Article Figures & Data
Figures
- Figure 1.
Transcriptomics analysis of nodose ganglion development reveals thousands of differentially expressed genes. A, Volcano plot showing the 1453 genes that significantly increased (red; adjusted p-values < 0.05, fold changes > 1.5) and 1734 genes that significantly decreased (blue; adjusted p-values < 0.05, fold changes less than −1.5) in adult male nodose ganglion samples compared with P7 samples (n = 10 P7 samples; n = 4 adult samples). B, Ingenuity pathway analysis of DEGs revealed the top canonical pathways and top upstream regulators in early postnatal versus adult samples. C, Heatmap of DEGs related to axon guidance signaling. Warmer colors indicate higher gene expression. Individual samples are represented across columns. Specific genes are organized across rows grouped using hierarchical clustering. D, Heatmap of DEGs related to synaptic LTP. E, Heatmap of DEGs regulated by BDNF. F, TMM-normalized counts of trophic factor receptors expressed in P7 nodose ganglion samples. Values highlighted in red were the top 5 highest expressed on P7 and were chosen for further testing in in vitro explant experiments.
- Figure 2.
BDNF and GDNF stimulate neurite outgrowth from cultured nodose ganglion explants. A–C, Representative images of nodose ganglion explants grown from Phox2bcre;tdTom mouse pups on P0 treated with vehicle (A), 5 ng/ml BDNF (B), or 5 ng/ml GDNF (C). Colored lines indicate average neurite lengths for each condition, as graphed in D. To focus specifically on neurite outgrowth and to reduce the effects of neurotrophic factors on neuronal survival, ganglia were collected on P0 after the peak in apoptosis (Extended Data Fig. 2-1). D, Quantification of average neurite length of nodose ganglion explants grown for 4 d with vehicle, BDNF, GDNF, NGF, or LIF. *p < 0.05 as analyzed by Kruskal–Wallis nonparametric ANOVA with Dunn’s correction for multiple comparisons. n = 6 vehicle, n = 5 BDNF, n = 5 GDNF, n = 3 NGF, n = 2 LIF. E, F, Representative images of multiplex in situ hybridization for Bdnf (green) and Gdnf (red), and DAPI counterstain (gray) in E15 mouse embryos. Dashed lines in E outline the boundaries of the right nodose ganglion. Arrow in F points to Gdnf transcript labeling in the muscular layers of the intestine.
- Figure 3.
GDNF receptor expression is enriched in Scn10a+ vagal afferent cell types that project to the GI tract. A–C, Representative images of the right nodose ganglia on E13 (A), P0 (B), and P7 (C) showing labeled Gfra1 (green) and Ntrk2 (magenta) transcripts, or the neuronal marker HuC/D (gray). D, Quantification of the proportion of neurons expressing Ntrk2 or Gfra1 on E13, P0, and P7 (n = 3 mice/group). *p < 0.05, ***p < 0.001. ns, Not significant, as analyzed by one-way ANOVA and Tukey’s correction for multiple comparisons. E, F, Representative images of labeled Scn10a (magenta), Gfra1 (green; E), and Ntrk2 (green; F) transcripts in the right nodose ganglion of P7 mouse pups. G, Quantification of the percentage of Scn10a+ and Scn10a– that coexpress Ntrk2 or Gfra1 on P7 (n = 3 mice/group). Extended Data Figure 3-1, quantification on E13, E15, and P0. *p < 0.05 as analyzed by two-sided t test. Statistics for Ntrk2 and Gfra1 were performed separately, then graphed together. H, Representative image of labeled Sst (magenta) and Gfra1 (green) transcripts in the right nodose ganglia of a P7 mouse pup. I, Quantification of the percentage of neurons expressing each cell type marker (x-axis) that coexpress Gfra1 on P7. Values for Scn10a+ and Scn10a– neurons also appear in G and are illustrated on the same graph for direct comparison (n = 3 mice/group). *p < 0.05 compared with Scn10a– percentage coexpression by one-way ANOVA and Tukey’s correction for multiple comparisons. #p < 0.05 compared with Scn10a+ percentage of coexpression by one-way ANOVA and Tukey’s correction for multiple comparisons.
- Figure 4.
