Expression of Claudin4 and evidence of taste pores suggests appropriate cell contacts are induced and maintained in mutant taste buds with stabilized β-catenin.

<p>Claudin4 is a tight junctional protein expressed specifically by control taste buds (A,B control). Claudin4 expression was maintained and expanded in mutant CVP taste epithelium (<b>A,</b> GOF 4 days, dotted line), and by ectopic taste buds in the FFP and non-taste epithelium (<b>B</b>, GOF 7 days, arrowheads). FFP in controls have a single taste pore shown via SEM (<b>C</b>, Control, red arrowhead), whereas in mutants treated with dox for 10 days, we encountered FFP with duplicated taste pores (<b>C</b>, GOF 10 days, red arrowhead). <i>NB</i>: red staining in the mesenchyme in <b>A</b> is due to non-specific secondary antibody binding. Nuclei were counterstained with DRAQ5 in blue. Three mice were used in each experimental group. Dotted line delineates the basement membrane. Scale bars = 20 μm in <b>A</b>,<b>B</b> and 30 μm in <b>C</b>.</p

Stabilized β-catenin increases the number of Shh⁺ precursors of all three taste cell types, but does not impact specific precursors of Type II or III cells.

<p><b>A</b>. In control mice, 1–3 Shh^<b>+</b> basal cells (green) are located in the basal compartment of each taste bud section (dashed circles), whereas in the CVP of mutant mice fed dox chow for 4 days, Shh^<b>+</b> cells (green) are increased and widespread in the expanded taste epithelium (dashed line). <b>B,B’</b>. In the anterior tongue of control mice, <i>Shh</i>^<b>+</b> cells are restricted to FFP taste buds. <b>C-F</b>. In stabilized β-catenin mutants following 7 days on dox, in addition to endogenous <i>Shh</i>^<b>+</b> cells (white arrows), ectopic <i>Shh</i>^<b>+</b> cells are detected in FFP epithelium (white arrowheads in <b>C,C’,D,E</b>), as well as in non-taste epithelium adjacent to filiform papillae (yellow arrowheads in <b>C,F</b>). MASH1 and Skn-1a are expressed in subsets of basal cells within taste buds, and regulate the Type III and Type II cell lineage, respectively. CVP sections of control and mutant mice after 4 days on dox showed that <i>Mash1</i>^{<b><i>+</i></b>} (<b>G</b>) and (<b>I</b>) Skn-1a^<b>+</b> (green) cell numbers were not altered by β-catenin stabilization. Real-time RT-PCR on the anterior tongue epithelium showed no difference in the expression of MASH1 between controls and mutants (<b>H</b>). <b>J</b>. After 7 days on dox chow, Skn-1a^<b>+</b> cells (green) were restricted to endogenous FFP taste buds in control and mutant mice (Krt8^<b>+</b> red, arrow). In mutants, Skn-1a^<b>+</b> cells were not detected in ectopic Krt8^<b>+</b> clusters in FFP epithelium (<b>J</b>, white arrowheads) or non-taste epithelium (<b>J</b>, yellow arrowheads). Nuclei were counterstained with DRAQ5 in blue. Dotted line indicates the basement membrane, and dashed circles indicate taste buds. Representative stack images and data from 3 control and 3 mutant mice. Data are represented as a scatter plot (individual symbols), and mean ± SEM (blue bars). Student’s t-test. