Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The emergence of transcriptional identity in somatosensory neurons

Abstract

More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments1,2,3,4. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific gene expression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: scRNA-seq of developing and mature DRG sensory neurons.
Fig. 2: Transcriptional development of subtypes of DRG neurons.
Fig. 3: The unspecialized sensory neuron compartment gives rise to most or all somatosensory neuron subtypes.
Fig. 4: Pou4f2 and Pou4f3 regulate select somatosensory neuron subtype maturation.
Fig. 5: The extrinsic cue NGF is required for subtype-specific gene expression patterns.

Data availability

Sequence data from this study have been deposited in the Gene Expression Omnibus with accession code GSE139088. The scRNA-seq data are also available for browsing and analysis on reasonable request or via the HTML interface at https://kleintools.hms.harvard.edu/tools/springViewer_1_6_dev.html?datasets/Sharma2019/all.

Code availability

The computational code used in the study is available at GitHub (https://github.com/wagnerde) or upon request.

References

  1. 1.

    Abraira, V. E. & Ginty, D. D. The sensory neurons of touch. Neuron 79, 618–639 (2013).

    CAS  PubMed  Google Scholar 

  2. 2.

    Julius, D. TRP channels and pain. Annu. Rev. Cell Dev. Biol. 29, 355–384 (2013).

    CAS  PubMed  Google Scholar 

  3. 3.

    Basbaum, A. I., Bautista, D. M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Julius, D. & Basbaum, A. I. Molecular mechanisms of nociception. Nature 413, 203–210 (2001).

    ADS  CAS  Google Scholar 

  5. 5.

    Le Douarin, N. The neural crest (Cambridge University Press, 1982).

  6. 6.

    Anderson, D. J. Lineages and transcription factors in the specification of vertebrate primary sensory neurons. Curr. Opin. Neurobiol. 9, 517–524 (1999).

    CAS  PubMed  Google Scholar 

  7. 7.

    Marmigère, F. & Ernfors, P. Specification and connectivity of neuronal subtypes in the sensory lineage. Nat. Rev. Neurosci. 8, 114–127 (2007).

    PubMed  Google Scholar 

  8. 8.

    Lallemend, F. & Ernfors, P. Molecular interactions underlying the specification of sensory neurons. Trends Neurosci. 35, 373–381 (2012).

    CAS  PubMed  Google Scholar 

  9. 9.

    Kitao, Y., Robertson, B., Kudo, M. & Grant, G. Neurogenesis of subpopulations of rat lumbar dorsal root ganglion neurons including neurons projecting to the dorsal column nuclei. J. Comp. Neurol. 371, 249–257 (1996).

    CAS  PubMed  Google Scholar 

  10. 10.

    Hasegawa, H., Abbott, S., Han, B. X., Qi, Y. & Wang, F. Analyzing somatosensory axon projections with the sensory neuron-specific Advillin gene. J. Neurosci. 27, 14404–14414 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Ozaki, S. & Snider, W. D. Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord. J. Comp. Neurol. 380, 215–229 (1997).

    CAS  PubMed  Google Scholar 

  12. 12.

    Mirnics, K. & Koerber, H. R. Prenatal development of rat primary afferent fibers: II. Central projections. J. Comp. Neurol. 355, 601–614 (1995).

    CAS  PubMed  Google Scholar 

  13. 13.

    Mirnics, K. & Koerber, H. R. Prenatal development of rat primary afferent fibers: I. Peripheral projections. J. Comp. Neurol. 355, 589–600 (1995).

    CAS  PubMed  Google Scholar 

  14. 14.

    Woodbury, C. J., Ritter, A. M. & Koerber, H. R. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the “hairy” skin of newborn mice: early maturation of hair follicle afferents. J. Comp. Neurol. 436, 304–323 (2001).

    CAS  PubMed  Google Scholar 

  15. 15.

    Woodbury, C. J. & Koerber, H. R. Widespread projections from myelinated nociceptors throughout the substantia gelatinosa provide novel insights into neonatal hypersensitivity. J. Neurosci. 23, 601–610 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Usoskin, D. et al. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat. Neurosci. 18, 145–153 (2015).

