Ribonuclease 4 Functions in Nociceptor-Mediated Nerve Homeostasis

Domain: biology
Published: 2026-03-24
Editorial outcome: major_revision

Abstract
The regulation of nociceptor identity and function is essential, as disruptions can significantly influence pain sensation, yet our understanding of the molecular mechanisms involved remains incomplete. In this study, we identified ribonuclease 4 (RNase 4) as selectively expressed in the unmyelinated nociceptor lineage. Analysis of RNase 4-deficient mice and single-cell transcriptomic data revealed a cell-autonomous role for RNase 4 in regulating nociceptor function. Moreover, in a neuropathic pain model, RNase 4 expression was upregulated in nociceptors during the pain and recovery phases, and its deletion altered mechanical sensation. Additionally, RNase 4 exerted non-cell-autonomous effects on the myelin structural organization of adjacent myelinated axons. Together, these findings implicate RNase 4 as a dual regulator of nociceptor biology and myelin integrity, revealing a molecular pathway for pain regulation and nerve repair.

Introduction
Nociceptors, the specialized pain-sensing neurons of the peripheral nervous system, are central to the detection and transmission of noxious stimuli to the central nervous system1. These neurons, located in the dorsal root and trigeminal ganglia, are equipped with a variety of receptors that detect thermal, mechanical, and chemical noxious stimuli2, highlighting the molecular complexity of pain perception and the need for cell-type-specific biomarkers that regulate nociceptor identity and function3,4.

Nociceptors are classified into two primary fiber types: Aδ fibers, which transmit fast, sharp pain, and C fibers, which convey slower, diffuse pain signals5. C fibers are further subdivided into distinct subpopulations characterized by the expression of specific molecular markers, including neuropeptides such as CGRP and substance P, receptors like TrkA, P2X3 purinergic receptors, Ret, and Mrg family receptors, as well as ion channels including TRPV1, TRPM8, TRPA1, and ASICs. These molecular signatures form critical cell-to-cell communication pathways that underpin the specialized functions of each nociceptor subtype, facilitating the detection and transmission of specific pain modalities. As a result, their modulation, or genetic deletion, in vivo has been shown to alter pain sensation1,6. The mechanisms regulating nociceptor function and their contribution to the progression of neuropathic pain, particularly following nerve injury, remain, however, less understood. Emerging evidence highlights the role of specific transcriptional regulators in these processes. For example, ATF37 and SOX118 have been implicated in axonal regeneration and pain sensitization, respectively, after peripheral nerve injury. Additionally, the transcriptional repressor G9a9, which is upregulated following nerve injury, has been associated with nociceptor plasticity underlying neuropathic pain.

There is still, however, a significant gap in understanding the molecular mechanisms that preserve the homeostatic state of nociceptors and how these are disrupted or contribute to neuropathic pain. This suggests that key regulatory pathways and their roles in nociceptor function and dysfunction remain to be elucidated. To address this, we adopted a broad approach and searched for molecular markers specific to unmyelinated neurons in the DRG, trigeminal ganglia (TG), and cochlea, and identified ribonuclease 4 (RNase 4) as a highly selective candidate.

Traditionally, ribonucleases are implicated in RNA metabolism10, yet they are also involved in a diverse array of physiological processes, including growth, neurogenesis, angiogenesis, immune modulation, and host defense10–12. Specifically, it has been shown that RNase 4 can be secreted into the extracellular matrix12. The restricted expression of RNase 4 in unmyelinated nociceptors highlights its potential role in regulating pain pathways and contributing to the functional specialization of these sensory neurons.

Here, we identify RNase 4 as a highly selective marker of unmyelinated nociceptors across mouse and human sensory ganglia and uncover an unexpected role for RNase 4 in linking nociceptor identity to peripheral nerve homeostasis. By combining single-cell transcriptomics, genetic loss-of-function approaches, behavioral analyses, and nerve injury models, we show that RNase 4 acts cell-autonomously to regulate nociceptor transcriptional programs, including potassium channels and pain sensitivity, while exerting non-cell-autonomous control over axonal regeneration, myelin remodeling, and immune-associated repair processes. In vitro analyses further indicate that RNase 4 can modulate PI3K–AKT signaling, suggesting a mechanistic pathway through which RNase 4 influences these effects. Together, these findings demonstrate that RNase 4-expressing nociceptors are not solely pain sensors but function as homeostatic and injury-responsive regulatory hubs that shape peripheral nerve stability, repair, and remodeling, thereby extending the role of nociceptors beyond sensory transduction.

Results
Rnase4 is selectively expressed in nociceptors
The identification of genes selectively expressed in nociceptors is crucial for understanding the regulation of pain perception and for developing targeted pain therapies. Using the iPain single-cell transcriptomic atlas13 to differentiate between various populations of nociceptors and mechanoreceptors14,15, including peptidergic (PEP), non-peptidergic (NP), somatostatin-positive (SST), and C-low threshold mechanoreceptors (c-LTMRs), in contrast to heavily myelinated fibers (NF, neurofilament positive fibers)(Fig. 1a), we fitted the negative binomial distribution with pyDESeq216 on the pseudo-bulk expression and identified differentially expressed genes (DEGs) amongst neuronal subtypes. The top selective genes for nociceptors included Grik1, Syt9, and Trpc3, while Slc16a6, Scn8a, and Kank4 were the leading genes for mechanoreceptors, aligning with prior findings17(Fig. 1b, Supplementary Data 1). Interestingly, RNase 4 was identified as a selective marker for nociceptor populations (Fig. 1b, c) and was absent from neurons in the CNS17. To further characterize this cell type–restricted expression, we performed RNAscope on cross-sections of dorsal root ganglia (DRG) from adult animals (Fig. 1d, j). Co-labeling used established markers: Calca (peptidergic nociceptors, PEPs), Sst (somatostatin-positive neurons), TH (cLTMRs), NF200/NF-H and PV (myelinated neurofilament neurons, NFs), peripherin (Prph; small-diameter, unmyelinated neurons), and Prdm12 (nociceptor lineage marker). Quantification of RNase 4+ DRG neuron soma size revealed high selectivity in small diameter neurons, with ~80% less than 20 microns and the remaining ~20% being between 20 and 35 microns (Fig. 1e).

RNase 4 showed complete overlap with Sst+ neurons and partial colocalization (~40–50%) with Calca+ peptidergic neurons (Fig. 1f, g), while TH+ cLTMRs, which share a developmental nociceptive lineage but mediate pleasant touch, were entirely RNase 4-negative (Fig. 1i). Minimal overlap occurred with NF200+ populations and none with PV+ myelinated neurons, with RNase 4 enriched in Prdm12+/peripherin+ small-diameter neurons (Fig. 1d, h, j). These findings establish RNase 4 as a selective marker of specific unmyelinated nociceptor subtypes.

Bulk RNA-seq analysis of human dorsal root ganglia (DRG)18 confirmed RNase 4 expression in human tissue, with a selective nociceptor-enriched pattern evident in a recent single-cell atlas of human somatosensory neurons19 (Fig. 1k, l). These findings demonstrate evolutionary conservation of RNase 4 expression across species, suggesting an important role in nociceptor biology.

Role of RNase 4 on nociceptor phenotype and pain sensation
To investigate how RNase 4 influences the identity and functionality of nociceptors, particularly in its absence, we developed a conditional Rnase4 knock out mouse line (Rnase4fl/fl) by inserting Short Conditional intrON (SCON) cassette20 containing LoxP recombination sites, within the Rnase4 exon and using the Easi-CRISPR technology, a streamlined method utilizing the CRISPR-Cas system for genome editing21(Fig. 2a). The Rnase4fl/fl mouse line was crossed with the Baf53bCre mouse line to delete Rnase4 in sensory neurons during embryonic development, after the neurogenesis of nociceptors (Fig. 2b, c). To explore the role of RNase 4 on the transcriptional phenotype of nociceptors, we performed RNA sequencing using Smart-seq3xpress on single nuclei from DRG neurons extracted from adult Rnase4 conditional knockout (cKO) mice (Baf53bCre;RNase4fl/fl, or cKO-BR) and control littermates. After filtering, we obtained 586 nuclei from the cKO-BR line and 514 nuclei from controls, totaling 1,113 neuronal nuclei. We applied cell type annotation using the pre-trained scANVI22 model from iPain13 to perform label transfer (Fig. 2d). Approximately 73% of the profiled nuclei were identified as belonging to the nociceptive lineage. A total of 765 genes were found to be significantly differentially expressed (either up- or downregulated) in the cKO-BR condition compared to controls within the nociceptive lineage neurons (Fig. 2e, f, Supplementary Data 2). Further analysis of the differentially expressed genes (DEGs) using the PANTHER database indicated substantial changes in key pathways and protein classes. Notably, C2H2 zinc finger proteins emerged as the most prominently altered group, with 42 proteins represented. This included various transcription factors such as Gm6710, Zfp433, and Maz. Additionally, increased expression was observed for Irf3 (interferon regulatory factor) and Rela (p65, a component of the NF-κB pathway), suggesting significant alterations in transcriptional regulation and immune response pathways in neurons within the conditional mutant.

The analysis also highlighted notable changes in cytoskeleton-related proteins and ribosomal components (Supplementary Fig. 1a, b). Alterations were observed in microtubule-binding motor protein Dync2h1, Mapt (microtubule-associated protein tau), and tubulin variant Tubd1. Furthermore, eleven ribosomal proteins were affected, including Mrpl4, Rpl9, Rps10, Rps27, and Mrps1. Significant changes were also noted in translation-related factors such as Eif2s1 (a translation initiation factor), Gspt1 (a translation termination factor), and Cpeb3, a negative regulator of translation with known function on Trpv1 expression23. These findings indicate widespread modifications in cellular architecture and protein synthesis machinery as a response to conditional mutation. Finally, focusing on ion channels within nociceptors, we identified differences in expression levels of potassium voltage-gated ion channels Kcnf1 (Kv5.1) and Kcng3 (Kv6.3) (Fig. 2e, f). These channels are integral to the potassium channel machinery that regulates membrane potential and facilitates the return to a negative resting potential following an action potential. Their reduction in RNase 4 cKO neurons may affect Kv channel composition and potentially influence neuronal excitability24,25. Interestingly, RNase 4 deletion altered gene expression in cLTMRs despite the absence of RNase 4 in this population, an observation that could be consistent with paracrine effects of RNase 4 on neighboring cells.

To determine whether the molecular alterations in cKO-BR mice are associated with changes in behavioral responses to noxious stimuli, we assessed acute responses to thermal and mechanical noxious stimuli. Our results revealed that early neuronal deletion of Rnase4 during development did not significantly alter mechanical or thermal sensory thresholds compared to control littermates (Fig. 2g). These findings suggest that, despite alterations in gene expression, baseline pain sensation under normal conditions remains unaffected by the absence of RNase 4 from early development.

Acute RNase 4 deletion in adults transiently increases mechanical pain sensitivity
To investigate the role of RNase 4 in adult nociceptors, we employed an intersectional genetic deletion approach. Specifically, we generated a Prdm12CreERT2 mouse line (see Fig. 3a and Methods for details), where PRDM12 serves as a specific marker for the nociceptive lineage26 to conditionally delete Rnase4 from nociceptors upon tamoxifen administration (Fig. 3a). The specificity and efficacy of the Prdm12CreERT2 line were first validated by crossing it with a Rosa26tdTOM (Ai14) reporter mouse. Following induction, tdTomato fluorescence was detected exclusively in Prdm12+ nociceptors and their projections: RFP+ cells and fibers show clear exclusion with PV (Fig. 3d) and colocalization with TrkA (Fig. 3d), IB4 and CGRP (Fig. 3e), indicating that they terminate within laminae associated with nociceptive afferents. This confirms the selective projection of PRDM12-lineage neurons to I-II regions of the dorsal horn. These findings were consistent across both developmental and adult stages (Fig. 3d, e, Supplementary Fig. 2), with a recombination efficiency estimated at ~42.5% (Fig. 3f). Finally, the successful removal of Rnase4 was confirmed in adult DRG cross sections from tamoxifen-treated Prdm12CreERT2; Rnase4fl/fl mice (Fig. 3g).

We therefore assessed the mechanical withdrawal threshold in inducible KO (cKO-PR) mice using the von Frey test. Following induction, cKO-PR mice exhibited a significant reduction in the paw withdrawal threshold (Fig. 3h), indicating an increased sensitivity to mechanical stimuli. Importantly, this change in threshold was transient: by day 14 post-tamoxifen injection, cKO-PR mice began to recover their baseline sensitivity, with full recovery observed by day 30. Thermal sensitivity, assessed with the Hargreaves test, showed no genotype-dependent differences at the intermediate time points; therefore, only the 30-day Hargreaves data are presented. These findings suggest RNase 4 contributes to adult nociceptor function, although its absence causes only transient effects (likely due to compensatory mechanisms). The data also suggest a role for RNase 4 in nociceptor homeostasis. This concept could be particularly relevant in models of chronic pain, where such homeostasis is disrupted.

To further characterize molecular changes underlying the cKO-PR phenotype, we performed single-nucleus RNA sequencing (snRNA-seq) using the 10x Genomics platform on dorsal root ganglia (DRG) from cKO-PR mice and wild-type controls (Fig. 4a, b). Dimensionality reduction and clustering revealed distinct neuronal subtypes, with nociceptors showing the most pronounced transcriptional alterations in cKO-PR samples (Fig. 4b). Differential expression analysis identified a core set of significantly downregulated genes shared across subtypes, including the nociceptor-specific transcription factor Prdm12 (Fig. 4c; Supplementary Data 2). Nociceptor-specific profiling revealed subtype-selective up- and downregulation of genes (Fig. 4d). Among the list of differentially expressed candidates (Supplementary Data 2), we were able to extract several ion channels such as Kctd1 and Kcnh8, that were upregulated in cKO-PR, consistent with potential mechanisms of neuronal hyperexcitability contributing to the behavioral phenotype.

