Sex-dependent differences in the genomic profile of lingual sensory neurons in naïve and tongue-tumor bearing mice

Abstract

Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: (1) FACS sorting obtained higher number of neurons from female trigeminal ganglia (TG) compared to males; (2) Naïve female neurons innervating the tongue expressed immune cell markers such as Csf1R, C1qa and others, that weren't expressed in males. This was validated by Immunohistochemistry. (3) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. (4) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, (5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.

Figure 1

Isolation and Estimation of Tongue-Innervating Sensory Neurons. (A, B and C) Male and Female mice were injected with 3 × 10^5 HSC3 cells in the tongue and at day 13 post-cell inoculation, tongue tissues were bilaterally injected with 1% WGA-488. Normal group received no HSC3 cells. Two days later, TG tissues were dissected to make single-cell suspension and subjected to flow sorting. Animals were grouped into male normal (MN), male tumor (MT), female normal (FN) and female tumor (FT). N = 3 samples per group. (A) Gating Strategy for flow sorting is shown. (B) Number of cells sorted for each group. Data points represent numbers of cells in each sample. (C) Percentage of WGA + neurons of total events in each group. Data points represent percentage of cells in each sample. Data are represented as mean ± SEM and analyzed by one-way ANOVA with Sidak’s post-hoc test.p < 0.05. (D and E) Naïve male and female mice were injected with 1%WGA-488 and 2 days later, TG tissues were harvested for immunohistochemistry. Images were taken at 20 × magnification using the Nikon C1 confocal microscope. N = 2 mice per group. (D) Percentage of TRPV1 + /WGA + and TRPV1-/WGA + TG neurons in naïve males and females. Each data point represents average percentage of neurons per mouse. A total of 10 images were taken per mouse. Data in bar graphs are represented as mean ± SEM and analyzed by one-way ANOVA with Sidak’s post-hoc test. (E) Representative images of WGA and TRPV1 staining of TG tissues in male and females are shown. Arrows indicate colocalization of TRPV1 and WGA.

Figure 2

Differentially Expressed Genes in Male versus Female Lingual Neurons. (A–D) Flow sorted neurons were subjected to RNA sequencing. DEGs were identified in normal male and female mice based on RPKM > 5, FC > 1.5 and p < 0.05. N = 3 per group. (A) Heatmap of genes that were expressed exclusively in tongue-innervating neurons of normal male and female mice. (B) Number of genes in males and females is depicted as bar graphs. (C) Heatmap of genes differentially expressed in female normal (FN) versus male normal (MN). (D) Bar graphs shows number of genes upregulated and downregulated in FN versus MN. (E–J) Validation of RNA sequencing data in normal male and female by Immunohistochemistry. N = 2 mice per group. Images taken with C1 Nikon Confocal Microscope at 20 × magnification. (E) Percentage of WGA + neurons expressing Csf1R in males and females. Each data point represents average percentage of neurons per mouse. 6–7 images were taken per mouse. Data are represented as mean ± SEM and analyzed by unpaired Student’s T Test at p < 0.05. (F) Percentage of Csf1R in lingual TRPV1 + and TRPV1- neurons. Each data point represents average percentage of neurons per mouse. Data are represented as mean ± SEM and analyzed by paired Student’s T Test at p < 0.05 (G) Representative images of immunostaining of Csf1R and TRPV1 in WGA + neurons in males and females. White arrows indicate colocalization of Csf1R and WGA whereas orange arrows indicate colocalization of Csf1R, WGA and TRPV1. (H) Percentage of WGA + neurons expressing C1qa in males and females. Each data point represents average percentage of neurons per mouse. 6–7 images were taken per mouse. Data are represented as mean ± SEM and analyzed by unpaired Student’s T Test at p < 0.05 (F) Percentage of C1qa in lingual TRPV1 + and TRPV1- neurons. Each data point represents average percentage of neurons per mouse. Data are represented as mean ± SEM and analyzed by paired Student’s T Test at p < 0.05 (G) Representative images of immunostaining of C1qa and TRPV1 in WGA + neurons in males and females. White arrows indicate colocalization of C1qa and WGA whereas orange arrows indicate colocalization of C1qa, WGA and TRPV1.

Figure 3

Effect of Csf1 in CAP-evoked calcium influx in male and female TG Neurons. (A), (B) and (C) Naïve female and (D), (E) and (F) naïve male mice were injected with 0.5% WGA and 2 days later, TG neuronal cultures were treated with either vehicle (Veh) or 100 ng/ml Csf1 overnight. Following treatment, neurons were recorded for 15 nM CAP-evoked calcium response. (A) and (D). Data are presented as mean ± SEM of percent neurons responsive to CAP in WGA + and WGA- neurons. Data analyzed by two-way ANOVA with Sidak post-hoc test at p < 0.05 (B) and (E). Data are presented as mean ± SEM of delta values calculated by subtracting baseline (BL) from peak CAP response in WGA + and WGA- neurons. Data analyzed by two-way ANOVA with Sidak post-hoc test at p < 0.05. (C) and (F). Representative traces of CAP response in Veh and CSF1 treated neurons is shown. Experiments were performed twice on two different days for each sex. 250–300 neurons were recorded per group.

Figure 4

Effect of tongue tumor on transcriptomic profile of lingual neurons in male and female mice. DEGs were identified by conducting two comparisons. (A). Volcanic Plots for all genes for MT versus MN comparison. DEGs identified are colored showing downregulated genes on the left and upregulated genes on the right. (B). Number of upregulated and downregulated genes are plotted as bar graph. (C). Top three upregulated and downregulated genes in MT versus MN are plotted as heatmap as well as tabulated for values of RPKM, Fold change (FC) and p-value. Data for heatmap plotted as fold change. Similarly, (D). Volcanic plots for FT versus FN. (E). Bar graph for upregulated and downregulated genes for FT versus FN. (F). Top 3 three upregulated and downregulated DEGs in FT versus FN as heatmaps and tabulated for RPKM, FC and p-values. Data in heatmap plotted as fold change. (G). Venn Diagram indicating number of overlapping and non-overlapping between MT versus MN and FT versus FN.

Figure 5

Biological Processes and Function of tongue-tumor controlled genes in males and females. Panther Pathway Analysis platform was used to elucidate biological processes from (A) DEGs upregulated in MT versus MN, (B) DEGs upregulated in FT versus FN and (C). DEGs downregulated in FT versus FN. For each analysis, the number of DEGs associated with each biological process is plotted as bar graphs. (D) Additional analyses was conducted to identify molecular function (MF) of all DEGs in males and females. Pie chart of the number of DEGs identified as transcription factors, ligands/receptors/growth factors (GFs), Channels/Receptors, Enzymes and Cytokines/Chemokines is shown. (E) Heatmap of select DEGs for each of the molecular function indicate the differences in expression between MT versus MN and FT versus FN. Data plotted as fold change. (F) Number of total DEGs obtained for each MF with MT versus MN and FT versus FN.