    CAS  PubMed  Google Scholar 

  18. 18.

    Zheng, Y. et al. Deep sequencing of somatosensory neurons reveals molecular determinants of intrinsic physiological properties. Neuron 103, 598–616.e597, (2019).

    CAS  PubMed  Google Scholar 

  19. 19.

    Nguyen, M. Q., Wu, Y., Bonilla, L. S., von Buchholtz, L. J. & Ryba, N. J. P. Diversity amongst trigeminal neurons revealed by high throughput single cell sequencing. PLoS One 12, e0185543 (2017).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    McInnes, L., Healy, J. & Melville, J. UMAP: Uniform manifold approximation and projection for dimension reduction. Preprint at https://arxiv.org/abs/1802.03426 (2018).

  21. 21.

    Kim, J., Lo, L., Dormand, E. & Anderson, D. J. SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron 38, 17–31 (2003).

    CAS  PubMed  Google Scholar 

  22. 22.

    Britsch, S. et al. The transcription factor Sox10 is a key regulator of peripheral glial development. Genes Dev. 15, 66–78 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Ma, Q., Fode, C., Guillemot, F. & Anderson, D. J. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 13, 1717–1728 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Zurborg, S. et al. Generation and characterization of an Advillin-Cre driver mouse line. Mol. Pain 7, 66 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Cao, J. et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566, 496–502 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Blanchard, J. W. et al. Selective conversion of fibroblasts into peripheral sensory neurons. Nat. Neurosci. 18, 25–35 (2015).

    CAS  PubMed  Google Scholar 

  27. 27.

    Mayer, C. et al. Developmental diversification of cortical inhibitory interneurons. Nature 555, 457–462 (2018).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Inoue, K. et al. Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons. Nat. Neurosci. 5, 946–954 (2002).

    CAS  PubMed  Google Scholar 

  29. 29.

    Levanon, D. et al. The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons. EMBO J. 21, 3454–3463 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Chen, C. L. et al. Runx1 determines nociceptive sensory neuron phenotype and is required for thermal and neuropathic pain. Neuron 49, 365–377 (2006).

    CAS  PubMed  Google Scholar 

  31. 31.

    Yoshikawa, M. et al. Coexpression of Runx1 and Runx3 in mechanoreceptive dorsal root ganglion neurons. Dev. Neurobiol. 73, 469–479 (2013).

    CAS  PubMed  Google Scholar 

  32. 32.

    Lawson, S. N. & Biscoe, T. J. Development of mouse dorsal root ganglia: an autoradiographic and quantitative study. J. Neurocytol. 8, 265–274 (1979).

    CAS  PubMed  Google Scholar 

  33. 33.

    Lawson, S. N., Caddy, K. W. & Biscoe, T. J. Development of rat dorsal root ganglion neurones. Studies of cell birthdays and changes in mean cell diameter. Cell Tissue Res. 153, 399–413 (1974).

    CAS  PubMed  Google Scholar 

  34. 34.

    Crowley, C. et al. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76, 1001–1011 (1994).

    CAS  PubMed  Google Scholar 

  35. 35.

    Patel, T. D., Jackman, A., Rice, F. L., Kucera, J. & Snider, W. D. Development of sensory neurons in the absence of NGF/TrkA signaling in vivo. Neuron 25, 345–357 (2000).

    CAS  PubMed  Google Scholar 

  36. 36.

    Miyamoto, T. et al. Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment. Dev. Cell 3, 137–147 (2002).

    CAS  PubMed  Google Scholar 

  37. 37.

    Hu, M. et al. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev. 11, 774–785 (1997).

    CAS  PubMed  Google Scholar 

  38. 38.

    Orkin, S. H. Diversification of haematopoietic stem cells to specific lineages. Nat. Rev. Genet. 1, 57–64 (2000).

    CAS  PubMed  Google Scholar 

  39. 39.

    Soldatov, R. et al. Spatiotemporal structure of cell fate decisions in murine neural crest. Science 364, eaas9536 (2019).

    CAS  PubMed  Google Scholar 

  40. 40.

