Editor's evaluation: scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Abstract Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using PirtEGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration. Editor's evaluation This study identifies injury-induced changes in transcriptomic signatures in peripheral sensory neurons at a single cell level, revealing key insights into sexual dimorphism as well as plasticity-related differences between injured and uninjured neurons. These results promote an understanding of the nature of molecular events involved in the establishment of neuropathic pain. https://doi.org/10.7554/eLife.76063.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The dorsal root ganglion (DRG) contains the somas of primary sensory neurons, which differ in size and axon myelination. Different gene expression profiles confer divergent neurochemical, physiologic, and functional properties on the various subtypes of DRG neurons (Gatto et al., 2019; Sharma et al., 2020; Usoskin et al., 2015; Zeisel et al., 2018; Zheng et al., 2019). Small-diameter neurons are important for transmitting nociceptive and thermal information, whereas large-diameter neurons are mainly non-nociceptive neurons, including mechanoreceptors and proprioceptors. Nerve injury induces various responses in DRG neurons, including cell stress, regeneration, hyperexcitability, and functional maladaptation. How these changes vary in functionally distinct neuronal subtypes and possibly affect nerve regeneration and neuropathic pain remains unclear. Recently, single-cell/single-nucleus RNA-sequencing (scRNA-seq/snRNA-seq) has begun to reveal transcriptomic perturbations in DRG neurons after transection or crush nerve injury (Hu et al., 2016; Renthal et al., 2020). Nevertheless, there remain many important questions which are not fully addressed, especially differential transcriptional changes in functionally distinct DRG neuronal subtypes related to neuropathic pain. The crush injury model used in some of previous studies is more suitable for studying nerve regeneration than for closely capturing the etiology of clinical neuropathic pain, which often involves chronic compression, neuroinflammation, and partial injury to a major nerve. Axotomized neurons may exhibit the most profound gene expression changes that are important for regeneration. Nevertheless, neighboring uninjured DRG neurons also show significant functional changes (e.g., hyperexcitability) and contribute to dysesthesia and evoked pain hypersensitivity as a result of the remaining peripheral innervations (Djouhri et al., 2012; Kalpachidou et al., 2022; Obata et al., 2003; Tran and Crawford, 2020). Thus, identifying and differentiating transcriptional changes in injured and uninjured neurons in a cell-type-specific manner will be important to search for new targets for nerve regeneration and pain treatment. So far, most previous scRNA-seq studies have mainly focused on changes in injured neurons, but details of possible cell-type-specific transcriptomic changes in uninjured DRG neurons under neuropathic pain conditions remain partially known. Moreover, increasing clinical and preclinical evidence suggests that males and females have differences in pain sensitivity and susceptibility to chronic pain (Fillingim et al., 2009). To optimize clinical treatment, it will also be important to delineate sex-related gene expression changes in functionally distinct subtypes of DRG neurons after nerve injury and determine how these changes underpin sexual dimorphisms in neuropathic pain. We established a highly efficient purification approach by using PirtEGFPf mice to enrich DRG neurons for scRNA-seq. Pirt is expressed in >83.9% of neurons in mouse DRG, but not in other cell types (Kim et al., 2008). Thus, green fluorescent protein (GFP) is selectively expressed in most DRG neurons in PirtEGFPf mice, driven by the Pirt promoter. GFP expression allows effective purification of DRG neurons from these mice. We then characterized perturbations of transcriptomes in DRG neurons at the single-cell level after chronic constriction injury (CCI) of the sciatic nerve. The CCI model represents a well-established neuropathic pain model that encompasses compression, ischemia, inflammation, and axonal