Non-cell-autonomous action of RNase 4: modulation of PI3K–AKT–mTOR signaling
To examine whether extracellular RNase 4 (R4) engages intracellular signaling pathways, ND7/23 cells were treated with RNase 4 alone or in combination with inhibitors of the PI3K–Akt–mTOR pathway, a central signaling axis linking extracellular cues to neuronal regulation. Pathway activation was assessed by measuring phosphorylation of Akt (Ser473) and mTOR (Ser2448), established readouts of pathway activity27.

In the first experimental dataset (Fig. 5a), inhibition of Akt (MK-2206) and PI3K (LY294002) markedly reduced p-Akt levels compared with vehicle control, confirming effective pathway suppression. When ND7/23 cells were co-treated with RNase 4, p-Akt levels remained similarly reduced in all inhibitor conditions, indicating that RNase 4 does not override pharmacological pathway blockade. Notably, RNase 4 decreased p-Akt/Akt ratios relative to DMSO alone, suggesting that extracellular RNase 4 can dampen basal Akt activation. This reduction corresponds to an estimated 60% (Fig. 5a, b) (P=0.0072; n=3 biological replicates) decrease in the p-Akt/Akt ratio, indicating a significant inhibitory effect of RNase 4. Interestingly, while LY294002 robustly and significantly suppressed p-Akt levels, wortmannin led to a decrease of p-Akt level, though it was not significant, and led to a moderate suppression of around 65% (not significant) when associated with RNase 4 (Fig. 5b), despite targeting the same pathway. Furthermore, no significant effect of inhibitor rapamycin alone was found at any concentration, though surprisingly, the Akt levels increased by 40% in line with rapamycin-mediated inhibition of mTORC1, which can activate PI3K/Akt signaling by relieving a negative feedback loop28, which may explain the upward trend in Akt levels observed in the same experiment. The addition of RNase 4 to rapamycin-treated cells diminished p-Akt levels to an estimate of 60% compared to rapamycin only (P=0.011 for 5 nM condition and P=0.018 for 10 nM condition, Fig. 5b). These data indicate that RNase 4 exerts a consistent Akt-suppressive effect on ND7/23 cells. Investigating the effect of RNase 4 on mTOR phosphorylation, particularly at Ser2448 (Fig. 5c, d), we found a subtle decrease of around 20%, though not significant. Expectedly, mTOR level was found to be significantly reduced (P=0.0082) by the PI3K/Akt pathway inhibitor LY294002 (Fig. 5c, d). βIII-tubulin levels were comparable across all conditions (Fig. 5e, f), verifying equal protein loading and supporting the reliability of the phosphorylation analyses.

Notably, while Akt and mTOR expression were detectable, Axl receptor protein, a known PI3K–Akt–mTOR modulator, was undetectable in both ND7/23 cells and primary sensory neuron cultures (Supplementary Fig. 4), indicating that the effects of RNase 4 on this pathway in the DRG-derived context are Axl-independent. Taken together, our results demonstrate that RNase 4 may also exert its effect through modulating the PI3K–AKT pathway, but its effects here may not be through the Axl receptor.

Dynamic of Rnase4 expression in a nerve injury model
To examine changes in Rnase4 expression following nerve injury in a neuropathic pain model, we utilized the iPain database13. This resource includes RNA datasets from animals both with and without injury, specifically focusing on those subjected to nerve crush up to four weeks post-injury, as well as control animals representing baseline conditions. Initial mapping of Rnase4 expression on the Pain dynamics (Reference, Pain, Recovery and Lasting, Fig. 6a) revealed a significant increase in Rnase4 levels across all nociceptor subtypes following injury (Fig. 6a–c). Interestingly, the increase in Rnase4 expression was most prominent during the first week post-injury and was particularly marked in nociceptor populations that initially expressed lower levels of Rnase4 (NP, PEP2, and cLTMRs). Notably, in these populations, Rnase4 levels returned to near baseline after two weeks, while in populations that initially expressed higher levels of Rnase4 (PEP1 and SST), overexpression persisted. We further extended our analysis to human DRG using bulk RNA-seq from 18 and found a similar increase in RNase 4 expression in donors diagnosed with pain (Fig. 6d). This trend was, however, not statistically significant, likely due to the limited number of samples.

To predict the interaction network of Rnase4 during Pain dynamics, we applied Spearman’s rank correlation analysis to identify genes whose expression was correlated with Rnase4. Gene Ontology (GO) analysis of the resulting network revealed distinct pathways associated with RNase 4. Under naïve conditions, the top three GO terms for positively correlated genes were “Regulation of Signal Transduction by P53 Class Mediator”, “Epithelial Cell Migration”, and “Positive Regulation of Transcription by RNA Polymerase II”. In contrast, negatively correlated genes were associated with “Chemical Synaptic Transmission”, “Cell Junction Assembly”, and “Cell-Cell Adhesion Mediated by Cadherin” (Supplementary Fig. 5a, Supplementary Data 3). Fourteen days after nerve crush injury, the GO terms for positively correlated genes shifted to “Cellular Response to Organic Cyclic Compound”, “Response to Axon Injury”, and “Nervous System Development”. For negatively correlated genes, the enriched terms included “Chemical Synaptic Transmission”, “Potassium Ion Transmembrane Transport”, and “Positive Regulation of Potassium Ion Transmembrane Transporter Activity” (Supplementary Fig. 5b, Supplementary Data 4). Additionally, Gene Set Enrichment Analysis (GSEA) comparing nerve crush to naïve conditions revealed that the top three upregulated pathways were “Axon Development”, “Positive Regulation of Programmed Cell Death”, and “Positive Regulation of Leukocyte Migration”, while no pathways were significantly downregulated (Supplementary Fig. 5c, Supplementary Data 5).

These analyses highlight RNase 4-associated gene networks linked to injury responses, suggesting RNase 4 maybe involved in processes related to tissue repair and immune modulation after nerve injury.

To further assess the role of RNase 4 in nociceptor function in a neuropathic pain model, we employed the sciatic nerve crush injury model. Sciatic nerve crush is a well-established model of Wallerian degeneration, characterized by axonal degeneration and myelin breakdown distal to the lesion, and is widely used to study mechanisms of neuropathic pain. Following nerve injury, cKO-BR and cKO-PR mice, along with their control littermates, exhibited a temporary reduction in mechanical or thermal sensation by 7 days after nerve injury, followed by recovery of sensory function. Von Frey testing revealed that both cKO-BR, cKO-PR (Fig. 6f, i), and control mice displayed the expected reduction in mechanical sensitivity following sciatic nerve crush, followed by a progressive recovery over time. In both knockout lines, recovery of mechanical thresholds occurred faster than in littermate controls, indicating that loss of RNase 4 facilitates restoration of tactile sensitivity after injury. In contrast, Hargreaves testing of thermal sensitivity showed an opposite trend between genotypes cKOs at 14 days post-injury, with cKO-BR mice displaying a delay in thermal recovery compared to controls, while cKO-PR fully recovered. This divergence suggests that RNase 4 modulates regeneration of mechanical and heat pathways in a differential, modality-specific manner, and highlights that accelerated recovery of tactile function in the knockouts does not necessarily generalize to thermal nociception at all stages of the regeneration process.

Evaluation of the role of RNase 4 on repair and remyelination
Our analysis revealed that RNase 4 expression in the peripheral nervous system is predominantly restricted to unmyelinated fibers, as observed in dorsal root ganglia (DRG; Fig. 1a–c), trigeminal ganglia (TG; Fig. 7a), and type II neurons of the spiral ganglion (Fig. 7b). These findings prompted us to investigate whether RNase 4 contributes to unmyelinated fiber sorting or facilitates interactions between C-fibers and Schwann cells under normal and injury conditions.

To address this, we collected sciatic nerve samples from control and Rnase4 cKO mice and processed them for electron microscopy (EM) to assess myelin ultrastructure and axonal morphology at ultrastructural resolution (Fig. 7c–o). Mice underwent unilateral sciatic nerve crush injury (ipsilateral, Ipsi) to examine Wallerian degeneration, demyelination, and remyelination processes.

In uninjured nerves, neither G-ratio (calculated per animal; Fig. 7d), axon diameter (Fig. 7e), nor axon density (axons/mm²; Fig. 7f) differed significantly between genotypes. However, in injured nerves, Rnase4 cKO mice exhibited a significant increase in axon density compared to controls (Fig. 7f), while the number of myelinated axons remained comparable between genotypes (Fig. 7g). These data indicate enhanced axon regeneration but no alteration in myelination rates in cKO-PR mice.

Notably, the incidence of aberrant myelin structures, including focal hypermyelination, myelin infolds, and degenerating myelin, was significantly elevated in Rnase4 cKO nerves compared to controls (Fig. 7h, i). This increase in myelin misfolding was predominantly observed in intermediate-diameter fibers, with both genotypes showing similar distributions of myelin defects (Fig. 7i). Analysis of Remak bundles in uninjured sciatic nerves revealed no differences between genotypes in bundle number or axons per bundle (Fig. 7j–l). Post-injury, cKO-PR mice exhibit similar C-fiber densities per Remak bundle comparable to uninjured levels, indicating accelerated axonal regeneration, whereas control nerves showed persistent reductions consistent with slower recovery post-injury (Fig. 7l).

cKO-PR mice also exhibited significantly reduced myelin debris enclosed by immune cells at 14 days post-injury (Fig. 7m–o), correlating with more advanced axonal regrowth. Efficient myelin debris clearance by macrophages and Schwann cells is a critical determinant of regeneration success. Schwann cell vacuolization, a hallmark of cellular stress, was observed exclusively in Rnase4 cKO nerves (Fig. 7h).

Importantly, western blot analysis revealed increased pAkt/Akt levels upon RNase 4 inhibition, indicating enhanced activation of the PI3K–AKT pathway (Fig. 5). Given the established role of AKT signaling in axonal growth and regeneration29,30 these data provide a mechanistic link between RNase 4 loss and the observed acceleration of axonal regrowth. Our findings suggest that RNase 4 normally restrains regenerative signaling, and that its absence promotes axonal regeneration, potentially through AKT-dependent pathways, while simultaneously perturbing Schwann cell homeostasis and myelin remodeling (Fig. 7p).

Taken together, our findings reveal a complex and unexpected role for nociceptors in nerve homeostasis and regeneration of surrounding nerve fibers (Fig. 7p, scheme). Under basal conditions, nociceptors expressing RNase 4 exert control over myelination, Remak bundle formation, and fiber wrapping in the sciatic nerve. Following injury, these nociceptors influence axon regeneration and remyelination during Wallerian degeneration. This dual effect, mediated through RNase 4, suggests that nociceptors act as critical modulators of myelin dynamics, likely by influencing Schwann cell activity and macrophage behavior. This dual mechanism highlights the complex role of nociceptors and RNase 4 in maintaining nerve tissue equilibrium.

These results not only expand our understanding of nociceptor function in the peripheral nervous system beyond pain sensation but also hint at the potential of targeting RNase 4 in nociceptors as a therapeutic strategy for promoting nerve repair and regeneration in pathological conditions.

Methods
Mice and ethics
Adult male and female wild type and transgenic mice of the C57BL/6J background (aged 2–4 months) and embryos (embryonic day 15.5–17.5) were used in this study. The animals were housed under a 12-hour light–dark cycle at 24°C, with ad libitum access to food and water. All animal care and experimental procedures were permitted by the Ethical Committee on Animal Experiments (Stockholm North committee) and conducted according to The Swedish Animal Agency’s Provisions and Guidelines for Animals Experimentation recommendations with ethical permit number 17396-2022.

Mice construction. EasyCRISPR
We developed two mouse models in this study, Rnase4fl/fl and Prdm12CreERT2, both using the Easi-CRISPR approach21,37. C57BL/6J mouse strain (Jax strain 000664) was used as zygote donor, and the transgenic mouse experiments were performed as described previously37,38.

Rnase4fl/fl mouse: The Rnase4 gene (ENSMUSG00000021876) has only one coding exon (ENSMUST00000022428). Creating a LoxP flanked (floxed) allele for single coding-exon genes would be challenging because inserting LoxPs may disrupt regulatory regions such as promoters and regulatory sequences in 5’ and 3’ ends, respectively. We used a split exon strategy to insert an ‘artificial intron with LoxP sites flanking a branching point’ at the split site to create a conditional knockout allele for RNAse420,39. One of the limitations of this approach is the unpredictable effect of splitting the exon (and the inserted cassette) on the normal expression of the conditional allele. Reasons for this could be many. Two important ones are: (a) the incomplete splicing out of the artificial intron (before Cre) and (b) the unknown effect on the normal expression of the transcript due to the presence of unnatural sequences within the exon. Because of this, the conditional alleles can diminish the gene expression (hypomorphic) or result in a complete loss of expression even before Cre-mediated excision of the branching point39. Using RNAscope technology, RNase 4 mRNA expression levels were comparable between the RNase 4fl/fl mice and their littermates (those without artificial intron insertion in the Rnase4 gene), while breeding with Cre mice or with CreERT2 mice followed by tamoxifen injection led to a decrease in the Rnase4 mRNA expression. Therefore, we presume that the split exon strategy for the Rnase4 locus did not affect the normal expression of the gene. Details of the Rnase4 SCON strategy and generating the mouse model are given in Supplementary Data 6.

The Prdm12CreERT2 mouse was generated by deleting the coding regions of the first two exons of Prdm12 and by inserting CreERT2-SV40 polyA cassette in place of it using the Easi-CRISPR approach. Details of the Prdm12CreERT2 mouse model design are given in the supplementary file 2. The Prdm12CreERT2 is a KI/KO mouse model. It can be used as a Cre driver mouse when it is used as a heterozygous and a Prdm12 knockout mouse when the allele is bred to homozygous or as a Cre driver mouse when it is used as a heterozygous. The knockout phenotype of the mouse is not characterized in this study. Details and sequences used to generate the mouse line is given in Supplementary Data 7.

Adult mice and embryos
Prdm12CreERT2 mice were crossed with Ai14 (Rosa26tdTOM, The Jackson Laboratory, 007914) for immunohistochemical and iDISCO staining characterization. Only heterozygote Prdm12CreERT2 were bred to avoid the possible generation of full Prdm12 knockouts. To characterize the effects of RNAse4 conditional knockout in neurons, the RNAse4fl/fl mice were crossed with Baf53bCre (The Jackson Laboratory, 02782640) for constitutive knockout during development or Prdm12CreERT2; Ai14 mice for timed knockout in adulthood. Both Cre lines were mated for heterozygote pups only.

Ear biopsies for genotyping were taken by facility technicians at weaning. DNA was extracted from tissue biopsies using the Phire Animal Tissue Direct PCR Kit (Thermo Scientific, F140WH) and genotyped with PCR gel electrophoresis using the primers found in Supplementary Table 1.

Tamoxifen treatment
On the day of injection, tamoxifen (Sigma, T5648-1G) was dissolved in corn oil (Sigma, C8267-500ML) at a concentration of 20 mg/ml.

For staining characterization of Prdm12CreERT2; Ai14 adult mice, tamoxifen was delivered via intraperitoneal injection (i.p.) for 3 or 5 consecutive days at a dose of 100 mg/kg, and were then allowed to recombine for 1 week. Similarly, 3 doses of 100 mg/kg tamoxifen were injected i.p. in Prdm12CreERT2; Rnase4fl/fl mice for behavior characterization according to the experiment timeline.

For characterization of Prdm12CreERT2; Ai14 in embryos, tamoxifen was injected i.p. in the pregnant mother at a dose of 100 mg/kg for 3 consecutive days prior to collection for E15.5–E17.5 embryos. Embryo collection was performed around 4 hours or the day after the final injection. Tail tips were also collected for genotype confirmation using the protocol and primers described above.

Sciatic nerve crush injury
As described in a reported method41,42, buprenorphine (0.1 mg/kg) and carprofen (5 mg/kg) were first administered subcutaneously, followed by the surgical exposure of the right sciatic nerve under isoflurane anesthesia. The nerve was then subjected to a controlled crush injury for 15 seconds using hemostatic forceps (Fine Science Instruments; 0.4 mm tip angled, Cat. No. 11063-07), followed by a second crush at the same site oriented orthogonally to the initial injury. After the procedure, the skin was sutured. Distal nerve stumps located 1 mm from the crush site were collected for electron microscopy (EM) analysis. A subsequent dose of carprofen (5 mg/kg) was administered subcutaneously 24 hours post-surgery.

Pain behavior
Mechanical sensitivity was assessed using calibrated von Frey filaments following the up–down method described previously43–46. Mice were individually placed in transparent chambers positioned on a metal mesh platform (6 × 6 mm grid) and allowed to habituate for 20–60 minutes before testing. The plantar surface of the hind paw was stimulated with a series of monofilaments (Stoelting, IL, USA) spanning forces from 0.008 to 2 g. A positive response was defined as a clear paw withdrawal or shake. Six responses were obtained for each paw per animal.

Thermal nociception was evaluated using the Hargreaves plantar test as previously reported43,44. Animals were acclimated for 20–60 minutes in Plexiglas chambers placed on a glass surface. A focused radiant heat source (IITC Inc., Woodland Hills, CA, USA) set at 30% intensity was directed at the central plantar region through the glass. Paw withdrawal latency was recorded, with a 20-second cutoff applied to prevent tissue injury.

RNAscope and immunohistochemistry
Adult mice were sacrificed by isoflurane overdose followed by transcardiac perfusion with chilled PBS and then 4% PFA, where sciatic nerve, spinal cord, and lumbar DRG were collected. Following dissection, tissues of interest were post-fixed in PFA for 1 hour for immunohistochemistry (IHC) or overnight for RNAscope at 4°C.

For embryo collection, the mother was sacrificed by CO2 overdose, and embryos were dissected out on ice. To process embryos for sectioning, the ventral organ mass was removed before post-fixation in PFA for 6 hours at 4°C.

After PFA fixation, samples were then transferred to 30% sucrose for cryoprotection overnight at 4°C. Embryo samples were further dissected to isolate the spinal column for further processing.

Dissected tissues were then embedded in optimal cutting temperature (OCT) medium and stored in −80°C. Blocks were sectioned using Epredia™ CryoStar™ NX70 cryostat at 14 µM and stored in −80°C. Sections were air-dried for at least 1 hour before use.

RNA in situ hybridization was performed using RNAscope® Multiplex Fluorescent Reagent Kit v2 according to manufacturer instructions (Advanced Cell Diagnostics, ACD). Target probes, Mm-Prdm12-C1 (ACD, 524371), Mm-Kcng3-C1 (ACD, 1230601-C1), Mm-Prdm12-C2 (ACD, 524371-C2), Mm-Sst-C2 (ACD, 404631-C2), Mm-Pvalb-C3 (ACD, 421931-C3), Mm-Rnase4-C1 (ACD, 1041951-C1), Mm-Kcnf1-C2 (ACD, 508731-C2), Mm-Calca-C3 (ACD, 578771-C3) and Mm-tdTomato-C3 (ACD, 317041-C3D) were used according to protocol.

IHC was performed on sectioned slides or following RNAscope using the following antibodies as necessary: 1:400 TH (Pel-Freez, P40101), 1:400 TrkA (R&D Systems, AF1056), 1:250 NF200 (Millipore, AB1991), 1:250 peripherin (Abcam, ab39374), 1:200 CGRP (Immunostar, AB_572217), 1:500 rabbit anti-PV (Swant, PV27a), and 1:500 rabbit anti-RFP (Rockland, 600-401-379). Isolectin B4 conjugated with fluorescein was added at 1:100 together with primary antibodies. Secondary antibodies were used at a concentration of 1:1000 for 1 hour at room temperature: donkey anti-rabbit IgG AF488 (Invitrogen, A-21206) or donkey anti-rabbit IgG 555 (Invitrogen, A-31572).

Slides were then counterstained with 1:200 NeuroTrace™ 435/455 Blue Fluorescent Nissl Stain (Invitrogen, N21479) or 1:1000 DAPI (Invitrogen, D1306) before sealing with DAKO fluorescent mounting medium (Agilent, S3023) and stored at 4°C.

Images were taken using a Zeiss LSM800 or Zeiss LSM800 Airyscan confocal microscope and analyzed in ImageJ.

Volume immunostaining and optical clearing of fixed mouse embryos
Embryos were collected from pregnant females euthanized by CO2 inhalation and fixed overnight at 4°C in modified Zamboni fixative (4% paraformaldehyde, 0.2% picric acid in phosphate buffer, pH 7.4). Samples were post-fixed for 1 hour at room temperature, washed, and stored in PBS containing 0.02% sodium azide at 4°C until processing. Whole-mount immunolabeling and clearing were performed using the iDISCO+ protocol47–49.

Genotyped E15.5 embryos were washed in 1× PBS and dehydrated in a graded methanol series (20–100%, 1 hour per step).

Autofluorescence was reduced by overnight incubation in 5% H2O2 in methanol at 4°C. Samples were rehydrated through a reverse methanol gradient and incubated sequentially in permeabilization buffer (0.2% Triton X-100, 20% DMSO, 0.3 M glycine in PBS with 0.02% sodium azide) and blocking solution (0.2% Triton X-100, 10% DMSO, 6% normal donkey serum), each for 48 hours at 37°C.

Primary antibodies (rabbit anti-RFP, 1:500, Rockland; goat anti-TrkA, 1:400, R&D Systems) were applied for 4 days at 37°C in antibody buffer (0.2% Tween-20, 10 mg/ml heparin, 5% DMSO, 3% normal donkey serum in PBS with sodium azide). Following extensive washes, embryos were incubated for 4 days with Alexa Fluor–conjugated secondary antibodies (donkey anti-rabbit AF555; donkey anti-goat AF647; 1:500, Invitrogen).

After staining, tissues were dehydrated in methanol, cleared in dichloromethane/methanol (2:1) followed by 100% dichloromethane. Finally, samples were transferred into 100% dibenzyl ether and stored in this medium until imaging.

Lightsheet fluorescence microscopy and 3D image reconstruction
Cleared samples were imaged using an Ultramicroscope II (LaVision Biotec, Bielefeld, Germany) operated with Imspector™ software49. The system was equipped with a 2×/0.5 Olympus objective, an MVX-10 zoom body (0.63–6.3×), a 6.5 mm working-distance spherical aberration–corrected dipping cap, and an Andor Neo sCMOS camera (2560×2160 pixels). Excitation was provided by Coherent OBIS lasers at 488 nm, 561 nm, and 640 nm with appropriate emission filters.

Acquisitions were performed in multicolor mode using single-sided illumination. Laser power was set to 60–80% with a 100 ms exposure time. The lightsheet numerical aperture was set to 0.156 with 70% sheet width. Z-stacks were collected at step sizes between 1.89 and 4.8 µM. No tiling was applied.

Images series were acquired as 16-bit TIFF stacks and converted to IMS format using Imaris File Converter (v9.7.2, Bitplane). Three-dimensional rendering and analysis were conducted in Imaris (v9.7.2). Background subtraction was applied during processing, and brightness and contrast were minimally adjusted for the figure.

Electron microscopy
Following behavioral assessment after nerve injury experiment, animals were mice were sacrificed by isoflurane overdose and transcardially perfused with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer50. A distal segment of the sciatic nerve (~1 mm distal to the crush site) was dissected and immersion-fixed for 1 hour at room temperature in 2.5% glutaraldehyde and 1% formaldehyde prepared in 0.1 M Sørensen phosphate buffer (pH 7.4), then stored at 4°C until further processing.

Samples were rinsed in 0.1 M phosphate buffer and post-fixed in 2% osmium tetroxide in the same buffer (pH 7.4) for 2 hours at 4°C. Tissues were dehydrated through a graded ethanol series, stained en bloc with 0.5% uranyl acetate, followed by acetone dehydration, resin infiltration, and embedding in LX-112 resin (Ladd Research).

Ultrathin sections (approximately 80–100 nm) were cut using a Leica EM UC7 ultramicrotome and mounted on formvar-coated slot grids. Sections were contrasted with uranyl acetate and lead citrate before examination on a Hitachi HT7700 transmission electron microscope operated at 80 kV. Images were captured using a 2k×2k Veleta CCD camera (Olympus Soft Imaging Solutions).

Image quantification
For immunohistochemistry: To examine the distribution of Rnase4 in DRG neuron sizes, Rnase4+ neurons were identified via RNAscope in ImageJ and their cell borders determined according to NeuroTrace™ Nissl Stain. The area of Rnase4+ neurons was measured, where the diameter was calculated as the square root of the area divided by p and then multiplied by 2 (Area=p*(Radius)2). A total of 4 sections taken at 20x magnification of different lumbar DRGs were quantified from 3 wild type mice. The number of Peripherin+ was counted manually using the multi-point tool from ImageJ and calculated accordingly in proportion to Nissl+ neurons in a minimum of 3 sections from 3 wild type mice.

To determine the recombination efficiency of Prdm12CreERT2; tdTomato (from crossing with the Ai14 Cre-dependent reporter line) and Prdm12 were labelled by RNAscope and counted using the multi-point tool in ImageJ. The number of tdTomato+ neurons was divided by the number of Prdm12+ neurons to obtain the proportion of Cre-dependent tdTomato expression in Prdm12+ DRG neurons from 4 lumbar DRG sections taken at 20x magnification of 4 wild type mice.

For electronic microscopy image analysis, g-ratio measurements were performed using the Napari software platform. Axons and myelin were segmented and selected automatically using deep learning algorithms integrated with morphological metric tools and the Napari GUI plugin. The g-ratio was calculated as the ratio of the inner axon diameter to the total diameter (axon plus myelin sheath), with diameters derived from the measured perimeters divided by π. Additional EM analyses in this study were conducted using ImageJ software. Axon counting and selection for these analyses were performed manually.

Western blot
ND7/23 cells (mouse neuroblastoma x rat dorsal root ganglion neurons) were obtained commercially and cultured according to protocol in high glucose DMEM media (Dutscher, 509053) containing 10% heat inactivated fetal bovine serum (FBS, Sigma, F9665), 2 mM L-glutamine (Cytiva, SH30034.01), 100 U/mL penicillin and 100 µg/mL streptomycin (Cytiva, SV30010) at 37°C, 5% CO2, in a humidified chamber with media change every 2-3 days. Prior to the experiment, cells were serum-starved overnight in DMEM with 2 mM L-glutamine, and then incubated for 1 hour with the presence of the following: DMSO (vehicle, Sigma), MK-2206 (Selleck Chemicals, S1078-5mg), Wortmannin (Sigma, 681676-1MG), Rapamycin (Cell Signalling Technologies, CST CST-9904S), or LY294002 (CST, CST-9901S). Mouse recombinant RNase 4 was obtained from OriGene (TP501018SE) and added at a concentration of 1 µg/mL for 5 minutes after inhibitor incubation. Pellets were then collected by aspiration in the same media and stored in −80°C until extraction. Three independent cell culture experiments were performed.

For primary culture, adult wild-type mice were anesthetized using isoflurane, and cardiac perfusion with chilled PBS was performed. DRGs were dissected in a laminar flow hood into DMEM medium (Gibco, 31966-021), followed by a 30-minute enzymatic digestion with 12.5 mg/mL collagenase IV (Sigma, C5138) at 37°C, and another 30-minute digestion with 6.25 mg/mL collagenase IV and 2.5 mg/mL dispase II (Gibco, 17105-041). Enzyme-containing media was then removed, and media with 10% FBS was added to stop the reaction. The solution was then serially titrated with 3 progressively smaller glass Pasteur pipettes (VWR, 612-1701) refined over a Bunsen burner (Integra), and coated with 0.2% bovine serum albumin (BSA, Sigma, A4503-10G). The solution was then filtered through a 70 mM cell strainer (Corning, CLS431751), and debris was removed by centrifugation into a bottom layer of 15% BSA. The supernatant was removed, and the pellet was then resuspended in Neurobasal media (Gibco, 21103-049) supplemented with 1X B-27 plus supplement (Gibco, A35828-01), GlutaMAX supplement (Gibco, 35050-061), 5 mg/mL cytosine arabinoside (AraC, Sigma, C6645), 50 ng/mL neural growth factor (NGF, PeproTech, 450-34), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were then seeded in pre-coated wells: 30 minutes at 37°C in poly-L-lysine (Sigma, P4707) followed by overnight at 37°C in recombinant human Laminin 521 (Gibco, A29248). The media was changed the following day and thereafter once every 2 days. One week after seeding, cells were stimulated with 1 mM capsaicin (Sigma, PHR1450) or an equivalent of ethanol (vehicle) for 60 seconds at 37°C, thereafter the cell pellet was collected for further analysis.

Protein was extracted from collected pellets using RIPA buffer (Sigma, R0278-50ML) supplemented with Halt™ protease inhibitor cocktail (ThermoFisher, 78439), phosphatase inhibitor cocktail 2 (Sigma, P5726-1ML), and phosphatase inhibitor cocktail 3 (Sigma, P0044-1ML) according to manufacturer recommendations. Quantification was performed using the Quick Start™ Bradford Protein assay (Bio-Rad, 500-0205) and measured with a NanoDrop spectrophotometer using reference standards diluted from 2 mg/mL BSA (ThermoFisher, 23209).

For Western blot, samples and Precision Plus Protein Dual Color Standards (Bio-Rad, 1610374) were processed with 10 mM dithiothreitol, and NuPAGE™ LDS Sample Buffer (ThermoFisher, NP0007). SDS-PAGE was performed using precast mini-PROTEAN TGX gels with a 4-20% gradient (Bio-Rad, 4561096) in NuPAGE™ MOPS SDS running buffer (ThermoFisher, NP0001). Transfer was performed for 1 hour with 100 V at 4°C using 20% methanol in Tris/Glycine Buffer (Bio-Rad, 1610734) onto PVDF membranes (GE Healthcare, RPN303F). Blocking was performed for 1 hour at room temperature using 5% BSA or nonfat dry milk (Bio-Rad, 1706404) in tris-buffered saline (TBS, ThermoFisher, J62938.K2) with 0.1% Tween-20 (TBS-T).

The following antibodies were applied overnight at 4°C at a concentration of 1:1000: rabbit anti-Axl (CST, 8661), rabbit anti-phospho-Axl Tyr702 (CST, 5724), rabbit anti-mTOR (CST, 2983), rabbit anti-phospho-mTOR Ser2448 (CST, 5536), rabbit anti-Akt (CST, 9271), rabbit anti-phospho-Akt (CST, 9272), and mouse anti-βIII tubulin (Promega, G712A). Subsequent washes were performed in 1% BSA in TBS-T or 0.5% nonfat dry milk. Secondary antibodies, HRP-linked anti-rabbit IgG (CST, 7074) or anti-mouse IgG (CST, 7076), were then applied at 1:3000 for 1 hour at room temperature. Membranes were then washed and incubated for 4 minutes with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher, 34577) and imaged with ImageQuant LAS4000 (GE Healthcare). Blots were stripped with Restore™ Western Blot Stripping Buffer (ThermoFisher, 21059) for 30 minutes at room temperature, to enable the probing of phosphorylated forms, followed by the total protein forms and then the loading control (tubulin) when relevant.

Bands were then analyzed and quantified in ImageJ using the ‘analyze gels’ function. The area under the integrated density curve under each lane profile was taken, while the background was considered by manually drawing a horizontal straight line across the peak base. In cases where the visible artifact signal was close to the band and merged with the peak signal on one side, another vertical straight line was drawn between the band peak and the visible artifact peak. Each band area was further normalized to the tubulin control, and then the ratio of the phosphorylated form to the total protein was taken for analysis. Original full scan blots are provided in the Source Data file for those included in Fig. 5 and the Supplementary Information file for those included in Supplementary Fig. 4 (Supplementary Figs. 7, 8).

DRG dissociation and single nuclei isolation51–53
Mice were deeply euthanized by isoflurane and perfused with 4% pre-cooled PBS. Sciatic nerve and L4-L6 DRGs were collected at 2 weeks after sciatic nerve crush injury, and then immediately put on dry ice. Frozen tissues stored at −80°C before further processing.

Nuclei were isolated from frozen DRGs following the protocol established by the Allen Institute for Brain Science54, with all procedures conducted at 4°C. Tissue samples were mechanically homogenized in 2 ml of ice-cold lysis buffer containing 10 mM Tris (pH 8.0), 250 mM sucrose, 25 mM KCl, 5 mM MgCl₂, 0.1 mM DTT, protease inhibitor cocktail (1×), and 0.1% Triton X-100. RNase inhibitors were included as appropriate: RNasin Plus (0.2 U/µl) for Smart-seq3xpress preparations or Protector RNase Inhibitor for 10x Genomics workflows.

The homogenate was sequentially passed through 70 µm and 30 µm strainers to remove large debris. Nuclei were pelleted by centrifugation at 900 × g for 10 minutes and resuspended in 250 µl of chilled homogenization buffer. To further purify nuclei and eliminate residual debris, samples were subjected to iodixanol gradient centrifugation (25%/29%). The final nuclei pellet was resuspended in 500 µl of blocking buffer (1× PBS, 1% BSA, 0.2 U/µl RNase inhibitor) prior to fluorescence-activated nuclei sorting and downstream Smart-seq3xpress or 10x Genomics library preparation.

Fluorescent-activated nuclei sorting and single nuclei RNA-sequencing (Smart-seq3xpress)51–54
To selectively isolate neuronal nuclei, the nuclei suspension was incubated on ice for 30 minutes with a PE-conjugated anti-NeuN antibody (1:500; FCMAB317PE, Merck). Nuclei were then collected by centrifugation at 400 × g for 5 minutes and resuspended in PBS supplemented with 0.1 µg/ml DAPI (Invitrogen D3571) and 0.2 U/µl RNase inhibitor.

Individual NeuN-positive and DAPI-positive nuclei were sorted into 384-well plates containing 0.3 µl of lysis reaction mix using a BD FACSAria Fusion or BD FACSAria III flow cytometer operated at 4°C. Sorting gates were defined based on DAPI fluorescence and PE signal intensity.

Immediately after sorting, plates were briefly centrifuged and stored at −80°C until processing. Single-nucleus RNA sequencing was subsequently performed using the Smart-seq3xpress protocol by the Rickard Sandberg laboratory at Karolinska Institutet.

Rnase4-centered RNA seq analysis of iPain
The expression matrices and metadata of iPain atlas13 were downloaded from CELLxGENE. The data was loaded and analyzed using Scanpy55 package for Python 3 with default parameters unless otherwise specified. For both iPain DRG and TG, neurons were selected and re-clustered. The nearest neighbor graphs were constructed using scANVI latent representation with “cosine” as a metric to calculate the neighbor distance. The UMAP projections of both tissues were computed with 0.3 as a minimum distance.

To perform differential gene expression analysis (DEA) between the nociceptors and NF neurons, cells from different cell types were binned into pseudo-bulk data by summing the gene expression of each cell type for each replicate. Genes found in fewer than 3 samples of the pseudo-bulk sample were removed. The DEA was performed using the DESeq2 pipeline, and Wald tests through pyDESeq216 package in Python 3. P-values were adjusted for multiple hypothesis testing with Benjamini-Hochberg (BH) correction. Genes were identified to be cell type specific when their log2 fold change was greater than 2.5, and their adjusted p-value was less than 0.05.

Genes correlated to RNase 4 were identified using Spearman’s rank correlation in Naïve and Crush at 14-day conditions. The p-values of the correlation test were corrected for multiple hypothesis testing with the BH correction. Genes with absolute correlation higher than 0.25 and adjusted p-values of less than 0.05 were kept. Gene set enrichment analysis (GSEA) was performed on these genes (both positively and negatively correlated) against Gene Ontology of biological process collection from 2023 with GSEApy56 package in Python 3.

RNAseq analysis of Baf53bCre;Rnase4fl/fl mouse line
The sequenced reads were aligned using the zUMIs pipeline version 2.9.7, which makes use of the STAR-SOLO57 alignment tool, and the reference genome was mouse (mm39).

The data was loaded and analyzed using Scanpy55 package for Python 3 with default parameters unless otherwise specified. Nuclei with unique gene count lower than 3000 genes, percent mitochondrial gene higher than 2, and percent ribosomal gene higher than 2 were removed from the data for Baf53bCre; Rnase4fl/fl mouse line. For the control line, nuclei with a unique gene count lower than 2000 genes, a percent mitochondrial gene higher than 2, and a percent ribosomal gene higher than 2 were removed.

After quality control, we performed the cell type annotation by using the pre-trained scANVI model from the iPain paper for the label transfer13,22. More specifically, we online updated the pre-trained model using the data from this study as a query. The query model was trained with 400 epochs, and the parameter “n_samples_per_label” was equal to 30.

DEA between the nociceptors from Baf53bCre;Rnase4fl/fl and control lines, cells from different lines were binned into pseudo-bulk data by summing the gene expression. Genes found in fewer than 3 samples of the pseudo-bulk sample were removed. The DEA was performed using DESeq258 pipeline, and Wald tests through the pyDESeq216 package in Python 3. P-values were adjusted for multiple hypothesis testing with the (BH) correction. Genes were identified as DE between the lines when their log2 fold change was greater than 0.5, and their adjusted p-value was less than 0.05. Gene set enrichment analysis (GSEA) was performed on top 15 up and down DE genes against Gene Ontology of biological process collection from 2023 with GSEApy56 package in Python 3.

RNAseq analysis of Prdm12CreERT2;Rnase4fl/fl mouse line
Libraries were sequenced on a NovaSeq 6000 platform (NovaSeq Control Software v1.7.5; RTA v3.4.4) using an S2 flow cell under the NovaSeq Standard workflow. The sequencing configuration consisted of 28 bp for Read 1, 10 bp for Index 1, 10 bp for Index 2, and 90 bp for Read 2.

Base call files were converted to FASTQ format using bcl2fastq (v2.20.0.422, Illumina/CASAVA suite), with quality scores encoded in Phred33 (Sanger/Illumina 1.8+ format). FASTQ files were processed using the Cell Ranger pipeline (v9.0.1, 10x Genomics), aligning reads to the mouse reference genome (mm10).

The expression matrices were loaded and analyzed using Scanpy55 and concatenated into a single matrix. Nuclei with a unique number of genes greater than 6000 and a percentage of mitochondrial counts greater than 0.5 were filtered out. The presence of ambient mRNAs was corrected using SoupX59. After the correction, the matrix was normalized and log-transformed using the normalize_total and log1p functions from Scanpy, respectively. We then identified the top 3000 highly variable genes using Seurat eversion 3 method on the corrected count matrix from SoupX. UMAP was computed after the construction of the nearest neighbor graph. Cell type annotation was done using the label transfer method from our pre-trained scANVI model after updating the weights of the model with the current dataset.

Cell types corresponding to the nociceptive lineage were selected for further analysis. UMAP was recomputed using the min_dist=0.3. We perform DEA at the conditional level using the rank_genes_groups function from Scanpy to compare cKO-PR to WT nociceptors, and the testing method was one-tail t-test with overestimated variance of each group. For the DEA of each nociceptive subtype comparing cKO-PR to WT groups, we created a pseudo-bulk with 5 pseudo-replicates for each subtype and condition to apply pyDESeq216 pipeline. P-values were adjusted for multiple hypothesis testing with BH correction. Genes were identified as DE between the lines when their log2 fold change was greater than 0.5, and their adjusted p-value was less than 0.25.

Statistical analysis
For immunohistochemistry and EM, the statistical analysis and tests were done in Python 3 with “stats” module from the SciPy v1.10.1 package. Data are presented as the mean±SEM or median ±SEM. Significant changes were identified using a two-tailed t-test, and multiple hypothesis testing was corrected with Benjamini-Hochberg. For single-cell data analysis, Python 3 with “stats” module from the SciPy v1.10.1 package was utilized. The data were imported as a DataFrame or Array respectively, through Pandas v2.0.3 or Numpy v1.22.4 package. In general, the t-test was applied when the data followed a normal distribution. Otherwise, the Mann-Whitney U test was performed. Paired t-tests were used for comparison of paired data from behavior experiments, whereas an independent t-test was used on unpaired data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript identifies RNase4 as selectively expressed in unmyelinated nociceptors and reveals its dual role in nociceptor function and nerve homeostasis. Through an integrated approach combining single-cell transcriptomics, genetic mouse models, and functional assays, the authors demonstrate that RNase4 regulates both nociceptor molecular identity and influences neighboring myelinated axons. While conditional deletion of Rnase4 during development alters gene expression profiles without affecting baseline pain sensitivity, adult-onset deletion causes transient mechanical hypersensitivity. Following nerve injury, Rnase4 deletion accelerates functional recovery and remyelination. The findings establish RNase4 as an important regulator of nociceptor biology with both cell-autonomous and non-cell autonomous effects. However, the molecular mechanisms underlying these effects remain inadequately explored.\nSpecific comments:\n1. The authors should perform co-localization studies using established nociceptor markers with RNase4 RNA FISH. Additionally, protein-level detection of RNase4 would strengthen the findings, as protein is the functional entity.\n2. In Fig 1g, comparative expression data showing differential expression between neuronal subtypes or tissues would provide better context for RNase4 specificity.\n3. Fig 2b and 2c rely on Nissl staining, which labels all neurons. Using DRG neuron-specific markers would provide better characterization of the cells expressing RNase4.\n4. The authors highlight differentially expressed genes in Figures 2e-f but fail to present comprehensive functional enrichment analysis of these genes. The manuscript mentions alterations in zinc finger proteins, transcription factors, and immune response pathways, but these findings are not adequately presented in figures or supplementary data.\n5. In Fig 2g, the nature of individual data points is unclear, and there appears to be inconsistency in the number of data points between left and right panels.\n6. The authors should examine whether RNase4 expression changes during the course of behavioral testing (von Frey and Hargreaves tests), which would help connect molecular changes with behavioral outcomes.\n7. Regarding the observed thicker myelin and smaller axon diameter in contralateral nerves of cKO-PR mice (Fig. 5c-f), it is important to clarify whether these are developmental effects or consequences of adult Rnase4 deletion. If the latter, the time course of these structural changes following deletion should be determined.\n8. The authors propose a compensatory relationship between RNase4 and RNase5 (angiogenin) in nerve regeneration but provide no experimental evidence. Examining RNase5 expression in Rnase4 knockout conditions would substantiate this hypothesis.\n9. As RNase4 is described as a secreted protein, rescue experiments administering recombinant RNase4 would demonstrate whether it has direct protective effects and strengthen the causal relationship between RNase4 and the observed phenotypes.\n10. The study lacks mechanistic explanation: Does RNase4 function through its enzymatic activity or through other molecular pathways? Is it modulating known signaling pathways such as AXL?", "reviewer_2": "RNase4 is known to be involved in a broad range of biological processes, including the regulation of intra- or extra-cellular RNA metabolism as well as antiviral, antibacterial, and antifungal activities, neurogenesis, angiogenesis, immune modulation and host defence, is selectively expressed in neurons of the nociceptive lineage. In this manuscript, the authors report that RNase4 is selectively expressed in sensory ganglia in the unmyelinated nociceptive lineage. They analysed the consequences of its loss of function using both a conditional KO and an inducible cKO strategy with a novel Prdm12CreERT2 line they have generated and may constitute a useful new tool in the field. Although the results obtained provide first evidence for a role of RNase4 in nociceptors and suggest its absence contributes to faster recovery after peripheral injury, we feel the study at the present stage substantially lacks quantified analyses to support working models and findings. Several claims lack unequivocal demonstration, particularly with regards to segregation between RNase4 autonomous and non cell-autonomous functions.\nMajor concerns and specific points that could be addressed to strengthen the study are as follow:\nFigure 1. In panel d-e, RNAscope is performed to confirm that RNase4 expression is selective of nociceptors. However, the quantification is performed by defining nociceptors solely based on their smaller soma size. C-LTMR are also of small diameter, which could bias the quantification. Therefore, further analysis is needed using bona fide markers of nociceptors, C-LTMR or myelinated LTMR subtypes (TrkA, CGRP, SST, TH, NF200, …). These, with the already performed costaining with PV, should also be quantified to allow the reader to evaluate the specificity of Rnase4 expression in situ.\nPanel b suggests that RNase4 is found in a small fraction of the entire nociceptive population. Whether this observation can be replicated in situ is also important to demonstrate before diving into subsequent results.\nPanel f shows that RNase4 also exists in human. The meaning of the indicated values in the graph is unclear.\nFigure 2. Panel b showing expression of RNase4 in section of adult mice appears redundant with the image shown in panel c.\nPanel e shows that many genes are misregulated in adult cKO-BR mice. Among the DEG identified, the authors cite Cpeb3 as a downregulated target whose deficiency is described in the literature to increase TrpV1 expression, however TrpV1 is not found among DEG in this study. Functional components of the nociceptors such as potassium voltage-gated channels are also misregulated, which is likely to impact the electrophysiological properties of nociceptors, but the behaviour tests in panel g show that cKO-BR mice behave as controls. Could it indicate that the cut-off chosen to define DEG was too loose?\nThe authors could examine by IF/ISH the expression of some of the identified DEG in cKO-BR mice to validate these scRNAseq data.\nIn panel f, genes such as Kcnf1 or Hcn3 appear deregulated in cLTMR neurons, and Kcng3 in PEP2 nociceptors. However, RNase4 appear not expressed in these cell types (Fig.1c and 5a). This should be mentioned here as RNAse4 is reported to act also as a secreted molecule and may thus have cell-autonomous and non-cell autonomous effects, and as the authors suggest it is the case during nerve injury.\nIn panel g, the authors show that mechanical and thermal sensitivity is unaltered in cKO-BR mice. The significance of this observation is hard to evaluate as RNase4 expression during development has not been approached.\nIn the very last sentence of the introduction (line 71), the authors state that the identity of nociceptors is affected. While transcriptional alterations are observed in cKO-BR, whether they are linked to a switched or aberrant identity is not demonstrated.\nFigure 3. The Prdm12CreERT2 line is presented as a line useful to conditionally selectively delete RNase4 in nociceptors at adulthood. However, as previously reported by the authors, scRNA-seq data in panel 3c show that Prdm12 is also expressed in C-LTMR. Co-staining of the tdTomato-reporter with a marker of C-LTMR should thus be performed and quantified to appreciate the degree of overlap within the C-LTMR neurons at adulthood. Similarly, as the scRNA-seq data reveal Prdm12 expression in NF1, co-staining with a NF marker would be useful to validate this unexpected observation.\nIn panel d, a Prdm12CreERT2;Ai14 E15,5 embryo whole-mount immunostained for tdTomato and TrkA is shown. As this procedure does not allow to fully appreciate the degree and specificity of the overlap between tdTomato and TrkA, a quantified analysis of this overlap on DRG cross-sections could be shown in addition to fully validate this Prdm12CreERT2 line.\nIn panels 3e and 3f, the authors show that Tomato staining is found in the spinal cord dorsal horn which receives nociceptive and mechanoreceptive inputs in dedicated lamina regions. To demonstrate that the Tomato-stained fibres selectively terminate in the dorsal horn region receiving nociceptive afferents, co-staining with appropriate lamina marker (PKCγ, IB4, …) should be performed.\nIn panel h, RNAscope images of DRG sections of adult cKO-PR mice are shown. Given the low level of recombination observed following tamoxifen injection (42.5%, panel g), the decrease of RNAse4 staining observed in this cKO-PR line following tamoxifen injection should be quantified.\nIn panel i, the authors assessed mechanical sensitivity using the Von Frey test, and thermal pain using the Hargreaves test. Their results show that cKO-PR mice exhibit a transient increase sensitivity to mechanical stimuli 7d days post tamoxifen injection. Why has the Hargreaves test, which is not described in the text, been performed at 30d post-tamoxifen injection and not 7d?\nFigure 4. In panel a-c, using the iPain database they have established previously, the authors show that RNase4 expression increases during the first week post-injury in the nerve crush injury model. Such finding would be stronger if experimentally validated.\nIn panels e-j, the authors perform Von Frey test and Hargreaves test following nerve crush injury on cKO-BR and cKO-PR mice. They show that both cKO and control mice exhibit as anticipated a reduction in sensory function following injury and that the absence of RNase4 accelerates the recovery in sensory function compared to controls. The results of the Hargreaves test show opposite trends at 14 days after injury. The authors should describe and comment this result in the text.\nFigure 5. ScRNA-seq data in panel a show that in trigeminal neurons RNase4 is excluded from myelinated PEP2 nociceptive neurons. ScRNA-seq data from DRG neurons shown in Fig.1a also suggest this pattern of expression. These data would be stronger if validated in situ.\nWhen characterizing by EM sciatic nerve features of control and cKO-PR mice 14 days after injury, the authors show in panel e that the axon diameter of neurons are smaller in the ipsilateral side, potentially due to the inclusion of newly formed, thinner axons. However, they also observe reduced axon diameter in the contralateral side, which does not allow this potential explanation to stand.\nIn panel h,I, the authors show that there is an increased presence of misfolded myelin structures in a subset (5%) of myelinated fibers in the cKO-PR mice compared to controls. Is there a selective type of myelinated fiber affected? Is it possible that these affected myelinated fibers represent Aδ-nociceptive fibers?\nPanel p shows a model of RNAse4 mode of action in nerve homeostasis and myelination, showing that it is released by the nerves to regulate nerve homeostasis and myelination. Is there any clue that RNAse4 is indeed found in the sciatic nerve environment or at least secreted by somatosensory neurons?\nDiscussion\nLine 266. The authors state that RNase4 plays a key role in maintaining the normal conductivity of unmyelinated nociceptors, however no experiment is performed to support this conclusion.\nThe discussion section extensively develops regarding the role of RNase4 in angiogenesis and its potential contribution to the nerve regeneration phenotype observed in cKO-PR mice. Could angiogenesis in the crush model in cKO-PR mice be quantified using EM images previously taken for analysis in figure 5?\nMinor points\n-Typo line 98 “Fig.1g”   “Fig.1f”\n-Typo line 103 “containg”  ”containing”\n-Typo figure 5k, remark should be replaced by remak.\n-In the methods section, lines 498 to 501 indicate that RNase4 is significantly decreased following TAM injection in cKO-PR mice, however no quantitative and statistical methods are shown to assess this reduction properly.\n-The sentence at lines 509 to 511 should be rephrased.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This study employs a multifaceted approach combining genetic knockout models, single-cell transcriptomic profiling, pain behavioral assays, and electron microscopy. These investigations culminate in the identification of RNase4 as a previously unrecognized regulator of nociceptor biology, uncovering its dual roles: (1) a cell-autonomous function in maintaining nociceptor function, and (2) non-cell-autonomous effects on the structural integrity of adjacent myelinated axons. While these findings provided a novel regulator of nociceptor biology, critical mechanistic insights and methodological and descriptive rigor are required to support and strengthen the broad claims.\nMajor concerns and recommendations\n1. Data-interpretation discrepancy\nThere is a fundamental contradiction between the textual description and graphical presentation of mechanical hypersensitivity in animal model of nerve crush injury. In lines 208-215, the manuscript states that \"Following nerve injury, cKO-BR and cKO-PR mice, along with their control littermates, exhibited the anticipated moderate and transient reduction in sensory function in response to mechanical stimuli.\", but Figure 4f and 4i demonstrate increased mechanical withdrawal thresholds (indicating hyposensitivity) in 7 days after nerve crush injury model compared to control mice. In lines 232-233, “However, the regenerated axon diameters were smaller (Fig. 5f), potentially due to the inclusion of newly formed, thinner axons”, but Fig. 5f demonstrates the number of axons. This inconsistency directly undermines the central claim regarding RNase4's role in pain modulation.\n2. Insufficient mechanism elucidation\nThe molecular mechanism by which RNase4, as an RNase, regulates nociceptor function remains unclear (e.g., target RNAs, downstream signaling). Although the authors performed scRNA-seq to find the role of RNase4 on the transcriptional phenotype of the nociceptor, they did not find the key downstream signaling due to they chose the wrong transgenic mice (Baf53bCre;Rnase4fl/fl (cKO-BR),). They should choose (Prdm12CreERT2;Rnase4fl/fl (cKO-PR)), which displays a transiently increases mechanical pain sensitivity. The authors provided a suppose that RNase5 serves as a compensatory factor for RNase4 in the discussion section. However, they do not identify the upregulation of RNase5 in cKO-BR mice. It is not even clear if RNase5 is expressed in nociceptors.