Figures and data
Antibodies used for histological validation of TRPV1-Cre/Ai9
Number of pools of each type used for the RNA-seq analysis
Sequences of the siRNAs used in the cultures
Primers used for qPCR analysis
Regeneration rate of peripheral neurons. A) Schematic representation of the transgenic mice and experimental design used in this study. B) Microscope images of longitudinal sections of regenerating nerves in the different Cre/Ai9 animals 7 days after injury. Images show a representative example of distal regeneration at 9 dpi. C) Quantification of the number of axons that regenerated at 12 mm (smooth bars) and 17 mm (stripped bars) at 7 or 9 dpi. Each color represents a different neuron subtype (purple: proprioceptors; blue: cutaneous mechanoreceptors; orange: nociceptors; green: motoneurons). *p<0.05, **p<0.01, ***p<0.001 vs each control group as calculated by 2−way ANOVA followed by Bonferroni’s correction for multiple comparisons. D) Relative number of regenerated axons normalized by the control number of axons in each neuron type. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as calculated by 2−way ANOVA followed by Bonferroni’s correction for multiple comparisons. Scale bar: 100 µm
Validation of the specificity of PV−Cre/Ribotag, Npy2r−Cre/Ribotag, TRPV1−Cre/Ribotag, and ChAT−Cre/Ribotag mice. A) Schematic representation of the mice and experimental design used in this experiment. B) Immunostaining against hemagglutinin (HA, green) shows the expression of tagged ribosomes in neuronal cells in the DRG (PV−Cre/Ribotag, Npy2r−Cre/Ribotag, and TRPV1−Cre/Ribotag) and in the motoneurons in the spinal cord (ChAT−Cre/Ribotag). In red, β−tubulin labels all cells in the DRG, and ChAT labels motoneurons in the spinal cord. C) Cre/Ribotag mice expresses HA (in green) in a similar pattern to the expression of TdTomato in Cre/Ai9 mice (in red). D) RT−qPCR reveals the enrichment of cell−type specific transcripts in each immunoprecipitate (Pv, Npy2r, Trpv1, ChAT) and the depletion of the glial transcript Fabp7 in immunoprecipitates from all neuron populations. E) Transcripts per million (TPM) of regeneration markers. The expression of the transcripts Gap43, Atf3, Tubb2b, and Stm2 is enriched in the immunoprecipitates from injured mice compared to control mice in all populations. Scale bar: 150 µm.
Differentially expressed genes (DEGs) vary between neuronal subtypes. A−B) Number of genes that were commonly upregulated or downregulated between populations or uniquely expressed in one of the neuron subtypes. C−F) Significantly up or downregulated genes in each population with a log2(fold change) (log2(FC)) above 4 or below −4. The genes shown in these graphs exclude those that are significantly regulated above |1| in the same direction in two or more neuron types. G−I) DEGs in groups of neuronal populations. DEGs with a log2(FC) above 2 or below −2 in cutaneous neurons (G), muscle neurons (H), or sensory neurons (I) are plotted from more upregulated to more downregulated.
Activation of relevant pathways in axon regeneration. A) Six of the most relevant Gene Ontology (GO) processes that are significantly activated by all the studied neurons after injury. B) Selection of relevant pathways enriched in neurons after injury according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. C) Some GO processes (semaphoring−plexin) and KEGG pathways (all the others) enriched in specific neuron groups. D) Examples of relevant pathways enriched in a specific neuron subtype. TGF: transforming growth factor, cAMP: cyclic adenosine monophosphate, MAPK: mitogen−activated protein kinases, VEGF: vascular endothelial growth factor, cGMP−PKC: cyclic guanosine monophosphate – protein kinase C, PPAR: peroxisome proliferator−activated receptor, AMPK: AMP−activated protein kinase, jnk: c−Jun N−terminal kinase.
Neurite extension in PV−Cre/Ai9, Npy2r-Cre/Ai9, and TRPV1-Cre/Ai9 DRG explants. A) Microscope images of the neurite outgrowth of each neuron population in explants with nerve growth factor (NGF), brain−derived neurotrophic factor (BDNF), and neurotrophin−3 (NT−3). B-D) Quantification of the longest neurite in each condition. *p<0.05 vs control as calculated by one−way ANOVA followed by Bonferroni’s multiple comparisons test (PV-Cre/Ai9 and Npy2r- Cre/Ai9) or by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (TRPV1-Cre/Ai9). D) Comparison of the increase in neurite length in proprioceptors, mechanoreceptors, and nociceptors. The increase is plotted as a fold−change vs each control to compare the effect of the neurotrophic factors. **p<0.01, ***p<0.001, ****p<0.0001 vs proprioceptors as calculated by a 2−way ANOVA and followed by a Tukey’s post hoc test. Scale bar: 200 µm.
Knockdown of Med12 in dissociated dorsal root ganglia (DRG) cultures. A) Microscope images from proprioceptors (in red) in cultures with scrambled or Med12 siRNA. βIII−tubulin was used as a pan−neuronal marker (in green). B) Med12 expression measured by qPCR and expressed as fold−change vs scrambled. C) Quantification of the neurite length in PV+ neurons (fluorescent neurons in PV−Cre/Ai9). The mean of the longest neurite and the total length per neuron in each culture are plotted. D) Quantification of neurite length in PV− neurons, labeled by βIII−tubulin. *p<0.05, ***p<0.001 as calculated by t−test. Scale bar: 100 µm.
