AP1 transcription factors are required to maintain the peripheral taste system

Abstract

The sense of taste is used by organisms to achieve the optimal nutritional requirement and avoid potentially toxic compounds. In the oral cavity, taste receptor cells are grouped together in taste buds that are present in specialized taste papillae in the tongue. Taste receptor cells are the cells that detect chemicals in potential food items and transmit that information to gustatory nerves that convey the taste information to the brain. As taste cells are in contact with the external environment, they can be damaged and are routinely replaced throughout an organism's lifetime to maintain functionality. However, this taste cell turnover loses efficiency over time resulting in a reduction in taste ability. Currently, very little is known about the mechanisms that regulate the renewal and maintenance of taste cells. We therefore performed RNA-sequencing analysis on isolated taste cells from 2 and 6-month-old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the activator protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells by 6 months and further declined at 12 months. We generated conditional c-Fos-knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by a decrease in proliferative marker, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance.

Figure 1

RNA-sequencing analysis of mouse CV/Fol taste cells from 2 and 6-month-old mice. (a) Schematic of RNA-sequencing workflow. Bioanalyzer profile of one of the RNA samples isolated from 6 month mice CV/Fol taste receptor cells. An RNA Integrity Number (RIN)⩾8 is recommended for RNA-sequencing library preparation. (b) Box plots of the expression RNA-seq read counts (FPKM) at the gene level and (c) the replicate genes level in CV/Fol taste cell RNA between 2 and 6-month-old mice taste cells. (d) Density plot shows the distribution of the FPKM values at the gene level in 2 and 6 month mice taste samples. (e) Variance of genes in 2 and 6-month-old mice taste samples. (f) Dispersion plots of 2 and 6-month-old mice taste samples. (g) Scatter plot shows differences in gene expression between 2 and 6-month-old mice taste cells. (h) Volcano plot identifying differentially expressed genes between 2 and 6-month-old mice taste samples (red is significant). Plots were generated using CummeRbund package v. 2.0

Figure 2

Multiple genes are differentially expressed in taste cells between 2 and 6 month age. (a) Heat map of differentially expressed genes in 2 and 6 month (Mo) mice taste cells. Higher expression is shown with darker color. (b) Plot shows the number of up- and downregulated genes in 6-month-old mouse taste cells. (c) Gene Ontology based classification of differentially expressed genes between 2 and 6 month mice. This analysis was performed using the Panther gene list analysis, molecular function. Bar graph depicts the number of genes associated with each functional category. The gene categories are also shown as pie chart (Inset). (d) String network analysis shows strong connections of AP1 transcription factors and potential target genes. The differentially expressed genes (see green/red arrows) in 6 month CV/Fol taste cells are shown in the ‘confidence view' of the string network analysis. Stronger associations are represented by thicker blue lines. (e) Relative FPKM values for c-Fos, c-Jun and Egr1 gene expression in 2, 6 and 12-month-old mice taste samples. Error bars denote s.d. of three independent experiments. c-Fos, 2–6 months (P<5.00E-05), 6–12 months (<5.00E-05), 2–12 months (<5.00E-05), c-Jun, 2–6 months (<5.00E-05), 6–12 months (NSD), 2–12 months (<5.00E-05), Egr1, 2–6 months (<5.00E-05), 6–12 months (NSD) 2–12 months (<5.00E-05). ***P<0.001

Figure 3

AP1 transcription factors downregulate in 6-month-old CV/Fol taste cells. (a) qRT-PCR analysis of the relative expression for different genes in CV/Fol taste cells from 2 and 6 month mice: c-Fos (P=0.033), c-Jun (P=0.0067), Egr1 (P=0.045). Taste cell receptor gene (Tas1r3, P>0.05), and olfactory gene (Olfr1, P>0.05). The olfactory-specific gene (Olfr1) is not expressed in taste cells and was used as a negative control. (b) qRT-PCR validation of selected genes: Lipf (P=0.0091), Lyg1 (P=0.0293), Slpi (P=0.0144), Dusp1 (P=0.011) and Zfp36 (P=0.0133) in 2 and 6 month mice. Statistical significance was determined using Student's t test. Error bars denote S.D. of three independent experiments. (c) Immunohistochemical analysis shows reduced labeling for c-Fos, c-Jun and acetylated histone H3 (AcH3) in CV taste bud in 6-month-old mouse compared with 2-month-old mice. Bar=20 μM. (d) Localization of c-Fos (red) and type I (NTPdase2, green), type II (Trpm5-GFP) and type III (GAD67-GFP) markers in CV taste papillae from 2-month-old mice. Nuclei were labeled with DAPI (blue). (e) Similarly, the localization pattern of c-Jun and different taste cell type markers. Bar=20 μM. *P<0.05; **P<0.01

Figure 4

Loss of c-Fos alters of taste bud structure. (a) c-Fos expression is reduced by 7 and 14 days post-tamoxifen treatment in c-Fos-KO taste cells. Immunohistochemical analysis shows reduced labeling for c-Fos (green) in CV taste papillae from a tamoxifen-treated c-Fos-KO mice compared with control mice. Bar=50 μM. (b) DIC image shows changes in the structural integrity of CV taste papillae in tamoxifen-treated c-Fos-KO mice after 14 days (right panel) compared with control mice (left panel). Bar=50 μM. (c) A distribution histogram of distances between the base and the tip of individual CV taste buds (see yellow arrows in b). WT and c-Fos-KO distance distributions were significantly different at 95% confidence level (non parametric Mann–Whitney test; ***P=0.0001). (d) Analysis of the number of taste receptor cells/bud in c-Fos-KO mice found a significant reduction in the number of taste cells/bud compared with controls (**P<0.01, WT=14 buds; KO=15 buds)

Figure 5

Loss of c-Fos results in reduced taste cell differentiation and increased apoptosis. (a) Relative expression of indicated genes: c-Fos (P=0.0056), Krt8 (P=0.0252), Ki67 (P=0.0202), Dusp14, Hmox1, Dcn and Ivl in CV/Fol taste cells from WT and c-Fos-KO mice. Statistical significance was determined using Student's t test. Error bars denote S.D. of three independent experiments. (b) Immunohistochemical analysis shows the expression level of cleaved-PARP in the CV taste cells of WT and c-Fos-KO mice, 14 days after tamoxifen treatment. (c) qRT-PCR analysis of taste cell type markers Glast (type I), Tas1R3 (type II) and Snap25 (type III). Error bars denote S.D. of three independent experiments. *P<0.05; **P<0.01