\nRecommendations:\n1) Perform scRNA-seq in the nociceptor of cKO-PR mice after TAM injection for 7 days.\n2) Assess RNA stability or protein levels of key pain-related genes (e.g., TRPV1, Nav1.8) in RNase4-deficient mice (cKO-PR) to link RNase4 activity to nociceptor function.\n3) They should explore the expression of RNase5 in their scRNA-seq dataset and iPain single-cell transcriptomic atlas.\n3. Inadequate statistical power in electron microscopic analysis\nThe ultrastructural analysis of myelinated axons in Figure 5 is based on data from only two cKO-PR mice (n = 2 biological replicates). This limited sample size precludes meaningful statistical inference, and renders the claimed \"altered myelinated axon organization\" biologically unreliable.\nMinor issues\n1. The author should explore the co-expression of Rnase4 and S100b or NF200, the broad marker genes of NFs because Pvalb just expressed in a kind of NFs.\n2. In line 103, “Short Conditional intrON (SCON) cassette20 containg LoxP recombination sites” containg should be “containing”"}, "author_response": "We thank the reviewers for their constructive feedback, which has helped improve the manuscript. Detailed responses to each comment are provided below in blue, with references to the corresponding revised sections of the text.\nReviewer #1 (Remarks to the Author):\nThis manuscript identifies RNase4 as selectively expressed in unmyelinated nociceptors and\nreveals its dual role in nociceptor function and nerve homeostasis. Through an integrated\napproach combining single-cell transcriptomics, genetic mouse models, and functional assays,\nthe authors demonstrate that RNase4 regulates both nociceptor molecular identity and\ninfluences neighboring myelinated axons. While conditional deletion of Rnase4 during\ndevelopment alters gene expression profiles without affecting baseline pain sensitivity, adult-\nonset deletion causes transient mechanical hypersensitivity. Following nerve injury, Rnase4\ndeletion accelerates functional recovery and remyelination. The findings establish RNase4 as\nan important regulator of nociceptor biology with both cell-autonomous and non-cell\nautonomous effects. However, the molecular mechanisms underlying these effects remain\ninadequately explored.\nSpecific comments:\n1. The authors should perform co-localization studies using established nociceptor markers with\nRNase4 RNA FISH. Additionally, protein-level detection of RNase4 would strengthen the\nfindings, as protein is the functional entity.\nWe agree with the reviewer that further characterization of RNase cell type–restricted\nexpression can be more thoroughly explored using mRNA hybridization techniques. To this\nend, we employed RNAscope, a highly sensitive and specific in situ hybridization (ISH) assay\nthat allows spatial detection of target RNA molecules with single-cell resolution RNAScope.\nThe following text has been added to the manuscript from line 90 to 100.\n“To further characterize this cell type–restricted expression, we performed RNAscope on cross-\nsections of dorsal root ganglia (DRG) from adult animals (Fig. 1). Co-labeling used established\nmarkers: Calca (peptidergic nociceptors, PEPs), SST (somatostatin-positive neurons), TH\n(cLTMRs), NF200/NF-H and PV (myelinated neurofilament neurons, NFs), peripherin (Prph;\nsmall-diameter, unmyelinated neurons), and Prdm12 (nociceptor lineage marker).\nRNase4 showed complete overlap with SST+ neurons and partial colocalization (~40-50%)\nwith Calca+ peptidergic neurons, while TH+ cLTMRs, which share a developmental\nnociceptive lineage but mediate pleasant touch, were entirely RNase4-negative (Fig. 1d-f).\nMinimal overlap occurred with NF200+ populations and none with PV+ myelinated neurons,\nwith RNase4 enriched in Prdm12+/peripherin+ small-diameter neurons. These findings\nestablish RNase4 as a selective marker of specific unmyelinated nociceptor subtypes.”\n2. In Fig 1g, comparative expression data showing differential expression between neuronal\nsubtypes or tissues would provide better context for RNase4 specificity.\nIn the previous version of our manuscript, the human DRG dataset was sufficient for bulk-level\nanalysis but did not allow for deconvolution into single cells into cell types. However, the\nrecently published single-nucleus DRG atlas (Bhuiyan et al., bioRxiv 2025, Figure 1) does\ncapture RNASE4 expression in human DRG neurons, confirming its presence and distribution\namong cell-types. Those data recapitulate the mouse RNase4 expression pattern we have\ncharacterized.\nThe following text has been added to the manuscript from line 101 to 104:\n“Bulk RNA-seq analysis of human dorsal root ganglia (DRG)19 confirmed RNase4 expression\nin human tissue, with a selective nociceptor-enriched pattern evident in recent single-cell atlas\nof human somatosensory neurons20 (Fig. 1k,l). These findings demonstrate evolutionary\nconservation of RNase4 expression across species, suggesting an important role in nociceptor\nbiology.”\nFor tissue-level expression, the Human Protein Atlas also reports RNASE4 diverse tissues\nRNASE4 protein expression summary - The Human Protein Atlas.\n3. Fig 2b and 2c rely on Nissl staining, which labels all neurons. Using DRG neuron-specific\nmarkers would provide better characterization of the cells expressing RNase4.\nThis comment has now been addressed and the results are presented in Figure 1.\n4. The authors highlight differentially expressed genes in Figures 2e-f but fail to present\ncomprehensive functional enrichment analysis of these genes. The manuscript mentions\nalterations in zinc finger proteins, transcription factors, and immune response pathways, but\nthese findings are not adequately presented in figures or supplementary data.\nThe authors apologize for the omission and now present in the supplementary file an enrichment\nanalysis.\n5. In Fig 2g, the nature of individual data points is unclear, and there appears to be inconsistency\nin the number of data points between left and right panels.\nWe are grateful to the reviewer for noticing this, this has now been corrected.\n6. The authors should examine whether RNase4 expression changes during the course of\nbehavioral testing (von Frey and Hargreaves tests), which would help connect molecular\nchanges with behavioral outcomes.\nThe authors have addressed this point from line 227 to 241 (Fig. 6a-c). Using the iPain atlas,\nwe report RNASE4 expression dynamics after nerve injury across 6 timepoints up to 28 days\npost-injury. In parallel, longitudinal behavioral assessments (von Frey for mechanical\nsensitivity, Hargreaves for thermal sensitivity) at 0, 7, 14, and 30 days pre- and post-RNASE4\nablation (Fig. 3h) directly link the changes in RNASE4 expression with alterations in sensory\nthresholds.\n7. Regarding the observed thicker myelin and smaller axon diameter in contralateral nerves of\ncKO-PR mice (Fig. 5c-f), it is important to clarify whether these are developmental effects or\nconsequences of adult Rnase4 deletion. If the latter, the time course of these structural changes\nfollowing deletion should be determined.\nFor the cKO-PR mice, RNASE4 deletion is temporally controlled and was induced in\nadulthood. In this study, recombination was performed in adult mice prior to injury, and the\nnerves were examined two weeks post-injury. Therefore, the structural changes observed in\nboth ipsilateral and contralateral nerves arise from acute adult deletion rather than\ndevelopmental effects.\nLonger or shorter time-course experiments were not feasible due to animal-number restrictions,\nbut the current design isolates the effect of adult RNASE4 loss.\n8. The authors propose a compensatory relationship between RNase4 and RNase5 (angiogenin)\nin nerve regeneration but provide no experimental evidence. Examining RNase5 expression in\nRnase4 knockout conditions would substantiate this hypothesis.\nWe thank the reviewer for this comment. We performed the suggested experiment and did not\ndetect RNase5 expression under RNase4 knockout conditions (Supplementary Fig. 6). In line\nwith this finding, we have corrected the statement suggesting compensation by RNase5.\n9. As RNase4 is described as a secreted protein, rescue experiments administering recombinant\nRNase4 would demonstrate whether it has direct protective effects and strengthen the causal\nrelationship between RNase4 and the observed phenotypes.\nWe thank the reviewer for this insightful suggestion. Rescue experiments using recombinant\nRNase4 would indeed provide an important test of causality. However, due to limited animal\navailability and the need for careful optimization of dosing and delivery, these experiments\ncould not be included in the present study. We agree that this represents an important direction\nfor future investigation and plan to pursue this approach in future work.\n10. The study lacks mechanistic explanation: Does RNase4 function through its enzymatic\nactivity or through other molecular pathways? Is it modulating known signaling pathways such\nas AXL?\nWe thank the reviewer for this important point. We have now investigated PI3K, AKT, mTOR\nand AXL in cell culture, treated with either inhibitor of the signaling pathways with and without\nRnase4.\nThe following text has been added to the manuscript from line 192 to 225:\n“RNase4 cell non-autonomous action: modulation of PI3K–AKT–mTOR signaling\nTo determine how RNase4 (R4), when present in the extracellular environment, influences\nintracellular signaling in surrounding cells, we treated ND7/23 cells with RNase4 alone or in\ncombination with inhibitors of the PI3K/Akt/mTOR pathway. We assessed phosphorylation of\nAkt (Ser473) and mTOR (Ser2448) as readouts of pathway activity28.\nIn the first experimental dataset (Fig. 5a), inhibition of Akt (MK-2206) and PI3K (LY294002)\nmarkedly reduced p-Akt levels compared with vehicle control, confirming effective pathway\nsuppression. When ND7/23 cells were co-treated with RNase4, p-Akt levels remained similarly\nreduced in all inhibitor conditions, indicating that RNase4 does not override pharmacological\npathway blockade. Notably, RNase4 decreased p-Akt/Akt ratios relative to DMSO alone,\nsuggesting that extracellular RNase4 can dampen basal Akt activation. This reduction\ncorresponds to an estimated 60% (Fig. 5a,b) (P=0.0072; n=3 biological replicates) decrease in\nthe p-Akt/Akt ratio, indicating a significant inhibitory effect of RNase4. Interestingly, while\nLY294002 robustly and significantly suppressed p-Akt levels, wortmannin led to a decrease of\np-Akt level though it was not significant, and lead to a moderate suppression of around 65%\n(not significant) when associated with Rnase4 (Fig. 5b), despite targeting the same pathway.\nFurthermore, no significant effect of inhibitor rapamycin alone was found at any concentration\nthough surprisingly the Akt levels increased by 40% in line with rapamycin-mediated inhibition\nof mTORC1 can activate PI3K/Akt signaling by relieving a negative feedback loop29, which\nmay explain the upward trend in Akt levels observed in the same experiment. The addition of\nRNase4 to rapamycin treated cells diminished p-Akt levels to an estimate of 60% compared to\nrapamycin only (P=0.011 for 5nM condition and P=0.018 for 10nM condition, Fig. 5b). These\ndata indicate that RNase4 exerts a consistent Akt-suppressive effect on ND7/23 cells.\nInvestigating the effect of RNase4 on mTOR phosphorylation, particularly at Ser2448 (Fig. 5c-\nd), we found a subtle decrease of around 20%, though not significant. Expectedly, mTOR level\nwas found to be significantly reduced (P= 0.0082) by the PI3K/Akt pathway inhibitor\nLY294002 (Fig. 5c-d). βIII-tubulin levels were comparable across all conditions (Fig. 5e–f),\nverifying equal protein loading and supporting the reliability of the phosphorylation analyses.\nNotably, while Akt and mTOR expression were detectable, Axl receptor protein, a known\nPI3K–Akt–mTOR modulator, was undetectable in both ND7/23 cells and primary sensory\nneuron cultures (Supplementary Fig. 4), indicating that the effects of RNase4 on this pathway\nin the DRG-derived context are Axl‑independent. Taken together, our results demonstrate that\nRNase4 may also exert its effect through modulating the PI3K–AKT–mTOR pathway in the\nDRG, in line with its previously reported effects in cancer cells30, but its effects here may not\nbe through the Axl receptor.”\nWe hope that the reviewer finds the revised manuscript satisfactory and suitable for publication\nin Nature Communications.\nReviewer #2 (Remarks to the Author):\nRNase4 is known to be involved in a broad range of biological processes, including the\nregulation of intra- or extra-cellular RNA metabolism as well as antiviral, antibacterial, and\nantifungal activities, neurogenesis, angiogenesis, immune modulation and host defence, is\nselectively expressed in neurons of the nociceptive lineage. In this manuscript, the authors\nreport that RNase4 is selectively expressed in sensory ganglia in the unmyelinated nociceptive\nlineage. They analysed the consequences of its loss of function using both a conditional KO\nand an inducible cKO strategy with a novel Prdm12CreERT2 line they have generated and may\nconstitute a useful new tool in the field. Although the results obtained provide first evidence for\na role of RNase4 in nociceptors and suggest its absence contributes to faster recovery after\nperipheral injury, we feel the study at the present stage substantially lacks quantified analyses\nto support working models and findings. Several claims lack unequivocal demonstration,\nparticularly with regards to segregation between RNase4 autonomous and non cell-autonomous\nfunctions.\nMajor concerns and specific points that could be addressed to strengthen the study are as follow:\nFigure 1. In panel d-e, RNAscope is performed to confirm that RNase4 expression is selective\nof nociceptors. However, the quantification is performed by defining nociceptors solely based\non their smaller soma size. C-LTMR are also of small diameter, which could bias the\nquantification. Therefore, further analysis is needed using bona fide markers of nociceptors, C-\nLTMR or myelinated LTMR subtypes (TrkA, CGRP, SST, TH, NF200, …). These, with the\nalready performed costaining with PV, should also be quantified to allow the reader to evaluate\nthe specificity of Rnase4 expression in situ.\nPanel b suggests that RNase4 is found in a small fraction of the entire nociceptive population.\nWhether this observation can be replicated in situ is also important to demonstrate before diving\ninto subsequent results.\nWe appreciate the reviewer’s critical evaluation and agree that defining nociceptor populations\nsolely based on smaller soma size can introduce quantification bias, as C-LTMRs are also of\nsmall diameter. To strengthen the study, we have performed further RNAscope analysis using\nbona fide molecular markers for distinct DRG neuron subtypes.\nThe following text has been added to the manuscript from line 90 to 100:\n“To further characterize this cell type–restricted expression, we performed RNAscope on cross-\nsections of dorsal root ganglia (DRG) from adult animals (Fig. 1). Co-labeling used established\nmarkers: Calca (peptidergic nociceptors, PEPs), SST (somatostatin-positive neurons), TH\n(cLTMRs), NF200/NF-H and PV (myelinated neurofilament neurons, NFs), peripherin (Prph;\nsmall-diameter, unmyelinated neurons), and Prdm12 (nociceptor lineage marker).