demyelination (Bennett and Xie, 1988). It has been suggested to mimic the etiology of clinical conditions and induces symptoms similar to those of post-traumatic neuropathic pain in humans (Challa, 2015; Costigan et al., 2010; Griffin et al., 2007; LaCroix-Fralish et al., 2011). Since cell interactions may occur within the same ganglion, the excitability and transcriptional changes in uninjured neurons can be greatly affected by satellite glial cells (SGCs) and neighboring injured neurons. Because only a portion of sciatic nerve fibers are injured after CCI, each of the lumbar DRGs contains a mixture of injured and uninjured neurons. Thus, in addition to characterizing injured neurons which express high levels of the injury marker gene Sprr1a, more importantly, we also examined transcriptomic changes in uninjured neurons (Sprr1a-) and investigated transcriptomic sexual dimorphism after CCI at the single-cell level. Our findings may be useful for developing DRG neuron subtype-specific treatment and sex-specific therapies for nerve regeneration and neuropathic pain. Results Enrichment of DRG neurons from PirtEGFPf mice for scRNA-seq Mice were randomly assigned to four groups (n=5 for each group): Male-CCI, Female-CCI, Male-Sham, and Female-Sham. Bilateral L4-5 DRGs were collected from mice on day 7 after bilateral sciatic CCI or sham surgery for scRNA-seq (Figure 1A, Figure 1—figure supplement 1A). In an animal behavior study conducted on day 6 after CCI, paw withdrawal frequencies to low-force (0.07 g) and high-force (0.4 g) mechanical stimulation at the hind paws (data averaged from both sides) were significantly increased (n=5/sex), as compared to the pre-injury frequency, indicating the development of mechanical hypersensitivity (Figure 1B). Paw withdrawal frequencies were not significantly changed after sham surgery (n=5/sex). Figure 1 with 1 supplement see all Download asset Open asset Single-cell RNA-sequencing (scRNA-seq) identified distinct clusters of cells in the dorsal root ganglion (DRG) of PirtEGFPf mice. (A) Schematic diagram showing the procedure for chronic constriction injury (CCI) of the sciatic nerve. (B) Paw withdrawal frequencies to low-force (0.07 g von Frey filament, left) and high-force (0.4 g, right) mechanical stimuli before and 6 days after CCI or sham surgery. n=10 per group (n=5/sex). Two-way mixed-model analysis of variance (ANOVA) followed by Bonferroni post hoc test. Data are expressed as mean ± SD, ***p<0.001 versus pre-injury. (C) Integration of four datasets visualized by uniform manifold approximation and projection (UMAP). (D) Seventeen distinct cell clusters were identified by Seurat, including SGC (1), NF (2), NP (3), PEP (6), cLTMR (1), and CCI-induced clusters (4). (E) Dot plot of subtype-specific marker genes in each cluster. Genes highlighted in the yellow, purple, pink, and blue zones are known markers for NF, NP, PEP, and cLTMR, respectively. Genes highlighted in the green zone are markers identified in CCI-induced (CCI-ind) clusters. The dot size represents the percentage of cells expressing the gene, and the color scale indicates the average normalized expression level in each cluster. (F) A heatmap shows the expression patterns of the top 50 marker genes in each cluster. DRGs contain a large number of non-neuronal cells, including SGCs, Schwann cells, immune cells, and fibroblasts. Previous studies have shown that scRNA-seq is advantageous for differentiating neurons from these non-neuronal cells (Renthal et al., 2020; Wang et al., 2021). Here, by using PirtEGFPf mice, we established a highly efficient purification process to further enrich DRG neurons for scRNA-seq (Figure 1—figure supplement 1A). After removing low-quality cells and doublets (Supplementary file 2), we recovered 3394 cells from the Male-Sham dataset; 5678 cells from the Female-Sham dataset; 2899 cells from the Male-CCI dataset; and 3681 cells from the Female-CCI dataset. Figure 1—figure supplement 1B showed the number of expressed genes in each dataset. We then utilized canonical correlation analysis embedded in Seurat 3.0 (1), a computational approach for minimizing experimental batch effect, to integrate cells from the four datasets for an unbiased cell clustering. The results showed that the integration worked well in our experiments, as clusters from each dataset aligned well regardless of different biological variations (Figure 1C). In total, we identified 16 neuronal clusters and one non-neuronal cluster (Figure 1D, Figure 1—figure supplement 1C). The non-neuronal cluster had fewer genes and unique molecular identifier (UMI) counts than did neuronal clusters (Figure 1—figure supplement 1D). It expressed SGC marker genes Fabp7 and Apoe (Renthal et al., 2020; Wang et al., 2021) but showed minimal expression of the pan-neuronal marker Tubb3. In contrast, all 16 neuronal clusters showed strong expression of Tubb3 but had very low levels of Fabp7 (Figure 1—figure supplement 1E). Although this non-neuronal cluster showed marker genes known to be expressed in both Schwann cells and SGCs (e.g., Mpz, Mbp, Plp1), it did not express any Schwann cell-specific genes such as Mag, Prx, or Ncmap (Avraham et al., 2020). Therefore, this non-neuronal cluster included mainly SGCs. No other non-neuronal cluster was detected in our datasets, suggesting a successful enrichment of neurons. Prominent new neuronal clusters appear after sciatic CCI We further validated the identities of the neuronal clusters by a list of known subtype markers (Figure 1E). Our findings confirmed the presence of 12 major standard neuronal clusters (Nppb+ non-peptidergic nociceptors [NP1], Mrgprd+/Cd55+ non-peptidergic nociceptors [NP2], Mrgpra3+/Cd55+ non-peptidergic nociceptors [NP3], Tac1+/Sstr2- peptidergic nociceptors [PEP1-2], Tac1+/Sstr2+ peptidergic nociceptors [PEP3-4], Trpm8+ peptidergic nociceptors [PEP5], Trpv1+ peptidergic nociceptors [PEP6], Nefh+/Scn1b+ Aβ low-threshold mechanoreceptors [NF1, NF2], and Fam19a4+/Th+ low-threshold mechano-receptive neurons with C-fibers [cLTMR]). Importantly, we also identified four new CCI-ind1-4 clusters that were prominent in CCI groups but minimal in sham groups (Figure 1D and E, and Figure 2A). CCI-ind1-4 clusters expressed high levels of genes like Cryba, Tgfbi, Fgf3, Tnfrsf12a, Ecel1, and Sema6a, and were segregated from all standard clusters. Expression profiles of the top 50 marker genes in the 16 neuronal clusters are shown in Figure 1F and listed in Supplementary file 1. Figure 2 Download asset Open asset New neuronal clusters are induced by chronic constriction injury (CCI) of the sciatic nerve. (A) Sciatic CCI induced four new clusters (marked in a red circle) of dorsal root ganglion (DRG) neurons in both female and male mice. These new clusters were named CCI-induced (CCI-ind) 1, 2, 3, and 4, and were not prominent in sham groups. x-axis: uniform manifold approximation and projection 1 (UMAP1), y-axis: UMAP2. (B) Percentage of cell population in 16 neuronal clusters present in each of the four treatment groups. (C) Feature heatmap shows the expression levels of injury-induced genes (Atf3, Sprr1a) in different clusters of CCI and sham groups. (D) The dot plot shows the top 30 marker genes of CCI-ind1-4 clusters, as compared to those in other clusters. (E) Top 25 biological processes enriched by the top 50 marker genes from CCI-ind1-4 clusters. Gene programs in CCI-ind1-4 clusters are important to both nerve regeneration and pain A much higher percentage of the cell population was contained in the CCI-ind1-4 clusters from CCI groups than from sham groups (Figure 2A and B). These clusters also showed high expression levels of injury-induced genes such as Sprr1a and Atf3 (Figure 2C; Bonilla et al., 2002; Tsujino et al., 2000). In line with previous findings (Nguyen et al., 2017), Atf3 was also frequently detected in the sequencing of isolated cells from sham or uninjured groups. Comparatively, Sprr1a was more selectively expressed in injured neurons of CCI mice (Figure 2C). Thus, Sprr1a may represent a more specific marker of injured neurons than Aft3 in scRNA-seq (Bonilla et al., 2002). Regeneration-associated genes, such as Anxa1, Flnc, Gadd45a, Inhbb, Itga7, Kif22, Plaur, Sema6a, Sox11, Tnfrsf12a, and Tubb6, were among the top 30 marker genes of CCI-ind1-4 clusters (Figure 2D; Chandran et al., 2016). Intriguingly, some of them may also be involved in neuropathic and inflammatory pain. For example, animal studies suggest that neurotensin (encoded by Nts) (Guillemette et al., 2012; Sarret et al., 2005) and annexin1 (encoded by Anxa1) may attenuate neuropathic pain and inflammatory pain, respectively (Pei et al., 2011; Zhang et al., 2021). Nnat and Sdc1 were exclusively increased in nociceptive DRG neurons after nerve injury (Chen et al., 2010; Murakami et al., 2015). Sox11 was identified by integrated bioinformatic analysis as a novel gene that is essential to neuropathic pain (Chen et al., 2021). Gene ontology (GO) analysis of the top 50 marker genes showed that CCI-induced transcriptomic changes were important for both nerve regeneration (e.g., nervous system development, axon guidance, cellular response to nerve growth factor stimulus) and neuronal excitability (e.g., positive regulation of calcium ion import, response to pain, regulation of sodium ion transport, and the neuropeptide signaling pathway; Figure 2E). NP1, PEP5, NF1, and NF2 clusters exhibit different transcriptional programs from other clusters after CCI Previous studies in different nerve injury models showed that no specific neuronal cluster was spared from injury. Injured neurons in all clusters lost their original subtype-specific marker genes beginning at day 1 after injury, and hence could no longer be categorized into original clusters (Hu et al., 2016; Nguyen et al., 2019; Renthal et al., 2020; Wang et al., 2021). Yet, these studies did not further examine the proportion of injured neurons in each cluster after injury. Our findings showed a large decrease of cell proportion in 8 of 12 standard neuronal clusters after CCI (Figure 3—figure supplement 1), suggesting that the injured neurons in these clusters were no longer categorized into their original clusters. Instead, they may be assigned to CCI-ind1-4 clusters (Figure 3A and B), as suggested by a previous study (Renthal et al., 2020). The remaining four clusters (NP1, PEP5, NF1, NF2) showed little decrease in cell proportion after CCI. Moreover, a portion of injured neurons (Sprr1a+) in these four clusters were still categorized into their original clusters after CCI (Figure 3A and B). Further analysis revealed two subpopulations of neurons in these clusters (Figure 3C–F). For example, in the NP1 cluster, the larger subpopulation showed high expression of Sprr1a and other injury-induced genes (Gal, Hspb1, Stmn4, Chl1, and Sox11) (Chandran et al., 2016), suggesting that these are injured neurons (Figure 3C). In contrast, the smaller subpopulation (Sprr1a-) showed little or no expression of injury-induced genes, but expressed other genes highly, such as Nppb and Sst, which are NP1 subtype-specific marker genes (Figure 1E). We found similar results in PEP5, NF1, and NF2 clusters (Figure 3D–F). Figure 3 with 1 supplement see all Download asset Open asset Transcriptional program changes in different neuronal clusters after sciatic nerve chronic constriction injury (CCI). (A) Left: The identities of 17 clusters of dorsal root ganglion (DRG) cells visualized by uniform manifold approximation and projection (UMAP). Right: UMAP displays distinct expression patterns of Sprr1a, an injury-induced gene, in cells of NP1, PEP5, NF1, and NF2 clusters (indicated by red circles). Each of these clusters contained Sprr1a+and Sprr1a- cells, resulting in two subpopulations. (B) Box plots show that Sprr1a expression was statistically different between sham and CCI groups in only four standard neuronal clusters (NP1, PEP5, NF1, and NF2), suggesting a subtype-specific expression profile in sham and CCI groups. We selected clusters (NF1, NF2, NP1, PEP5, CCI-ind1, CCI-ind2, CCI-ind3, CCI-ind4) with normalized unique molecular identifier (UMI) expression >1 in sham and CCI conditions for statistical analysis. n=330 (NF1), 180 (NF2), 190 (NP1), 806 (PEP5), 28 (CCI-ind1), 70 (CCI-ind2), 138 (CCI-ind3), and 64 (CCI-ind4) for clusters in sham condition, and n=202, 215, 258, 631, 369, 351, 1188, 638 for these clusters in CCI condition. Student’s t-test, ****p<0.0001 versus sham. (C–F) Left: Two subpopulations (Sprr1a+, Sprr1a-) of cells in NP1 (C), PEP5 (D), NF1 (E), and NF2 (F) clusters were separated based on the expression of Sprr1a. Right: Top 10 DEGs in Sprr1a+and Sprr1a- subpopulations of NP1, PEP5, NF1, and NF2 clusters. Transcriptional changes in injured neurons of NP1, PEP5, NF1, and NF2 clusters after CCI Because a significant portion of injured neurons (Sprr1a+) in NP1, PEP5, NF1, and NF2 clusters maintained their identities after CCI (Figure 3), we were able to determine transcriptomic changes in these neurons by comparing them to uninjured neurons of the same clusters in the sham group. After CCI, 197, 67, 41, and 79 differentially expressed genes (DEGs) were generated from NP1, PEP5, NF1, and NF2 clusters, respectively (Supplementary file 3), with 19 shared DEGs (Supplementary file 4). Most were common regeneration-associated genes induced by nerve injury (Chandran et al., 2016), and some (Cacna2d1, Gal, Gap43, Gadd45a, Atf3, Sprr1a) were also significantly regulated under chronic pain conditions (LaCroix-Fralish et al., 2011; Perkins et al., 2014). GO analysis showed that these four clusters shared many common pathways, including those related to nervous system development, axon guidance, neuron projection development, microtubule-based process, neuropeptide signaling pathway, and cell differentiation (Figure 4A). In addition, we observed CCI-induced changes that affect neuronal excitability (e.g., downregulation of potassium and sodium channels, upregulation of calcium channel Cacna2d1, dysregulation of genes encoding neuropeptide, and G-protein-coupled receptors; Figure 4—figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset Gene ontology analysis of chronic constriction injury (CCI)-induced differentially expressed genes (DEGs) and pain-related protein-protein interaction (PPI) networks in NP1, PEP5, NF1, and NF2 clusters. (A) Gene ontology analysis of biological processes enriched by CCI-induced DEGs in NP1, PEP5, NF1, and NF2 clusters. (B–E) The neuropathic pain-specific PPI networks of CCI-induced DEGs in NP1, PEP5, NF1, and NF2 clusters. Colored edges mark the type of interaction. Colored nodes mark the expression changes after CCI. Node size indicates the number of interactions against pain interactome. We further examined pain-related protein-protein interaction (PPI) networks within the pain interactome, a comprehensive network of 611 interconnected proteins specifically associated with pain (Jamieson et al., 2014). Examining 197 DEGs of the NP1 cluster revealed an interconnected network of 93 genes (Figure 4B). Among them, Calca, Prkca, Sst, and Tac1 were hub genes that were significantly downregulated after CCI, whereas Tgfb1 and Gal were hub genes that were significantly upregulated. Intriguingly, the top marker gene of the NP1 cluster, Sst, is also a key gene for neuropathic pain (Zhu et al., hence it may be an important new target for pain we examined an interconnected network of genes from DEGs in the PEP5 cluster, we identified Gal, and Tac1 as hub genes that changed significantly after CCI (Figure of DEGs from the NF1 cluster revealed an interconnected network of genes (Figure with Calca, Gal, and as hub an of 79 DEGs from the NF2 cluster revealed an interconnected network of genes (Figure including hub genes Gal, and Among these hub genes, and Tac1 were downregulated whereas Gal, and were in multiple clusters. was identified as a key and target of neuropathic pain et al., and and may play roles in both neuropathic pain and nerve regeneration et al., 2020). For example, of neurotensin (encoded by Nts) induced pain in animal models of neuropathic pain (Guillemette et al., 2012; Sarret et al., It is possible that the upregulation of may represent a after injury to the of neuropathic pain. studies are to delineate the roles of each of these shared hub genes in neuropathic pain A of neuronal clusters shows subtype-specific transcriptional changes in uninjured neurons of our study was to transcriptional changes in uninjured neurons (Sprr1a-) of different clusters under neuropathic pain many DEGs were identified in Sprr1a- neurons from a of clusters including NP1 PEP5 NF1 and NF2 after CCI (Figure Supplementary file Comparatively, only a DEGs were present in Sprr1a- neurons from other clusters, suggesting subtype-specific transcriptional changes in uninjured neurons. Figure Download asset Open asset Gene ontology analysis of chronic constriction injury (CCI)-induced differentially expressed genes (DEGs) in the Sprr1a- subpopulation of NP1, PEP5, NF1, and NF2 clusters. (A) The shows the number of DEGs induced by CCI in Sprr1a- neurons