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The emergence of transcriptional identity in somatosensory neurons

Nature volume 577, pages 392–398 (2020)Cite this article

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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.

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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.

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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.

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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.

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Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Nikhil Sharma, Kali Flaherty, Karina Lezgiyeva & David D. Ginty

  2. Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    Nikhil Sharma, Kali Flaherty, Karina Lezgiyeva & David D. Ginty

  3. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Daniel E. Wagner & Allon M. Klein

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  1. Nikhil Sharma
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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.

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Correspondence to David D. Ginty.

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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. a–e, 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.

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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

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