Early prenatal expression of vagal afferent cell type markers becomes progressively more mature by P7. A, Quantification of all 12 transcripts measured using HiPlex RNAscope and the SCAMPR for analysis at single-neuron resolution. The mRNAs that were probed are indicated on the x-axis. The percentage of neurons expressing each transcript is indicated on the y-axis. Each point represents percentages calculated from a single mouse, in which expression was quantified from several hundred neurons in the right nodose ganglion (n = 3 mice/group; 324–795 neurons measured per sample). *p < 0.05 comparing all ages for each transcript by one-way ANOVA and Tukey’s correction for multiple comparisons. B–D, Representative images of the right nodose ganglion on E13, P0, and P7 labeled for Sst (magenta) and Gpr65 (green) transcripts and the neuronal marker HuC/D. Scale bar, 100 μm. E–G, Representative images of the right nodose ganglion on E13, P0, and P7 labeled for Calca (magenta) and Vip (green) transcripts and HuC/D. Scale bar, 100 μm. H–J, Representative images of the right nodose ganglion on E13, P0, and P7 labeled for Oxtr (magenta) and Glp1r (green) transcripts and HuC/D. Scale bar, 100 μm. For representative images of Htr3b and Cckar transcripts refer to Extended Data Figure 4-1.
- Figure 5.
Expression of some vagal afferent cell type markers continues to increase after the first postnatal week. A, TMM normalized counts of vagal afferent cell type markers expressed in P7 male and adult male and female nodose ganglion samples (n = 10 P7 male samples, n = 4 adult male samples, n = 3 adult female samples). B, Schematic depicting each cell type marker and its respective subtype definition as projecting to the stomach versus intestine, and as an IGLE versus mucosal afferent neuron type. C, TMM normalized counts of other notable cell-defining markers expressed in P7 male and adult male and female nodose ganglion samples. The markers graphed are the same ones probed in the HiPlex RNAscope experiments described in Figure 4 (n = 10 P7 male samples, n = 4 adult male samples, n = 3 adult female samples). *p < 0.05 comparing all groups by one-way ANOVA and Tukey’s correction for multiple comparisons.
Extended Data
Table 1-1
Canonical pathways identified from RNA sequencing comparing nodose ganglia from P7 and 12-week-old mice. Differentially expressed genes (in “Molecules” field of the table) related to each Ingenuity canonical pathway are listed along with -log(p-values). Download Table 1-1, XLS file.
Table 1-2
Upstream pathways identified from RNA sequencing comparing nodose ganglia from P7 and 12-week-old mice. Differentially expressed genes (in “Target molecules in dataset” field of the table) related to each upstream pathway are listed along with -log(p-values). Download Table 1-2, XLS file.
Table 1-3
DEGs identified from RNA sequencing comparing nodose ganglia from male and female 12-week-old mice. Cutoffs of fold change >1.5, false discovery rate <0.05, and TMM-normalized counts >5 were used to identify high-confidence DEGs. Download Table 1-3, CSV file.
Figure 2-1
Ontogeny of cell death in the nodose ganglion. A–D, Representative images of the right nodose ganglion on E13, E15, P0, and P7 from Phox2bcre;Tomato mice. Immunohistochemistry was used to label the apoptotic marker, activated caspase 3 (magenta), together with endogenous tdTomato labeling (green) of all VSNs. E, Quantification of the number of activated caspase-positive neurons per ganglion on E13, E15, P0, and P7 (n = 3–4 mice/group). *p < 0.05, **p < 0.01 comparing all groups by one-way ANOVA and Tukey’s correction for multiple comparisons. Download Figure 2-1, TIF file.
Figure 3-1
Neurotrophic factor receptor expression in Scn10a+ and Scn10a– neurons across development. The percentage of neurons coexpressing either Ntrk2 or Gfra1 was measured on E13, E15, and P0 (n = 3 mice/group). *p < 0.05 as analyzed by two-sided t-test at each age. Statistics for Ntrk2 and Gfra1 were performed separately, then graphed together. Download Figure 3-1, TIF file.
Figure 4-1
Early specification of subpopulations of vagal neurons expressing Htr3b. A–C, Representative images of the right nodose ganglion on E13, P0, and P7 labeled for Htr3b (magenta) and Cckar (green) transcripts and HuC/D. Scale bar, 100 μm. Download Figure 4-1, TIF file.
In this issue
