A subpopulation of itch‐sensing neurons marked by Ret and somatostatin expression
2016; Springer Nature; Volume: 17; Issue: 4 Linguagem: Inglês
10.15252/embr.201540983
ISSN1469-3178
AutoresKalina K Stantcheva, Loredana Iovino, Rahul Dhandapani, Concepción Martínez, Laura Castaldi, Linda Nocchi, Emerald Perlas, Carla Portulano, Martina Pesaresi, Kalyanee Shirlekar, Fernanda de Castro Reis, Triantafyllos Paparountas, Daniel Bilbao, Paul A. Heppenstall,
Tópico(s)Circadian rhythm and melatonin
ResumoArticle29 February 2016Open Access Transparent process A subpopulation of itch-sensing neurons marked by Ret and somatostatin expression Kalina K Stantcheva Kalina K Stantcheva EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Loredana Iovino Loredana Iovino EMBL Mouse Biology Unit, Monterotondo, Italy Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany Search for more papers by this author Rahul Dhandapani Rahul Dhandapani EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Concepcion Martinez Concepcion Martinez EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Laura Castaldi Laura Castaldi EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Linda Nocchi Linda Nocchi EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Emerald Perlas Emerald Perlas EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Carla Portulano Carla Portulano EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Martina Pesaresi Martina Pesaresi EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Kalyanee S Shirlekar Kalyanee S Shirlekar EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Fernanda de Castro Reis Fernanda de Castro Reis EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Triantafillos Paparountas Triantafillos Paparountas IRCCS Santa Lucia, Rome, Italy Search for more papers by this author Daniel Bilbao Daniel Bilbao EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Paul A Heppenstall Corresponding Author Paul A Heppenstall EMBL Mouse Biology Unit, Monterotondo, Italy Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany Search for more papers by this author Kalina K Stantcheva Kalina K Stantcheva EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Loredana Iovino Loredana Iovino EMBL Mouse Biology Unit, Monterotondo, Italy Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany Search for more papers by this author Rahul Dhandapani Rahul Dhandapani EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Concepcion Martinez Concepcion Martinez EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Laura Castaldi Laura Castaldi EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Linda Nocchi Linda Nocchi EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Emerald Perlas Emerald Perlas EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Carla Portulano Carla Portulano EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Martina Pesaresi Martina Pesaresi EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Kalyanee S Shirlekar Kalyanee S Shirlekar EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Fernanda de Castro Reis Fernanda de Castro Reis EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Triantafillos Paparountas Triantafillos Paparountas IRCCS Santa Lucia, Rome, Italy Search for more papers by this author Daniel Bilbao Daniel Bilbao EMBL Mouse Biology Unit, Monterotondo, Italy Search for more papers by this author Paul A Heppenstall Corresponding Author Paul A Heppenstall EMBL Mouse Biology Unit, Monterotondo, Italy Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany Search for more papers by this author Author Information Kalina K Stantcheva1, Loredana Iovino1,2, Rahul Dhandapani1, Concepcion Martinez1, Laura Castaldi1, Linda Nocchi1, Emerald Perlas1, Carla Portulano1, Martina Pesaresi1, Kalyanee S Shirlekar1, Fernanda Castro Reis1, Triantafillos Paparountas3, Daniel Bilbao1 and Paul A Heppenstall 1,2 1EMBL Mouse Biology Unit, Monterotondo, Italy 2Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany 3IRCCS Santa Lucia, Rome, Italy *Corresponding author. Tel: +39 690091233; Fax: +39 690091272; E-mail: [email protected] EMBO Reports (2016)17:585-600https://doi.org/10.15252/embr.201540983 The copyright line of this article was changed on 25 August 2016 after original online publication PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::AviliDTR mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens. Synopsis This study shows that a subset of DRG neurons expressing the tyrosine kinase Ret and somatostatin function as itch receptor and mediate 5HT1f receptor agonist-induced scratching in mice. Flow cytometric analysis of peripheral sensory neurons is used to isolate