Fig. 4

scRNA-seq analysis reveals heterogeneity of mesencephalic trigeminal nucleus (MTN) neurons. a Schematic diagram of MTN neuron preparation for scRNA-seq. b Representative fluorescence images for a couple of MTN neurons, which were retrogradely labeled with DiI (red) from the masseter muscle, in the brainstem. A scale bar: 100 µm. Insets represent the high-magnification images showing DiI (red) and Nissl staining (white). Scale bars: 20 µm. n = 1 mouse (P21). c Representative immunofluorescence images showing Advillin positivity (green) in cultured DiI-labeled MTN neurons. A scale bar: 20 µm. n = 1 mouse (P21). d Heatmap representing the distribution of two major subgroups of MTN neurons: those with high levels of both Ntrk3 and Ntrk2 (designated as Ntrk2^high) and those with high levels of Ntrk3 but low levels of Ntrk2 (designated as Ntrk2^low). e–h Heatmaps showing genes categorized according to intrinsic properties, including neurotropic factors and receptors, proprioceptor/mechanoreceptors, glutamate release and receptors, and acetylcholine receptors. Data represent the log transform of mean DESeq2-normalized counts of transcripts in each group (refer to Table S3). i–k Volcano plots showing the DE genes with a minimum log2 (fold change; FC) of 0.5 and an adjusted P-value < 0.05 that are enriched in the DPA neurons (left side) or MTN neurons (right side). For analysis, gene lists from Gene Ontology (GO) categories including sensory perception (GO:0007600), response to stimuli (GO:0050896), and a custom gene list of mechanosensitive ion channels were used. Genes of interest within the top 20 DE genes indicated with text labels), ranked by log2 FC in each GO term, are highlighted in blue (refer to Table S4)