    Dasen, J. S., Tice, B. C., Brenner-Morton, S. & Jessell, T. M. A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell 123, 477–491 (2005).

    CAS  PubMed  Google Scholar 

  41. 41.

    Dasen, J. S., Liu, J. P. & Jessell, T. M. Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature 425, 926–933 (2003).

    ADS  CAS  PubMed  Google Scholar 

  42. 42.

    Briscoe, J., Pierani, A., Jessell, T. M. & Ericson, J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435–445 (2000).

    CAS  PubMed  Google Scholar 

  43. 43.

    Hoppe, P. S. et al. Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios. Nature 535, 299–302 (2016).

    ADS  CAS  PubMed  Google Scholar 

  44. 44.

    Wende, H. et al. The transcription factor c-Maf controls touch receptor development and function. Science 335, 1373–1376 (2012).

    ADS  CAS  PubMed  Google Scholar 

  45. 45.

    Ichikawa, H., Deguchi, T., Nakago, T., Jacobowitz, D. M. & Sugimoto, T. Parvalbumin, calretinin and carbonic anhydrase in the trigeminal and spinal primary neurons of the rat. Brain Res. 655, 241–245 (1994).

    CAS  PubMed  Google Scholar 

  46. 46.

    Zheng, Y. et al. Deep sequencing of somatosensory neurons reveals molecular determinants of intrinsic physiological properties. Neuron 103, 598–616.e7 (2019).

    CAS  PubMed  Google Scholar 

  47. 47.

    Bai, L. et al. Genetic identification of an expansive mechanoreceptor sensitive to skin stroking. Cell 163, 1783–1795 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Rutlin, M. et al. The cellular and molecular basis of direction selectivity of Aδ-LTMRs. Cell 159, 1640–1651 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Li, L. et al. The functional organization of cutaneous low-threshold mechanosensory neurons. Cell 147, 1615–1627 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Kobayashi, K. et al. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with aδ/c-fibers and colocalization with trk receptors. J. Comp. Neurol. 493, 596–606 (2005).

    CAS  PubMed  Google Scholar 

  51. 51.

    Rosenfeld, M. G. et al. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304, 129–135 (1983).

    ADS  CAS  PubMed  Google Scholar 

  52. 52.

    Dong, X., Han, S., Zylka, M. J., Simon, M. I. & Anderson, D. J. A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 106, 619–632 (2001).

    CAS  PubMed  Google Scholar 

  53. 53.

    Zylka, M. J., Dong, X., Southwell, A. L. & Anderson, D. J. Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family. Proc. Natl Acad. Sci. USA 100, 10043–10048 (2003).

    ADS  CAS  PubMed  Google Scholar 

  54. 54.

    Zylka, M. J., Rice, F. L. & Anderson, D. J. Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 45, 17–25 (2005).

    CAS  PubMed  Google Scholar 

  55. 55.

    Arber, S., Ladle, D. R., Lin, J. H., Frank, E. & Jessell, T. M. ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons. Cell 101, 485–498 (2000).

    CAS  PubMed  Google Scholar 

  56. 56.

    de Nooij, J. C., Doobar, S. & Jessell, T. M. Etv1 inactivation reveals proprioceptor subclasses that reflect the level of NT3 expression in muscle targets. Neuron 77, 1055–1068 (2013).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Stantcheva, K. K. et al. A subpopulation of itch-sensing neurons marked by Ret and somatostatin expression. EMBO Rep. 17, 585–600 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Mishra, S. K. & Hoon, M. A. The cells and circuitry for itch responses in mice. Science 340, 968–971 (2013).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Bautista, D. M. et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204–208 (2007).

    ADS  CAS  PubMed  Google Scholar 

  60. 60.

    McKemy, D. D., Neuhausser, W. M. & Julius, D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52–58 (2002).

    ADS  CAS  PubMed  Google Scholar 

  61. 61.

    Hockley, J. R. F. et al. Single-cell RNAseq reveals seven classes of colonic sensory neuron. Gut 68, 633–644 (2018).

    PubMed  PubMed Central  Google Scholar 

  62. 62.