multiple Ret-positive subsets. Transcriptional profiling of sensory neurons with low levels of Ret and an absence of IB4 binding reveals co-expression of somatostatin (Sst), interleukin-31 (IL-31) and serotonin receptor 5HT1f. Ablation of Sst-positive neurons reduces scratching responses to IL-31 and 5HT1f agonists in vivo. Introduction The perception of physical and chemical stimuli through the skin is initiated by peripheral sensory neurons that have their cell body in the DRG. The complexity of somatosensation is reflected by the fact that a myriad of sensations including touch, pain, itch, and temperature are recognized by the peripheral nervous system. It has long been debated whether this functional complexity arises from activation of specific subtypes of sensory neuron for each stimulus modality, or from encoding and summation of neuronal activity generated by neurons that can detect a broad range of stimuli 1. Recently, it has been proposed that the sensation of itch is a discrete sensory modality that utilizes a dedicated neuronal pathway tuned to perceive only this sensation 234. Itch, the sensation that elicits a desire to scratch, serves a protective function against potentially harmful environmental irritants 5. Although unpleasant, itch is inherently different from pain, both in its sensory quality and its behavioral outcome (scratching compared to withdrawal). The neuronal pathways that mediate itch versus pain also appear to be distinct. In the spinal cord, ablation of neurons expressing receptors for the neuropeptides, gastrin-releasing peptide (Grp) 4 or natriuretic polypeptide b (Nppb) 3, reduces itch responses to multiple pruritogens but does not affect nociceptive behavior. Similarly, subpopulations of C-fiber primary afferents are activated by pruritogens 678 and ablation or selective activation of DRG neurons positive for the Mas-related G protein-coupled receptor A3 (MrgprA3) impacts upon scratching behavior but not pain 2. Itch is further categorized by its dependence upon histaminergic or non-histaminergic mechanisms. Histamine-dependent itch is elicited through activation of the H1 receptor (HRH1) and signaling through phospholipase-β3 (PLCβ3) and the ion channel TRPV1 910. The existence of further histamine-independent pathways is supported by observations that many chronic pruritic syndromes such as atopic dermatitis are resistant to antihistamine therapy 11. Mechanistically, histamine-independent itch is likely to be mediated by activation of the ion channel TRPA1. For example, injection of the antimalarial agent chloroquine induces itch 12 via MrgprA3 receptors 13 functionally coupled to TRPA1 14, and TRPA1 is also required for itch produced by oxidative stress and leukocyte accumulation 1516. Other antihistamine-resistant itch responses include those elicited by cytokines such as interleukin-31 (IL-31) released from T cells during allergic itch 1718. IL-31 induces severe pruritus and may be a key mediator in atopic dermatitis 19. Further information of the molecular profile of itch-sensing neurons and identification of molecular markers for different subtypes of itch neuron would be valuable for understanding how different pruritogens activate itch pathways. Intriguingly, a recent study which took an unbiased approach to classify sensory neuron subtypes identified three populations of putative itch receptors 20. Each of these populations was enriched for transcripts associated with itch. However, from their gene expression profile, it was not clear whether they all signal only itch or can also contribute to other sensations such as pain. To distinguish different populations of sensory neuron and ultimately define their function, we examined the expression pattern of the glial-derived neurotrophic factor (GDNF) receptor Ret in mouse DRG. Almost every DRG neuron expresses at least one neurotrophic factor 21, and approximately 60 percent of cells are marked by Ret 22. The Ret tyrosine kinase is the signaling receptor for GDNF family ligands GDNF, neurturin, artemin, and persephin which bind via GPI-anchored co-receptors termed GFRα1–4 to initiate signaling through Ret 23. Two distinct waves of Ret expression arise during development with the first occurring prior to E11.5 and the second emerging subsequently 2224. Early Ret-positive neurons develop into rapidly adapting mechanoreceptors and express the co-receptor GFRα2 and high levels of Ret 2526. Late Ret-positive neurons form a large heterogeneous group of non-peptidergic nociceptors that can be distinguished by their binding of the plant lectin IB4 2224. A further population of Ret-positive neuron co-expresses the enzyme tyrosine hydroxylase (TH) 2728 and forms C-fiber low threshold mechanoreceptors which have been implicated in the affective component of touch 29. Finally, a fourth population of Ret-positive neurons that expresses the neurotrophin receptor TrkA in adult mice has also been described 2230. This population is rare, corresponding to around 10% of all Ret cells, and its role in vivo is unknown. We sought to determine the function of this unique population of Ret-positive sensory neuron. To this end, we took a genetic approach and generated mice where eGFP expression was driven from the Ret locus exclusively in peripheral sensory neurons 3132. We identified multiple subpopulations of Ret-positive neurons in DRG which were quantified using flow cytometry. Microarray analysis of Ret-expressing neurons that were negative for IB4 uncovered a sparse population of cells enriched in transcripts for TrkA, neuropeptides such as somatostatin (Sst), and pruritogen receptors. We validated the expression of Sst in this population using an Sst-Cre driver line and generated a new mouse line to selectively ablate these neurons in vivo. Mice displayed normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by several classes of pruritogen was significantly reduced. Thus, Ret marks a population of itch receptors characterized by their co-expression of Sst and multiple pruritogen receptors. Results Ret-eGFP expression in primary sensory neurons To examine Ret expression in the adult peripheral nervous system, ReteGFP/+ mice 31 were crossed with Avilcre/+ 32 mice to obtain heterozygote Avil-Cre::ReteGFP/+ mice. This approach allowed us to target all DRG neurons and avoid extraneous GFP expression in surrounding tissues. Heterozygous mice were viable, exhibited no overt behavioral phenotype, and displayed robust eGFP fluorescence in peripheral sensory ganglia. We investigated Ret-eGFP distribution in DRG by co-staining sections from Avil-Cre::ReteGFP/+ mice with a selection of markers for different subtypes of sensory neuron. Ret-eGFP was present in 55 percent of neurons (Fig 1M) and displayed a broad range of fluorescence intensities across different cells. We examined expression with IB4 and NF200, markers of non-peptidergic nociceptors and myelinated neurons, respectively, and observed overlap with the majority of Ret-eGFP-positive neurons (Fig 1A–D), reflecting the early and late Ret neurons described previously 2224. We further investigated Ret-eGFP co-expression with TH, a marker of C-fiber low threshold mechanoreceptors 2728. eGFP fluorescence was evident in many TH-positive neurons and these cells were not co-labeled with NF200 or IB4 (Fig 1E–L). Our analysis also indicated that a small proportion of Ret-eGFP-positive neurons were not marked by either IB4, NF200, or TH, suggesting the existence of a novel subtype of Ret-expressing neuron. We quantified the overlap between Ret-eGFP and each marker and determined that 14% of Ret-eGFP-positive neurons were negative for all markers (Fig 1N), supporting the idea that this population may reflect a functionally uncharacterized subset of primary afferent neuron. Figure 1. Ret-eGFP is expressed in multiple sensory neuron subsets A–L. Ret-positive neurons largely overlap with markers for myelinated neurons (NF200), non-peptidergic nociceptors (IB4), and C-fiber low threshold mechanoreceptors (TH). However, some Ret-positive neurons are negative for these markers (indicated by the arrows in D, H, and L) suggesting the existence of a further subset of Ret+ neurons. Scale bars, 50 μm. M. Quantification of the proportion of Ret-eGFP-positive neurons in DRG (n = 2,278 cells from three mice). N. Quantification of Ret-eGFP co-expression with other markers (n = 2,278 cells from three mice). Download figure Download PowerPoint We utilized Avil-Cre-driven Ret-eGFP expression to examine the peripheral and central projections of Ret-positive sensory neurons. In the skin, Ret-eGFP fluorescence was broadly distributed and present in free nerve endings terminating in the dermis and epidermis (Appendix Fig S1), and in lanceolate endings encircling hairs (Appendix Fig S1). Similarly in spinal cord sections, Ret-eGFP was widely expressed across the dorsal horn. This was evident as a dense plexus of expression in lamina IIo corresponding to IB4-positive non-peptidergic nociceptors (Appendix Fig S2) and more diffusely through laminae III to V overlapping with NF200-labeled mechanoreceptor inputs (Appendix Fig S2). Notably, we also detected Ret-eGFP expression immediately ventral to IB4-positive terminals that coincided with PKCγ, a marker for lamina IIi/III interneurons (Appendix Fig S2), and in lamina I co-expressed with CGRP. Thus, RET-eGFP expression distinguishes multiple populations of peripheral sensory neuron that are likely to be functionally distinct. To obtain quantitative data on the distribution of Ret-positive neuronal populations in DRG, we applied flow cytometric analysis to acutely dissociated neurons. We focused on levels of IB4 binding and native Ret-eGFP fluorescence as this would allow for quantitative measurements in live cells. In line with histological data, we observed both IB4-positive and IB4-negative populations of Ret-eGFP neuron (Figs 2A and EV1). Importantly, however, flow cytometric analysis revealed multiple well-defined subpopulations delineated by their levels of eGFP fluorescence and IB4 binding (termed Ret-eGFPLo:IB4Neg (A), Ret-eGFPHi:IB4Neg (B), Ret-eGFPLo:IB4Lo (C), Ret-eGFPHi:IB4Lo (D), and Ret-eGFPHi:IB4Hi (E)). There was a broad distribution in cell size across all populations as determined by forward scatter values, and no correlation between size and levels of IB4 binding or fluorescence (Fig 2B). We further validated flow cytometric analysis using fluorescent microscopy and observed that in both sensory neuron cultures (Fig 2C) and sections of DRG (Fig 2D) from Avil-Cre::ReteGFP/+ mice, native eGFP fluorescence varied by an order of magnitude across IB4-positive and IB4-negative cells. Figure 2. Multiple Ret-positive subpopulations as determined by flow cytometric analysis of dissociated sensory neurons from Avil-Cre::Ret+/eGFP mice A. Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret-eGFP expression and IB4 binding (also indicated in Fig EV1). There are 5 well-defined subsets, 2 of which do not bind to IB4 but display a differential level of eGFP intensity (termed Ret-eGFPLo:IB4Neg, A, and Ret-eGFPHi:IB4Neg, B, respectively). The other 3 subsets bind IB4 and display a range of eGFP intensities, termed Ret-eGFPLo:IB4Lo, C, Ret-eGFPHi:IB4Lo, D, and Ret-eGFPHi:IB4Hi, E. Percent of total events are indicated in each box. B. No correlation between the median cell size of different Ret+ subsets and eGFP intensity or IB4 binding. The graph shows the forward scatter values plotted against the normalized number of cells, displayed as the percent of Max. C, D. Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy. (C) Cultured neurons and (D) DRG section from Avil-Cre::Ret+/eGFP mice displaying native eGFP fluorescence and stained with IB4. Different levels of eGFP intensity (high, indicated by arrows, and low, indicated by arrowheads) are detected across IB4+ and IB4− cells. Scale bar, 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Avil-Cre::Ret+/eGFP DRG cell sorting strategyHierarchical gating strategy for the isolation of live Ret+ sensory neurons. DRG cells were isolated from Avil-Cre::Ret+/eGFP mice, quantified, and selected according to their size and complexity. Dead cells and immune cells were excluded using the living dye Sytox Blue and an antibody against CD45. Live sensory neurons were defined by their levels of eGFP fluorescence and IB4 binding. The bottom left plot is identical to that in Fig 2A. Download figure Download PowerPoint Transcription profiling of Ret-eGFP- and IB4-negative neurons We reasoned that by defining the molecular composition of Ret-eGFP populations, we may be able to gain clues as to their function. We focused on IB4-negative neurons because flow cytometric data indicated that these cells formed two well-defined and potentially homogeneous populations. Moreover, while one of these populations presumably corresponds to RA mechanoreceptors 2526, the other may reflect an as yet uncharacterized population of Ret-positive neurons. We performed differential microarray screening on sorted Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Neg cells and determined that these two populations do indeed cluster into distinct, homogeneous subsets. We further identified several functional markers in each population that gave a first indication as to their identity. For example, Ret-eGFPLo:IB4Neg cells were enriched in transcripts for TRPV1, CGRP (Calca), and the Ret co-receptors GFRα1 and GFRα3 (Fig 3A), while Ret-eGFPHi:IB4Neg neurons expressed significantly higher levels of Ret, GFRα2, and the mechanosensitive ion channel Piezo 2 (Fam38b) (Fig 3A). Intriguingly, neither population was enriched for TH. We thus speculate that Ret-eGFPHi:IB4Neg neurons may correspond to RA mechanoreceptors, while Ret-eGFPLo:IB4Neg cells represent a novel, functionally distinct subtype of nociceptor. Figure 3. Transcriptional profiling of Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Neg sensory neurons Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Neg