\nRNase4 showed complete overlap with SST+ neurons and partial colocalization (~40-50%)\nwith Calca+ peptidergic neurons, while TH+ cLTMRs, which share a developmental\nnociceptive lineage but mediate pleasant touch, were entirely RNase4-negative (Fig. 1d-f).\nMinimal overlap occurred with NF200+ populations and none with PV+ myelinated neurons,\nwith RNase4 enriched in Prdm12+/peripherin+ small-diameter neurons. These findings\nestablish RNase4 as a selective marker of specific unmyelinated nociceptor subtypes.”\nPanel f shows that RNase4 also exists in human. The meaning of the indicated values in the\ngraph is unclear.\nThis has now been addressed (Figure 1k in the updated manuscript)\nFigure 2. Panel b showing expression of RNase4 in section of adult mice appears redundant\nwith the image shown in panel c.\nPanel has been removed.\nPanel e shows that many genes are misregulated in adult cKO-BR mice. Among the DEG\nidentified, the authors cite Cpeb3 as a downregulated target whose deficiency is described in\nthe literature to increase TrpV1 expression, however TrpV1 is not found among DEG in this\nstudy. Functional components of the nociceptors such as potassium voltage-gated channels are\nalso misregulated, which is likely to impact the electrophysiological properties of nociceptors,\nbut the behaviour tests in panel g show that cKO-BR mice behave as controls. Could it indicate\nthat the cut-off chosen to define DEG was too loose? The authors could examine by IF/ISH the\nexpression of some of the identified DEG in cKO-BR mice to validate these scRNAseq data.\nThe changes identified in the single-cell data were confirmed using RNAscope for the\npotassium ion channels Kcnf1 and Kcng3 (Figure 2f in the updated manuscript). We agree with\nthe reviewer that the behavioral results do not necessarily reflect the transcriptional changes.\nWe also added the corresponding text 130 to 145:\n“The analysis also highlighted notable changes in cytoskeleton-related proteins and ribosomal\ncomponents (Supplementary Fig. 1a,b). Alterations were observed in microtubule-binding\nmotor protein Dync2h1, Mapt (microtubule-associated protein tau), and tubulin variant Tubd1.\nFurthermore, eleven ribosomal proteins were affected, including Mrpl4, Rpl9, Rps10, Rps27,\nand Mrps1. Significant changes were also noted in translation-related factors such as Eif2s1 (a\ntranslation initiation factor), Gspt1 (a translation termination factor), and Cpeb3, a negative\nregulator of translation with known function on Trpv1 expression24. These findings indicate\nwidespread modifications in cellular architecture and protein synthesis machinery as a response\nto conditional mutation. Finally, focusing on ion channels within nociceptors, we identified\ndifferences in expression levels of potassium voltage-gated ion channels Kcnf1 (Kv5.1) and\nKcng3 (Kv6.3) (Fig. 2e, f). These channels are integral to the potassium channel machinery that\nregulates membrane potential and facilitates the return to a negative resting potential following\naction potential. Their reduction in RNase4 cKO neurons may affect Kv channel composition\nand potentially influence neuronal excitability25,26. Interestingly, RNase4 deletion altered gene\nexpression in cLTMRs despite the absence of RNase4 in this population, an observation that\ncould be consistent with paracrine effects of RNase4 on neighboring cells.”\nIn panel f, genes such as Kcnf1 or Hcn3 appear deregulated in cLTMR neurons, and Kcng3 in\nPEP2 nociceptors. However, RNase4 appear not expressed in these cell types (Fig.1c and 5a).\nThis should be mentioned here as RNAse4 is reported to act also as a secreted molecule and\nmay thus have cell-autonomous and non-cell autonomous effects, and as the authors suggest it\nis the case during nerve injury.\nWe thank the reviewer for this constructive comment and have now introduced the secreted\nnature of RNase4 earlier in the text from line 143 to 145.\n“Interestingly, RNase4 deletion altered gene expression in cLTMRs despite the absence of\nRNase4 in this population, an observation that could be consistent with paracrine effects of\nRNase4 on neighboring cells.”\nIn panel g, the authors show that mechanical and thermal sensitivity is unaltered in cKO-BR\nmice. The significance of this observation is hard to evaluate as RNase4 expression during\ndevelopment has not been approached.\nWe thank the reviewer for this constructive comment. We had attached the expression profile\nof Rnase4 during development as shown in the plots below:\nPanel legend: The expression dynamics of Rnase4 and Baf (Actl6b) along the embryonic days\nextracted from Faure et al., 2020.\nPanel legend: The expression dynamics of Rnase4 and Baf (Actl6b) along the embryonic days\nextracted from Sharma et al., 2020.\nIn the very last sentence of the introduction (line 71), the authors state that the identity of\nnociceptors is affected. While transcriptional alterations are observed in cKO-BR, whether they\nare linked to a switched or aberrant identity is not demonstrated.\nWe thank the reviewer for pointing this out and have removed the sentence to avoid\noverinterpretation.\nFigure 3. The Prdm12CreERT2 line is presented as a line useful to conditionally selectively\ndelete RNase4 in nociceptors at adulthood. However, as previously reported by the authors,\nscRNA-seq data in panel 3c show that Prdm12 is also expressed in C-LTMR. Co-staining of\nthe tdTomato-reporter with a marker of C-LTMR should thus be performed and quantified to\nappreciate the degree of overlap within the C-LTMR neurons at adulthood. Similarly, as the\nscRNA-seq data reveal Prdm12 expression in NF1, co-staining with a NF marker would be\nuseful to validate this unexpected observation.\nWe now provide data showing co-localization of RFP with TH (C-LTMRs) and absence of co-\nlocalization with PV (Supplementary Figure 2 in the updated manuscript).\nIn panel d, a Prdm12CreERT2;Ai14 E15,5 embryo whole-mount immunostained for tdTomato\nand TrkA is shown. As this procedure does not allow to fully appreciate the degree and\nspecificity of the overlap between tdTomato and TrkA, a quantified analysis of this overlap on\nDRG cross-sections could be shown in addition to fully validate this Prdm12CreERT2 line.\nWe now show that TrkA co-localizes with RFP in DRG cross-sections, confirming the overlap\n(Fig. 3d).\nIn panels 3e and 3f, the authors show that Tomato staining is found in the spinal cord dorsal\nhorn which receives nociceptive and mechanoreceptive inputs in dedicated lamina regions. To\ndemonstrate that the Tomato-stained fibers selectively terminate in the dorsal horn region\nreceiving nociceptive afferents, co-staining with appropriate lamina marker (PKCγ, IB4,…)\nshould be performed.\nWe agree that defining the laminar termination is important. We have now performed co-\nstaining of RFP fibers with IB4 and CGRP (Fig. 3e).\nThe corresponding text was added in the manuscript from line 161 to 164.\n“RFP+ cells and fibers show clear exclusion with PV (Fig. 3d) and co-localization with TrkA\n(Fig. 3d), IB4 and CGRP (Fig. 3e), indicating that they terminate within laminae associated\nwith nociceptive afferents. This confirms the selective projection of Prdm12-lineage neurons to\nI-II regions of the dorsal horn.”\nIn panel h, RNAscope images of DRG sections of adult cKO-PR mice are shown. Given the\nlow level of recombination observed following tamoxifen injection (42.5%, panel g), the\ndecrease of RNAse4 staining observed in this cKO-PR line following tamoxifen injection\nshould be quantified.\nThis has now been quantified (Fig. 3g). Number of RNase4 / Nissl neurons were around 55%\nin control and 19% in cKO-PR as quantified from n = 3 mice per group, significance tests\nshowed p < 0.001.\nIn panel i, the authors assessed mechanical sensitivity using the Von Frey test, and thermal pain\nusing the Hargreaves test. Their results show that cKO-PR mice exhibit a transient increase\nsensitivity to mechanical stimuli 7d days post tamoxifen injection. Why has the Hargreaves test,\nwhich is not described in the text, been performed at 30d post-tamoxifen injection and not 7d?\nThe Hargreaves test was performed at the same timepoints as the von Frey tests, including 7\ndays post-tamoxifen, but did not show significant differences between genotypes at that\ntimepoint. Therefore, only the 30-day data are presented.\nA sentence clarifying this point has now been added to the manuscript from line 173 to 177:\n“Thermal sensitivity, assessed with the Hargreaves test, showed no genotype-dependent\ndifferences at the intermediate time points; therefore, only the 30-day Hargreaves data are\npresented. These findings suggest RNase4 contributes to adult nociceptor function, although its\nabsence causes only transient effects (likely due to compensatory mechanisms). The data also\nhint at a role for RNase4 in nociceptor homeostasis.”\nFigure 4. In panel a-c, using the iPain database they have established previously, the authors\nshow that RNase4 expression increases during the first week post-injury in the nerve crush\ninjury model. Such finding would be stronger if experimentally validated.\nWe understand the reviewer’s advice; however, the iPain resource is based on previously\npublished single-cell transcriptomic datasets generated from injured tissue, not on predictive\nmodels. These datasets originate from multiple laboratories and injury models and therefore\nalready represent experimentally derived measurements. We developed iPain in part to reduce\nduplication of animal experiments in pain research, in line with the 3R principles, and we hope\nthe reviewer supports this approach.\nIn panels e-j, the authors perform Von Frey test and Hargreaves test following nerve crush injury\non cKO-BR and cKO-PR mice. They show that both cKO and control mice exhibit as\nanticipated a reduction in sensory function following injury and that the absence of RNase4\naccelerates the recovery in sensory function compared to controls. The results of the Hargreaves\ntest show opposite trends at 14 days after injury. The authors should describe and comment this\nresult in the text.\nWe thank the reviewer for the comments.\nThe following text has been added to the manuscript from line 265 to 275:\n“Von Frey testing revealed that both cKO-BR, cKO-PR (Fig. 6f,i), and control mice displayed\nthe expected reduction in mechanical sensitivity following sciatic nerve crush, followed by a\nprogressive recovery over time. In both knockout lines, recovery of mechanical thresholds\noccurred faster than in littermate controls, indicating that loss of RNase4 facilitates restoration\nof tactile sensitivity after injury. In contrast, Hargreaves testing of thermal sensitivity showed\nan opposite trend between genotypes cKOs at 14 days post-injury, with cKO-BR mice\ndisplaying a delay in thermal recovery compared to controls while cKO-PR fully recovered.\nThis divergence suggests that RNase4 modulates regeneration of mechanical and heat pathways\nin a differential, modality-specific manner, and highlights that accelerated recovery of tactile\nfunction in the knockouts does not necessarily generalize to thermal nociception at all stages of\nthe regeneration process.”\nFigure 5. ScRNA-seq data in panel a show that in trigeminal neurons RNase4 is excluded from\nmyelinated PEP2 nociceptive neurons. ScRNA-seq data from DRG neurons shown in Fig.1a\nalso suggest this pattern of expression. These data would be stronger if validated in situ.\nAlthough we agree that in-situ validation of PEP2 neurons would be informative, this analysis\nis technically challenging because no single reliable marker labels the entire PEP2 population.\nAvailable markers such as Penk and Trpm8 identify only subsets of these neurons to our\nknowledge, which would limit the interpretability of such an experiment. Importantly, the\nexclusion pattern of RNase4 is supported by two independent scRNA-seq datasets from DRG\nand trigeminal ganglia, which already provides strong convergent evidence. We hope the\nreviewer finds this clarification satisfactory.\nWhen characterizing by EM sciatic nerve features of control and cKO-PR mice 14 days after\ninjury, the authors show in panel e that the axons diameter of neurons are smaller in the\nipsilateral side, potentially due to the inclusion of newly formed, thinner axons. However, they\nalso observe reduced axon diameter in the contralateral side, which does not allow this potential\nexplanation to stand.\nWe characterized both ipsilateral and contralateral nerves to distinguish between effects related\nto injury and those related to the conditional deletion. The reduction in axon diameter observed\non the ipsilateral side may reflect the presence of newly formed axons after injury. However,\nthe reduced axon diameter on the contralateral side indicates that this phenotype is not solely\ninjury-dependent and is instead attributed to the mutation itself.\nIn panel h, the authors show that there is an increased presence of misfolded myelin structures\nin a subset (5%) of myelinated fibers in the cKO-PR mice compared to controls. Is there a\nselective type of myelinated fiber affected? Is it possible that these affected myelinated fibers\nrepresent Aδ-nociceptive fibers?\nWe thank the reviewer for this interesting point. We examined whether a specific class of\nmyelinated fibers was selectively affected. While we cannot definitively assign subtype identity\nto the affected fibers, we analyzed the axons by diameter range. This showed that the increase\nin myelin misfolding is predominantly present in intermediate-diameter axons, in both\ngeneotypes. Therefore, the effect does not appear in uniform across all myelinated fibers. This\ncould indeed be some Aδ fibers. We have included this point in our updated manuscript (Figure\n7i).\nThe following text has now been added to the manuscript from line 293 to 296:\n“Notably, the incidence of aberrant myelin structures, including focal hypermyelination, myelin\ninfolds, and degenerating myelin, was significantly elevated in cKO-Rnase4 nerves compared\nto controls (Fig. 7h,i). This increase in myelin misfolding was predominantly observed in\nintermediate-diameter fibers, with both genotypes showing similar distributions of myelin\ndefects (Fig. 7i).”\nPanel p shows a model of RNAse4 mode of action in nerve homeostasis and myelination,\nshowing that it is released by the nerves to regulate nerve homeostasis and myelination. Is there\nany clue that RNAse4 is indeed found in the sciatic nerve environment or at least secreted by\nsomatosensory neurons?\nRNase4 is a secreted ribonuclease by structural domain, and multiple transcriptomic datasets\nshow robust expression in sensory neurons. The functional phenotype in the sciatic nerve after\nRNase4 deletion further supports its presence and role locally. We also added a new figure\n(Fig.5) showing that RNase4 activates the AKT and mTOR pathways, further supporting its\nfunctional role in nerve homeostasis.\nAccordingly, we added the following text line 307 to line 313:\n“Importantly, western blot analysis revealed increased pAkt/Akt levels upon RNase4 inhibition,\nindicating enhanced activation of the PI3K–AKT pathway (Fig.5). Given the established role\nof AKT signaling in axonal growth and regeneration31,32 these data provide a mechanistic link\nbetween RNase4 loss and the observed acceleration of axonal regrowth. Together, our findings\nsuggest that RNase4 normally restrains regenerative signaling, and that its absence promotes\naxonal regeneration, potentially through AKT-dependent pathways, while simultaneously\nperturbing Schwann cell homeostasis and myelin remodeling (Fig. 7q).”\nDiscussion\nLine 266. The authors state that RNase4 plays a key role in maintaining the normal conductivity\nof unmyelinated nociceptors, however, no experiment is performed to support this conclusion.\nWe thank the reviewer for this comment. The sentence has now been reformulated to avoid\noverinterpretation and to reflect only the experimental observations.\nThe discussion section extensively develops regarding the role of RNase4 in angiogenesis and\nits potential contribution to the nerve regeneration phenotype observed in cKO-PR mice. Could\nangiogenesis in the crush model in cKO-PR mice be quantified using EM images previously\ntaken for analysis in figure 5?\nUnfortunately, the images obtained are not suitable for an angiogenesis analysis.\nMinor points\n-Typo line 98 “Fig.1g” to “Fig.1f” Those have been addressed\n-Typo line 103 “containg” to ”containing” Those have been addressed\n-Typo figure 5k, remark should be replaced by remak. Those have been addressed\n-In the methods section, lines 498 to 501 indicate that RNase4 is significantly decreased\nfollowing TAM injection in cKO-PR mice, however no quantitative and statistical methods are\nshown to assess this reduction properly. Those have been addressed\n-The sentence at lines 509 to 511 should be rephrased. Those have been addressed.\nWe are grateful to the reviewer for their comments and hope that they are satisfied with the\nchanges made to the manuscript.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThis study employs a multifaceted approach combining genetic knockout models, single-cell\ntranscriptomic profiling, pain behavioral assays, and electron microscopy. These investigations\nculminate in the identification of RNase4 as a previously unrecognized regulator of nociceptor\nbiology, uncovering its dual roles: (1) a cell-autonomous function in maintaining nociceptor\nfunction, and (2) non-cell-autonomous effects on the structural integrity of adjacent myelinated\naxons. While these findings provided a novel regulator of nociceptor biology, critical\nmechanistic insights and methodological and descriptive rigor are required to support and\nstrengthen the broad claims.\nThe authors appreciate and are thankful for the reviewer’s efforts to improve the manuscript.\nMajor concerns and recommendations\n1.Data-interpretation discrepancy\nThere is a fundamental contradiction between the textual description and graphical presentation\nof mechanical hypersensitivity in animal model of nerve crush injury. In lines 208-215, the\nmanuscript states that \"Following nerve injury, cKO-BR and cKO-PR mice, along with their\ncontrol littermates, exhibited the anticipated moderate and transient reduction in sensory\nfunction in response to mechanical stimuli.\", but Figure 4f and 4i demonstrate increased\nmechanical withdrawal thresholds (indicating hyposensitivity) in 7 days after nerve crush injury\nmodel compared to control mice. In lines 232-233, “However, the regenerated axon diameters\nwere smaller (Fig. 5f), potentially due to the inclusion of newly formed, thinner axons”, but\nFig. 5f demonstrates the number of axons. This inconsistency directly undermines the central\nclaim regarding RNase4's role in pain modulation.\nWe believe there is a misunderstanding in the interpretation of the data. We agree that increased\nwithdrawal thresholds indicate reduced sensory function (hyposensitivity), which is the\nexpected and classical outcome of the nerve crush model. This is consistent with our results at\n7 days post-injury.\nRegarding the axon analysis, the reviewer is correct that the axon diameter was quantified at 14\ndays post-injury, not at 7 days. The reference in the previous version of the manuscript\nmistakenly pointed to Fig. 5f instead of Fig. 5e. We have now corrected the figure references\nand revised the text to be in accordance with our results.\n2. Insufficient mechanism elucidation\nThe molecular mechanism by which RNase4, as an RNase, regulates nociceptor function\nremains unclear (e.g., target RNAs, downstream signaling). Although the authors performed\nscRNA-seq to find the role of RNase4 on the transcriptional phenotype of the nociceptor,\nthey did not find the key downstream signaling due to they chose the wrong transgenic mice\n(Baf53bCre;Rnase4fl/fl (cKO-BR),). They should choose (Prdm12CreERT2;Rnase4fl/fl (cKO-\nPR)), which displays a transiently increase in mechanical pain sensitivity. The authors provided\na suppose that RNase5 serves as a compensatory factor for RNase4 in the discussion section.\nHowever, they do not identify the upregulation of RNase5 in cKO-BR mice. It is not even clear\nif RNase5 is expressed in nociceptors.\nRegarding the sequencing data, we did not choose the wrong mice. We performed sequencing\nfor both transgenic lines, but the cKO-PR dataset failed and at that time we did not have\nsufficient animals to repeat the experiment. Since the original submission, we have re-\nestablished the line and obtained enough animals to repeat the sequencing. We have now\nsuccessfully generated the cKO-PR dataset at 7 days post-tamoxifen injection and include the\nanalysis in a new Figure 4.\nRegarding RNase5, new analysis and staining were performed and are now added to the text\nfrom line 366 to 373 and the in the supplementary Fig.6.\n“Although no overt pain phenotype was observed under normal conditions in the absence of\nRNase4 when deleted during embryogenesis, a notable mechanical allodynia emerged several\ndays after acute deletion of RNase4 in adult mice. This phenotype was transient, resolving\nwithin a month, suggesting that nociceptors activate compensatory mechanisms to restore\nhomeostasis. RNase5 (angiogenin), a structurally related RNase A superfamily member with\nestablished roles in neuroprotection 36,37, was undetectable in sensory neurons (Supplementary\nFig. 6) in basal condition or in cKO-Rnase4 mice, excluding simple RNase superfamily\ncompensation. “\nRecommendations:\n1) Perform scRNA-seq in the nociceptor of cKO-PR mice after TAM injection for 7 days.\nThis experiment has been done, and we have now created a new figure with the analysis of the\ncKO PR neurons.\n2) Assess RNA stability or protein levels of key pain-related genes (e.g., TRPV1, Nav1.8) in\nRNase4-deficient mice (cKO-PR) to link RNase4 activity to nociceptor function.\nWe have performed the RNAscope for TRPV1 and could not detect any obvious variation with\nthe removal of RNAse4 in the cKO-PR.\n3) They should explore the expression of RNase5 in their scRNA-seq dataset and iPain single-\ncell transcriptomic atlas.\nWe have examined RNase5 expressions in the iPain datasets, in our single-cell RNA-seq data,\nand by RNAscope. RNase5 is not expressed in DRG neurons in control nor in CKO conditions.\nWe have added this information to the revised manuscript from line 369 to 373.\n“This phenotype was transient, resolving within a month, suggesting that nociceptors activate\ncompensatory mechanisms to restore homeostasis. RNase5 (angiogenin), a structurally related\nRNase A superfamily member with established roles in neuroprotection 33,34, was undetectable\nin sensory neurons (Supplementary Fig. 6) in basal condition or in cKO-Rnase4 mice, excluding\nsimple RNase superfamily compensation.”\n3. Inadequate statistical power in electron microscopic analysis\nThe ultrastructural analysis of myelinated axons in Figure 5 is based on data from only two\ncKO-PR mice (n = 2 biological replicates). This limited sample size precludes meaningful\nstatistical inference, and renders the claimed \"altered myelinated axon organization\"\nbiologically unreliable.\nThe reviewer notes that the ultrastructural analysis in Figure 5 was based on two biological\nreplicates. We agree that this limited the statistical power. We have now increased the number\nof animals per group (n=4 control and n=3 cKO-PR) and updated the figure and statistical\nanalysis accordingly.\nMinor issues\n1. The author should explore the co-expression of Rnase4 and S100b or NF200, the broad\nmarker genes of NFs because Pvalb just expressed in a kind of NFs.\nWe agree with the reviewer that further characterization of RNase cell type–restricted\nexpression can be more thoroughly explored using mRNA hybridization techniques. To this\nend, we employed RNAscope, a highly sensitive and specific in situ hybridization (ISH) assay\nthat allows spatial detection of target RNA molecules with single-cell resolution RNAScope.\nThe following text has been added to the manuscript from line 90 to 100.\n“To further characterize this cell type–restricted expression, we performed RNAscope on cross-\nsections of dorsal root ganglia (DRG) from adult animals (Fig. 1). Co-labeling used established\nmarkers: Calca (peptidergic nociceptors, PEPs), SST (somatostatin-positive neurons), TH\n(cLTMRs), NF200/NF-H and PV (myelinated neurofilament neurons, NFs), peripherin (Prph;\nsmall-diameter, unmyelinated neurons), and Prdm12 (nociceptor lineage marker).\nRNase4 showed complete overlap with SST+ neurons and partial colocalization (~40-50%)\nwith Calca+ peptidergic neurons, while TH+ cLTMRs, which share a developmental\nnociceptive lineage but mediate pleasant touch, were entirely RNase4-negative (Fig. 1d-f).\nMinimal overlap occurred with NF200+ populations and none with PV+ myelinated neurons,\nwith RNase4 enriched in Prdm12+/peripherin+ small-diameter neurons. These findings\nestablish RNase4 as a selective marker of specific unmyelinated nociceptor subtypes.”\n2. In line 103, “Short Conditional intrON (SCON) cassette20 containg LoxP recombination\nsites” containg should be “containing”\nThis has been changed.\nWe hope that the reviewer finds the revised manuscript satisfactory and suitable for publication\nin Nature Communications.\nRebuttal Second revision: Response to reviewer comments,\nWe would like to sincerely thank all the reviewers for their careful evaluation of our\nmanuscript, for their supportive assessment of the study, and for recognizing the interest and\nsignificance of the findings.\nREVIEWERS' COMMENTS\nReviewer#1 (Remarks to the Author):\nThe authors have adequately addressed my concerns and made appropriate revisions to\nstrengthen the manuscript. I have no further questions.\nThank you for your careful evaluation of our manuscript and for your constructive feedback\nthroughout the review process. We sincerely appreciate your time and thoughtful assessment.\nReviewer#2 (Remarks to the Author):\nWe acknowledge that the authors have effectively addressed most of our previous concerns\nregarding their study on the role of RNAse4 in nociceptor homeostasis. The inclusion of novel\nsnRNA-seq data from the cKO-PR mice, together with the evidence indicating that RNase4\nmay exert its effects through modulation of the PI3K–AKT pathway, substantially strengthens\nthe manuscript. Overall, these additions significantly enhance the impact of the study, and we\ntherefore support its publication.\nWe sincerely thank the reviewer for the careful re-evaluation of our manuscript and for the\nthoughtful and constructive feedback throughout the review process.\nWe are grateful that the reviewer recognizes the value of the newly included snRNA-seq data\nfrom the cKO-PR mice and the mechanistic insights linking RNase4 to modulation of the\nPI3K–AKT pathway. We agree that these additions significantly strengthen the study and\nclarify the role of RNase4 in nociceptor homeostasis.\nWe appreciate the reviewer’s support for publication and the engagement in improving the\nimpact of this work.\nMinor points to be fixed:\n1. Fig. 1, panels g, h, and i are not referenced in the text.\nThese panels are now explicitly referenced and described in the Results section.\n2. Fig. 1, panel j is not described and not referenced in the text.\nPanel j has now been described and appropriately referenced in the Results section.\n3. Line 168: Fig. 3h should be Fig. 3g.\nThis typographical error has been corrected.\n4. Line 197: The reasoning behind testing the effect of RNase4 on the PI3K–Akt pathway\nshould be better introduced this should be explained.\nWe have revised the text to explicitly introduce the rationale for examining the PI3K–\nAkt–mTOR pathway, clarifying why this signaling axis was tested in the context of\nextracellular RNase4 activity.\n5. Fig3, panel F. The legend of the figure indicates that this is an RNAse4 staining. This\nis not visible in this panel.\nThe figure legend has been corrected to accurately reflect the content of the panel.\n6. Fig. 7C is not referenced in the text.\nFig. 7C is now explicitly referenced and discussed in the Results section.\n7. Line 313: There is no panel “q” in Fig. 7.\nThis has been corrected, and the reference now corresponds to the correct panel.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for this information. We appreciate the transparency regarding the co-review\nprocess and fully support initiatives that promote training and recognition of Early Career\nResearchers in peer review.\nReviewer#4 (Remarks to the Author):\nThe authors have provided clear and detailed responses to each of my concerns, and they have\nmade appropriate adjustments to the manuscript. These modifications have improved the\nwork's impact and clarity while thoroughly and persuasively addressing my earlier criticisms.\nThe revisions they have made significantly improve the manuscript. I have no\nmorecomments on this work, and I believe it is now well-suited for publication in Nature\nCommunications.\nWe sincerely thank the reviewer for the thorough evaluation of our revised manuscript and\nfor the constructive feedback throughout the review process.\nWe are grateful for the positive assessment and are pleased that the revisions have improved\nthe clarity, and impact of the work. We appreciate the reviewer’s thoughtful engagement,\nwhich has helped strengthen the manuscript.\nReviewer#4 (Remarks on code availability):\nThe code provide a README file. And i could install the dependencies of this code.\nHowever, due to the absence of the original files required to run the code, it is not possible to\nconfirm whether the script can be reproduced.\nWe thank the reviewer for carefully assessing the code availability.\nWe would like to clarify that the input files are not hosted on GitHub because they are\npublicly available through established repositories. The README has now been updated to\ninclude direct links and accession numbers to the original datasets required to run the\nanalysis.\nIn addition, the scripts rely primarily on established and publicly available packages, and the\nspecific package versions used are now clearly listed to ensure reproducibility."}]

Decision letter