    Li, C. L. et al. Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity. Cell Res. 26, 967 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Wagner, D. E. et al. Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo. Science 360, 981–987 (2018).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank all members of the Ginty laboratory for discussions and critical feedback during the course of this work. We thank L. Yap, A. Rodrigues, A. Shyer, B. Shrestha, C. Santiago, C. Harwell, D. Paul, G. Fishell, L. Orefice, L. Goodrich, M. Pecot, and R. Wolfson for feedback and critical evaluation of the data and manuscript. We thank L. Yap and M. Greenberg for providing the base construct for AAV-mediated shRNA delivery. We thank M. Greenberg for access to the NextSeq 500 sequencing platform. This work was supported by NIH grant NS97344 (D.D.G.), Howard Hughes Medical Institute–Life Sciences Research Foundation postdoctoral fellowship (D.E.W.), NIH grant 1K99GM121852 (D.E.W.), NIH grant 5R33CA212697 (A.M.K.), the Bertarelli Foundation (D.D.G.), a Fix Fund Postdoctoral Fellowship (N.S.), and the Edward R. and Anne G. Lefler Center for Neurodegenerative Disorders. D.D.G. is an investigator of the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Contributions

N.S. and D.D.G. conceived and designed the project. N.S. designed, executed and analysed all experiments with assistance and guidance from D.E.W. and A.M.K. on the STITCH/SPRING analysis. N.S., K.F. and K.L. designed, prepared, and validated AAV constructs. N.S. and D.D.G. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to David D. Ginty.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Jeremy Dasen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Quality control metrics for DRG sensory neuron scRNA-seq data and canonical correlation analysis.

a–e, Distribution of the number of genes discovered in each cell (individual points) in each population of sensory neuron (underlying violin plot) in adult mice (a), P5 (b), P0 (c), E15.5 (d), and E12.5 (e). Individual cells with fewer than 1,000 genes (considered to be low quality) or more than 10,000 genes (considered likely to be doublets) were eliminated from subsequent analysis. Individual cells with fewer than 1,000 UMIs (considered to be low quality) were excluded from subsequent analysis. f, Integration of adult/P5 (first plot), P5/P0 (second plot), P0/E15.5 (third plot), and E15.5/E12.5 (fourth plot) using canonical correlation analysis to find common sources of variation between time points. Single cells are labelled as individual points, with colour representing identified cell types and grey representing cells in the preceding time point. For n values, see Methods.

Extended Data Fig. 2 Somatosensory neuron subtype composition varies across axial levels.

a, Left, schematic representing which axial levels were quantified. Right, quantification of smRNA-FISH to determine the percentage of C6/7, T7/8, and L4/5 DRG neurons that corresponds to each transcriptionally defined somatosensory neuron subtype. Black dotted lines highlight the subtypes present at different percentages at different axial levels. b, Example images of smRNA-FISH for transcriptionally distinct somatosensory neuron subtypes in C6/7 (top), T7/8 (middle) and L4/5 (bottom) DRG. For n values, see Methods.

Extended Data Fig. 3 Dorsal root ganglia and trigeminal ganglia comprise similar subtypes of somatosensory neurons.

a, t-SNE visualization of trigeminal ganglia scRNA-seq data obtained from adult (P28–42) mice. Colours denote principal cell types and dotted circles were added to aid in visualization of principal cell types. b, Distribution of the number of genes discovered in each population of sensory neuron in adult trigeminal ganglia displayed as violin plots. c, Heat map depicting expression of genes that are enriched in somatosensory neuron subtypes resident in DRG as well as their expression levels in cognate subtype counterparts in trigeminal ganglia. d, Heat map depicting expression of genes that are enriched in somatosensory neuron subtypes resident in the trigeminal ganglia as well as their expression levels in cognate subtype counterparts in DRG. c, d, Boxes represent IQR, whiskers represent minimum and maximum values, and notches represent the 95% confidence interval of the median. *P < 0.01, two-sided Wilcoxon rank-sum test with Bonferroni correction. For n values, see Methods.