neurons display distinct expression profiles. A volcano plot of fold change expression in Ret-eGFPLo:IB4Neg versus Ret-eGFPHi:IB4Neg against probability. Ret has higher expression in the Ret-eGFPHi:IB4Neg subset, which also shows an upregulation of the Ret co-receptor Gfra2 and Fam38b, encoding for the mechanosensitive ion channel Piezo 2. The Ret-eGFPLo:IB4Neg subset displays an array of molecules previously associated with itch (marked in red). Itch-associated transcripts are enriched in the Ret-eGFPLo:IB4Neg population compared to all DRG neurons. Volcano plot of fold change expression in Ret-eGFPLo:IB4Neg neurons versus sorted neurons from Avil-Cre::R26tdRFP mice against probability. Molecules linked to itch perception are significantly upregulated in the Ret-eGFPLo:IB4Neg subset. Gene expression within the Ret-eGFPLo:IB4Neg population is confirmed by triple comparison between Ret-eGFPLo:IB4Neg, Ret-eGFPHi:IB4Neg, and Avil-Cre::R26tdRFP datasets. Validation of differential microarray screening by quantitative RT–PCR. Transcripts encoding ion channels, receptors, neuropeptides, signaling molecules, and transcription factors were selected for quantitative RT–PCR. Differential expression between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations correlates with microarray analysis (n = 3). Download figure Download PowerPoint We sought to define the function of the novel Ret-eGFPLo:IB4Neg population and observed that this subset was highly enriched in molecules previously implicated in itch (Fig 3A, red dots). For example, transcripts for histamine-dependent itch mediators HRH1, PLCβ3, and TRPV1 were all differentially expressed in Ret-eGFPLo:IB4Neg neurons, as were itch-associated neurotransmitters such as Nppb 3 and neuromedin B (Nmb) 33, and the co-receptors for IL-31, Il31ra, and Osmr. However, other molecules implicated in itch such as GRP, MrgprC11, and endothelin receptors were not over-expressed in Ret-eGFPLo:IB4Neg neurons, suggesting that these cells may contribute to a subtype of itch receptor. To further investigate whether itch-associated receptor transcripts were indeed specifically expressed in Ret-eGFPLo:IB4Neg neuron, we performed a second microarray screen where we assessed differential expression in this population with respect to all DRG neurons. We sorted DRG neurons from Avil-Cre::R26tdRFP mice in which the majority of peripheral sensory neurons are marked by RFP fluorescence (Appendix Fig S3) and subjected them to microarray analysis. Similar to differential screening between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations, this dataset was also enriched in itch-associated molecules (Fig 3B). Fold change levels were lower than for comparisons with Ret-eGFPHi:IB4Neg neurons (Fig 3C), presumably reflecting the mixed molecular profile of all DRG neurons, and the fact that some transcripts such as Calca (CGRP) mark large populations of Ret-negative neurons. To validate the microarray analysis, we performed parallel quantitative RT–PCR analysis of 38 transcripts in Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations using a microfluidic platform (Fluidigm). We selected genes that represented not only itch-associated transcripts but also ion channels, signaling molecules, and transcription factors with a demonstrated role in the peripheral nervous system. In agreement with microarray data, high differential expression between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg neurons was evident for itch-related genes such as the neuropeptide Nppb, the membrane receptors Il31ra, Osmr, Cysltr2, MrgprA3, and Hrh1, and the signaling molecule Plcb3. We also observed higher expression in this population for other markers such as Ntrk1 (TrkA), Calca (CGRP), and Trpv1 transcripts, marking it as a novel population of Ret-positive neurons, as well as the ion channels Trpm6, Trpc6, Trpm2, and P2rx3 and the serotonin receptors Htr1f and Htr1a (Fig 3D and Appendix Fig S4). In the Ret-eGFPHi:IB4Neg population, we detected almost 10-fold higher expression of Ret and eGFP, validating the flow cytometry analysis. In addition, Gfra2 and the mechanosensitive ion channel Piezo 2 (Fam38b) (but not Piezo 1 (Fam38a)) were upregulated, supporting the assumption that these neurons function as RA mechanoreceptors. Intriguingly, we also observed that this population was enriched in transcripts involved in chloride transport including the GABA channel subunits α1 and γ2 (Gabra1, Gabrg2) and the putative chloride channel anoctamin 6 (Ano6) (Fig 3D and Appendix Fig S4). SstCre as a surrogate marker for Ret-eGFPLo:IB4Neg neurons In order to investigate the function of Ret-eGFPLo:IB4Neg neurons, we required a molecular tool with which to selectively manipulate this population. From the microarray and quantitative RT–PCR analysis, the neuropeptide somatostatin (Sst) was among the highest enriched transcripts in Ret-eGFPLo:IB4Neg cells, displaying a 1,126-fold higher expression compared to the Ret-eGFPHi:IB4Neg population. Moreover, an SstCre driver line is available which has been well characterized in the CNS 34. We thus examined the expression pattern of SstCre-mediated recombination in the PNS to determine whether it coincides with the Ret-eGFPLo:IB4Neg population and could be used to genetically target these cells. We first assessed the anatomical properties of SstCre-positive neurons by crossing the SstCre driver line with ReteGFP mice to generate heterozygote Sst-Cre::ReteGFP/+ mice. In DRG sections from these animals, we detected weak eGFP fluorescence in a sparse population of cells corresponding to 1.3% of all neurons (Fig 4I). We explored this expression pattern in more detail by co-staining sections with IB4, NF200, and TH (Fig 4A–H). SstCre-driven Ret-eGFP expression was most evident in neurons not expressing any of these markers (85% of all Ret-eGFP-positive cells, Fig 4J), suggesting that SstCre may indeed mediate recombination in the Ret-eGFPLo:IB4Neg population. Figure 4. SstCre-driven expression in DRG and skin A–H. SstCre-mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co-express NF200 or TH. Triple immunostaining of DRG from Sst-Cre::ReteGFP/+ mice with RetGFP (A), IB4 (B), NF200 (C), RetGFP (E), IB4 (F), and TH (G). Scale bars, 50 μm. I. Quantification of SstCre::Ret-eGFP expression in DRG (n = 8,827 cells from three mice). J. Quantification of co-expression of SstCre::Ret-eGFP with neuronal markers (n = 8,827 cells from three mice). K−M. Peripheral projections of sensory neurons from Sst-Cre::Rosa26SnapCaaX mice terminate in the hairy skin as free nerve endings. Non-innervated hair follicles are indicated by arrows. Double labeling using TMR-Star to label SNAP tag (K) and DAPI (L). Scale bars, 50 μm. Download figure Download PowerPoint To investigate whether SstCre is selective for Ret-eGFPLo:IB4Neg neurons, we crossed mice with a ubiquitous RFP reporter driven from the Rosa26 locus (Sst-Cre::Rosa26RFP mice) and performed immunohistochemistry for neuronal markers on DRG sections. We again observed a low number of RFP-positive cells (1.8% of total neurons, Fig EV2) that were mostly negative for IB4 and NF200 (87% of all Rosa26RFP-positive cells, Fig EV2). These values were not significantly different from the number of Sst-Cre::ReteGFP-positive neurons (P = 0.36 for total cells and P = 0.7 for marker negative cells) implying that SstCre does not drive substantial recombination beyond the Ret-eGFPLo:IB4Neg population. Click here to expand this figure. Figure EV2. Sst-Cre::Rosa26-driven expression in sensory neurons A–F. SstCre-mediated recombination of the Rosa26 locus drives RFP expression in a small population of sensory neurons. Triple immunostaining of DRG from Sst-Cre::Rosa26RFP/+ mice with RFP (A), IB4 (B), and NF200 (C). (E) Quantification of SstCre:: Rosa26RFPexpression in DRG. (F) Quantification of co-expression of SstCre::Rosa26RFP with neuronal markers (n = 2,592 cells from 3 mice). Scale bar, 50 μm. G–I. SNAP-tag labeling of skin from Sst-Cre::Rosa26SNAPCaaX/+ mice. Hair follicle innervation, often occurring in 3 hairs together, is occasionally observed in skin from Sst-Cre::Rosa26SNAPCaaX mice. Scale bar, 50 μm. Download figure Download PowerPoint We further examined the peripheral projections of SstCre-positive sensory neurons using a Rosa26SNAPCaaX reporter mouse line that allows for highly sensitive detection of Cre-positive cells 35. We labeled skin from Sst-Cre::Rosa26SNAPCaax mice with fluorescent TMR-Star SNAP substrate and observed prominent fluorescence mainly confined to neurons that formed free nerve endings and ran parallel to the dermal/epidermal border of the skin (Fig 4K–M). In occasional sections, we also detected a rare population of hair follicles that were innervated by SstCre-positive neurons (1.9 ± 1% of hair follicles in 3 out of 13 sections, Fig EV2). Importantly, this staining pattern was absent from skin taken from control mice not expressing SstCre. To obtain a more quantitative assessment of the degree of intersection between SstCre-positive neurons and the Ret-eGFPLo:IB4Neg population, we applied flow cytometric analysis to acutely dissociated neurons from Sst-Cre::Ret-eGFP mice. We followed an identical preparation protocol to that performed previously for Avil-Cre::ReteG
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