Combined in silico and in vivo analyses reveal role of Hes1 in taste cell differentiation

Abstract

The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction), the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP), we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1(-/) (-) mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds.

Figure 1. Colocalization of TRCSMs in early developing CVP.

Immunohistochemical analyses of PLCβ2 (green signal) and IP3R3 or gustducin (red signal) in developing taste buds in CVP from stages E17 to P5. PLCβ2 and IP3R3 or gustducin colocalize within the same cells, at least until P5. Scale bar, 10 µm.

Figure 2. In silico analysis of the upstream region of TRCSMs in mammals.

(A) Venn diagram representing the results of in silico analysis of the 5 kb upstream of TRCSM genes, including Plcβ2, gustducin, Ip3r3, Trpm5, and Ggamma13 in mouse, human, and rat. Ninety-four transcription factors were identified as putative transcription regulators. (B) Summary of the putative HES1 binding sites in the 5 kb upstream sequence of each TRCSM. The putative binding sites on the mouse, human, and rat sequences are indicated with differently colored arrows (mouse, red; human, blue; and rat, green) on the horizontal lines, which represent the 5 kb upstream sequences of the TRCSMs.

Figure 3. Binding of HES1 to TRCSM promoter sequences.

(A) Position of HES1 binding sites within the promoter regions of Plcβ2 and Ip3r3 are indicated by green squares. Arrowheads indicate primer pairs used for ChIP assays. Primer pairs p1, ip1, and ip2 amplified the DNA fragment that included HES1 binding sites, while fragments amplified by p2C and ip3C primer sets did not contain the HES1 binding sequence. (B) ChIP results using P19 embryonal carcinoma cells as chromatin substrate. HES1 antibody efficiently precipitated sequences containing Plcβ2 and Ip3r3 promoter HES1 binding sites.

Figure 4. Immunohistochemical analysis of HES1 and IP3R3 in developing CVP at P0 and W3.

HES1 immunoreactivity exhibited rather uniform distribution in CVP from P0 animals, whereas a few nuclei showed reduction of HES1 immunoreactivity. The cells with reduced nuclear HES1 immunoreactivity exhibited IP3R3 expression at P0. In W3 animals, most of the cells in the taste buds displayed cytoplasmic localization of HES1, suggesting that it was nonfunctional as a transcription regulator. The very few cells retaining HES1 in the nucleus are indicated by white arrows; these cells did not express IP3R3 or SNAP25. However, some of the cells with reduced HES1 reactivity in the nucleus expressed IP3R3, SNAP25, or blood type H antigen (arrowheads).

Figure 5. Gene dosage effect of Hes1 on taste cell differentiation.

(A) CVP from E18 embryos of wild-type and Hes1 ^−/− mutant littermates were stained with antibodies against PLCβ2 and IP3R3. The developing taste buds from the oral epithelium of Hes1 ^−/− mutants (lower panels) exhibited many more PLCβ2 (green) and/or IP3R3 (red) immunoreactive cells than did their wild-type littermates (upper panels), which displayed very few PLCβ2 and IP3R3 immunoreactive cells at this stage. Scale bar, 10 µm. (B) PLCβ2 and IP3R3 immunoreactive cells in Hes1 mutant CVP at E18 and P0. Serial sections of entire CVP from wild-type, Hes1 ^+/−, and Hes1 ^−/− littermates were immunostained with the PLCβ2 and IP3R3 antibodies, and immunoreactive cells were counted. The results represent the mean of more than five specimens. (C) The table shows the average and standard deviation (S.D.) of PLCβ2 and IP3R3 immunoreactive cells at E18 and P0 obtained from sections of entire CVP from wild-type, Hes1 ^+/−, and Hes1 ^−/− littermates. The table is graphically displayed in Figure 5B. More than five specimens of each genotype and stage were used for counting the cells.

Figure 6. Schematic illustration of the transcriptional regulation of TRCSMs during differentiation of taste cells.

HES1 activity is required to maintain the undifferentiated state and to repress the transcription of TRCSMs in developing immature taste cells. Loss of Hes1 is accompanied by differentiation of taste cells expressing TRCSMs such as PLCβ2 and IP3R3.