Extended Data Fig. 4 Neural crest progenitors, sensory neuron progenitors and unspecialized sensory neurons express highly distinct gene programs.

a, Heat map depicting cell cycle (S/G2/M)-associated genes for the principal subtypes identified at E11.5. b, Heat map depicting expression of genes enriched in USNs in both mature somatosensory neuron subtypes and USNs. c, Left, heat map depicting expression of genes enriched in USNs as well as their expression in NCPs and SNPs. Right, violin and box plots depicting example genes enriched in USNs. d, Left, heat map depicting expression of genes enriched in NCPs as well as their expression in SNPs and USNs. Right, violin and box plots depicting example genes enriched in NCPs. e, Left, heat map depicting expression of genes enriched in SNPs as well as their expression in NCPs and USNs. Right, violin and box plots depicting example genes enriched in SNPs. ae, Boxes represent IQR, whiskers represent minimum and maximum values, and notches represent the 95% confidence interval of the median. *P < 0.01, two-sided Wilcoxon rank-sum test with Bonferroni correction. For n values, see Methods.

Extended Data Fig. 5 Force-directed layout of putative subtype-restricted transcription factors.

a, Force-directed layout representation of DRG with expression patterns displayed for the remaining putative subtype-restricted transcription factors. b, t-SNE visualization of expression of Runx1, Runx3, Pou4f2 and Pou4f3 in the adult DRG. c, Left, smRNA-FISH for Runx1 and Runx3 in E11.5, P0 or adult DRG. For E11.5, the spinal cord and DRG are labelled as references. Right, smRNA-FISH for Pou4f2 and Pou4f3 in E11.5, P0 or adult DRG. For E11.5, the spinal cord and DRG are labelled as references. Bottom, quantification of the smRNA-FISH. For n values, see Methods.

Extended Data Fig. 6 Expression of somatosensory neuron subtype-specific genes during development.

a, Box plots representing subtype-specific genes at E12.5, E15.5, P0, P5 and adult (P28–42) for each identified somatosensory neuron subtype. Boxes represent IQR, whiskers represent minimum and maximum values, and notches represent the 95% confidence interval of the median. *P < 0.01, two-sided Wilcoxon rank-sum test with Bonferroni correction. b, Normalized line plots showing what percentage of adult levels of subtype-specific gene expression are detected at E12.5, E15.5, P0, and P5. The black line represents the median of each time point with adult being defined as 100%. Upper and lower bands represent 95% confidence intervals (defined as ±1.87 × IQR/√n, where n is sample size). For n values, see Methods.

Extended Data Fig. 7 DRG counts and TF analysis in Pou4f2 and Pou4f3 mutants.

a, Representative images of Avil smRNA-FISH from T7/8 DRG in Pou4f3WT/WT (left) or Pou4f3KO/KO (right) littermate DRG. Right of images, quantification of estimated cell count per DRG. b, Representative images of Avil smRNA-FISH from T7/8 DRG in Pou4f2KO(Cre)/WT (left) or Pou4f2KO(Cre)/KO(Cre) (right) littermate DRG. Right of images, quantification of estimated cell count per DRG. c, Box plots displaying the expression of subtype-restricted TFs in each somatosensory neuron subtype in Pou4f3WT/WT (left) or Pou4f3KO/KO (right) littermates. d, Box plots displaying the expression of subtype-restricted TFs in each somatosensory neuron subtype in Pou4f2WT/WT (left) or Pou4f2KO(Cre)/KO(Cre) (right) littermates. c, d, Boxes represent IQR, whiskers represent minimum and maximum values, and notches represent the 95% confidence interval of the median. For n values, see Methods.

