented by the color intensity. Exp describes whether the expression in the TG was previously known (+) or unknown (-). To the best of our knowledge, among the 30 most highly expressed genes, 14 GPCRs have not been previously described as expressed in the TG. Most of the detected GPCRs have unknown functions.
Figure 4.
In situ hybridization experiments in the mouse TG.
A Mrgprd (Mas-related Receptor D) B Mrgpre (Mas-Related Receptor E), C Mrgprx1 (Mas-Related Receptor X1) D Cnr (Cannabinoid Receptor 1) E Gpr35 (G-Protein Coupled Receptor 35) F Gpr126 (G-Protein Coupled Receptor 126) G Gpr155 (G-Protein Coupled Receptor 155) H Gpr158 (G-Protein Coupled Receptor 158) I Darc (Duffy/Antigen/Chemokine Receptor) J: Fzd3 (Frizzled 3) K Paqr (Progestin and AdipoQ Receptors) L Tac3 (Tachykainin 3) M Oprmd (mu-Opioid Receptor) N Tbx2 (Thromboxane A2 Receptor) O Drd3 (Dopamin Receptor D3) P O3far1 (Omega 3 Fatty Acid Receptor) Q Olfr78 (Olfactory Receptor 78/PSGR) R Olfr420 (Olfactory Receptor 420) S Olfr1417 (Olfactory Receptor 1417) T Ntrs2 (Neurotensin Receptor 2) (Scale for A, C-T 200 µm, B 100 µm).
Figure 5.
GPCR genes are ranked according to their specific expression in the TG, which is calculated by the quotient of the FPKM values of TG and the mean FPKM values of brain (B), liver (L), olfactory epithelium (OE), and skeletal muscle (SM). Members of the Mrgprs are the most specific GPCRs that have been detected in the TG and DRG. Among the 30 most specific GPCRs, seven newly detected GPCRs in the TG were identified. Twelve of the most specifically detected GPCRs are still orphans and, based on their specific expression patterns in the TG and DRG, may be important for tissue-specific functions.
Figure 6.
Comparison of cumulative FPKM values (sFPKM).
sFPKM values for some prominent ion channels and GPCRs where compared to the sFPKM values of the ORs. The expression of potassium channels is most pronounced in the TG (sFPKM 1551) and DRG (sFPKM 1230), followed by the VGSCs (TG: 963 sFPKM, DRG: 695 sFPKM). Mrgprs, iGlut, P2X, and GABA(A) are expressed at a similar level. Nevertheless, the cumulated FPKM of ORs (TG: 24 sFPKM, DRG: 10 sFPKM) can be compared with the cumulated FPKM of Cnr (TG: 22 sFPKM, DRG: 10 sFPKM), mGlut (TG: 41 sFPKM, DRG: 29 sFPKM), and P2y (TG: 38 sFPKM, DRG: 51 sFPKM). Mrgprs are expressed almost 3-fold higher in the DRG (197 sFPKM) than in the TG (76 sFPKM).
Figure 7.
Ranking of the most highly expressed ion channels in the TG.
We investigated the expression in the brain (B), liver (L), olfactory epithelium (OE), skeletal muscle (SM), DRG, and TG. Seven of the 30 most highly expressed ion channels have never been described as expressed in sensory ganglia in previous studies (marked with (-)). Many of the most highly expressed ion channels in the TG and DRG are involved in the sensation of pain.
Figure 8.
In situ hybridization for some ion channel in mouse TG.
A Trpv1 (Transient Receptor Potential Channel 1) B Trpm7 (Transient Receptor Potential Channel M7) C Trpm8 (Transient Receptor Potential Channel M8) D Trpc6 (Transient Receptor Potential Channel C6) E Scn1a (Voltage-Gated Sodium Channel 1A) F Scn9a (Voltage-Gated Sodium Channel 9A) G GlyRb (Glycine Receptor Beta) H Ano3 (Anoctamin 3) I Kcnk3 (Potassium Channel K3) J Kcnk9 (Potassium Channel K9) K Kcnk18 (Potassium Channel 18) L Kcna4 (Potassium Channel A4) M Grik2 (Glutamate Receptor, Ionotropic, Kainate 2) N Pgr (Progesterone Receptor) O Pirt (Phosphoinositide-Interacting Regulator of TRP) P Tlr1 (Toll-Like Receptor 1) Q Grik2 (Glutamate Receptor, Ionotropic, Kainate 2) R Ano3 (Anoctamin 3) S Trpm7 (Transient Receptor Potential Channel M7). Weak signals are marked by arrows. Scale for A-P 250 µm, Q-S 75 µm.
Figure 9.
Ranking of TG- and DRG-specific ion channels.
We compared the expression levels of the most specific ion channels of the TG and DRG with those in the brain (B), liver (L), olfactory epithelium (OE), and skeletal muscle (SM). Four previously unreported ion channels could be identified among the 30 most specific ion channels in the TG (marked with (-)). Most ion channels are involved in TG thermosensation, mechanosensation and pain perception.
Figure 10.
Ranking of potassium channels that are most specifically expressed in the TG and DRG.
A comparison of the most specific potassium channels in the TG and DRG compared with the brain (B), liver (L), olfactory epithelium (OE), and skeletal muscle (SM). For the TG, newly detected channels are marked with (-).
Figure 11.
Comparison of expression profiles of the TG and DRG.
A Differences in the gene expression pattern of the TG and DRG. Of all the genes detected in the TG and DRG, 98.6% were similar (> 1 FPKM), 8113 genes were detected in neither the DRG nor the TG (< 1 FPKM), and 0.5% of the genes were TG- and 0.9% DRG-specific (> 1 FPKM). B FPKM distribution for both tissues is highly similar when plotting FPKM values against the number of detected genes. C The regression graph visualizes the correlation of the expression patterns for all detected transcripts in the TG and DRG. R2= 0.73.
Figure 12.
Genes expressed differentially in the TG and DRG.
A 65 genes are expressed at least 10-fold higher in the TG than in the DRG. Brain (B), liver (L), olfactory epithelium (OE), and skeletal muscle (SM) were used to visualize the global expression patterns for the selected genes. The expression of genes that are marked with (*) is significantly different between the TG and DRG. Interestingly, 15 of the trigeminally-expressed genes are also expressed in the OE and have a function in olfaction. B In the DRG, many of the 117 specifically expressed genes play a role in the development or regulation of gene expression, such as Ampd1 or Cfd. Of the 117, 23 genes were significantly expressed at higher levels in the DRG than in the TG. In contrast to the TG, we found several Hox genes with higher expression in the DRG than in the TG.