of each cluster. (B) Top 10 marker genes of Sprr1a- neurons in NP1, PEP5, NF1, and NF2 clusters of CCI and sham groups. (C–F) Gene ontology analysis of CCI-induced DEGs in Sprr1a- neurons in NP1 (C), PEP5 (D), NF1 (E), and NF2 (F) clusters. GO analysis showed that top shared by Sprr1a- neurons in NP1, PEP5, NF1, and NF2 clusters are related to protein including transport, and (Figure Supplementary file related to nerve regeneration and neuropathic pain were also found in these clusters but were much These findings suggest that these uninjured neurons in NP1, PEP5, NF1, and NF2 clusters may a in response to nerve injury. differences in transcriptional changes of different DRG neuronal subtypes after CCI and animal models suggest the presence of differences in pain sensitivity and chronic pain (Fillingim et al., et al., 2016). The peripheral neuronal these sexual dimorphisms remain and studies have compared transcriptional changes of DRG neurons at the single-cell level, especially under neuropathic pain is a specific expressed exclusively by the in female et al., with previous we did not expression in cells from our male mice (Figure supplement 1A). subtype were similar between female and male mice in both sham and CCI groups (Figure Female-Sham and Male-Sham groups showed correlation in cluster indicating a of transcriptional programs under conditions (Figure supplement 1B). Nevertheless, our findings suggest differences in the transcriptional changes that after CCI. compared to the sham the Male-CCI group and the Female-CCI group and within which there were 79 and DEGs (Figure Supplementary file Top were further identified by GO analysis of sex-specific and shared DEGs (Figure Figure 6 with 1 supplement see all Download asset Open asset of transcriptional changes between female and male mice after chronic constriction injury (CCI). (A) The diagram shows the number of genes that were differentially expressed between CCI and sham in male and female mice. Gene ontology that are associated with differentially expressed genes (DEGs) in male mice female mice (C), and both male and female mice (E) based on the of DEGs after CCI in clusters, NP, PEP, NF, and represent genes that showed differences between female and male mice. (F) Gene ontology analysis of the genes from correlation analysis was also based on the of the Most neuronal clusters showed a correlation of DEGs between males and females (Figure indicating and a minimal batch Yet, the cLTMR cluster had a Intriguingly, et that of a factor which is highly expressed in cLTMR, behavior and pain only in male mice et al., 2020). Calca, which is a gene that is highly in of male mice et al., showed differences between female and male in our dataset. These findings suggest that may play an important role in the sexual dimorphism of pain. all clusters, genes showed differences between female and male CCI mice (Figure Supplementary file The top 10 from the GO analysis of these DEGs included nervous system development, neuropeptide signaling pathway, response to pain, sensory of pain, and response to (Figure these findings suggest differential transcriptomic changes in DRG neurons after CCI between the two of increased Fgf3 expression in DRG neurons after CCI Fgf3 is one of the top genes in CCI-ind1-4 clusters which express high levels of injury-induced gene Sprr1a as shown in our scRNA-seq study 1 and 2, Supplementary file 1). Previous studies suggested that Fgf3 may play a role in pain and For example, mice in Schwann cells pain hypersensitivity and Fgf3 and Sprr1a expression in DRG neurons et al., 2020). we conducted RNAscope in to further examine the expression and of Fgf3 in DRG and compared findings between and CCI mice. Fgf3 and Sprr1a were minimal in the sham but increased after CCI (Figure In CCI mice, a large portion of cells were suggesting the expression of Fgf3 in injured neurons. In contrast, Sprr1a- neurons which express different cell subtype markers were in both CCI and sham groups (Figure Figure 7 with 2 see all Download asset Open asset The and functional of Fgf3 in mouse dorsal root ganglion (DRG) neurons. RNAscope in of lumbar DRGs from and chronic constriction injury (CCI) mice with against Fgf3 Sprr1a an injury and different cell subtype markers and Nppb (C)