Extended Data Fig. 8 Generation and validation of Bmpr1bT2a-Cre and Avpr1aT2a-Cre mouse lines.

a, Targeting strategy for inserting a T2a-Cre-TGASTOP codon; Frt-PGK:NeoR-pA-Frt cassette immediately upstream of the stop codon in Bmpr1b. b, smRNA-FISH for both Bmpr1b and GFP in Bmpr1bT2aCre AAV-CAG:FLEX-GFPP14 I.V mice to confirm the specificity and utility of the Bmpr1bT2a-Cre allele. c, Targeting strategy for inserting a T2a-Cre-TGASTOP codon; Frt-PGK:NeoR-pA-Frt cassette immediately upstream of the stop codon in Avpr1a. d, smRNA-FISH for both Avpr1a and tdTomato in Avpr1aT2a-Cre(ΔNeo) Rosa26 LSL-tdTomato/WT mice to confirm the specificity and utility of the Avpr1aT2-aCre allele. e, Top left, t-SNE representation of transcriptionally mature DRG overlaying the expression pattern of Avpr1a. Remaining images, representative immunostaining images of tdTomato and CGRP in skin sections obtained from Avpr1aT2a-Cre Rosa26LSL-tdTomato animals. f, Top left, t-SNE representation of transcriptionally mature DRG overlaying the expression pattern of Bmpr1b. Remaining images, representative immunostaining images of GFP and CGRP in skin sections obtained from Bmpr1bT2a-Cre AAV-CAG:FLEX-GFPP14 I.V animals. g, Representative immunostaining images of GFP in skin sections obtained from Pou4f2KO(Cre);AAV-CAG:FLEX-GFPP14 I.V animals. h, Quantification of ending morphology for CGRP-α (Avpr1aT2a-CreRosa26LSL-tdTomato) and CGRP-η (Bmpr1bT2a-CreAAV-CAG:FLEX-GFPP14 I.V) somatosensory neuron subtypes, as well as Pou4f2 subtypes. i, Schematic representation of the skin with the distinct morphological ending types of CGRP-α and CGRP-η neurons displayed, as well as Pou4f2 subtypes. j, Representative images of CGRP immunostaining in skin samples from 2–3-week-old Pou4f3WT/WT (left) or Pou4f3KO/KO (right) littermate controls. *P < 0.01, two-tailed t-test. k, Representative images of GFP immunostaining in skin samples from 3–4-week-old Pou4f2KO(Cre)/WT (top left) or Pou4f2KO(Cre)/KO(Cre) (right) littermates; representative RNA-FISH for GFP in Pou4f2KO(Cre)/WT and Pou4f2KO(Cre)/KO(Cre) littermate controls are displayed below the skin immunostaining images. *P < 0.01, two-way ANOVA with Tukey’s HSD post-hoc analysis (h); two-sided t-test (j, k). Bar graphs in h, j, k show mean ± s.e.m. For n values, see Methods.

Extended Data Fig. 9 Subtype-restricted TF expression profiles in Ngf−/− Bax−/− cell clusters.

a, Heat map depicting expression of the subtype-restricted TFs in P0 somatosensory neuron subtypes (left) and clusters from Ngf−/−Bax−/− mutants (right). b, smRNA-FISH for pairs of subtype-restricted TFs in Bax−/− (top) or littermate Ngf−/−Bax−/− mutants (bottom). c, Quantification of the smRNA-FISH data showing the number of Pou4f3/Shox2 double-positive, Pou4f3/Hopx double-positive, Bcl11a/Hopx double-positive, Neurod1 single-positive or Neurod6 single-positive neurons. d, Schematized model of gene expression programs as cells traverse development milestones. Transcriptionally unspecialized sensory neurons that emerge from Sox10+ and Neurog1+ progenitors co-express multiple TFs, which become restricted to select subtypes as neurons mature. These TFs are responsible for establishing the transcriptional specializations found in each neuronal subtype. c, *P < 0.01, two-sided t-test. For n values, see Methods.

Supplementary information

Supplementary Data 1

Subtype-specific genes in DRG sensory neuron subtypes.This table includes the gene name, p value (two-sided Wilcoxon rank-sum test), percentage of cells expressing the indicated gene within the subtype of interest, and percentage of cells expressing the gene outside the subtype of interest. Note that these are the genes used to populate the heatmaps in Fig. 1 and the genes are presented in the same order (top to bottom) in both the heatmaps and this table.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sharma, N., Flaherty, K., Lezgiyeva, K. et al. The emergence of transcriptional identity in somatosensory neurons. Nature 577, 392–398 (2020). https://doi.org/10.1038/s41586-019-1900-1

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing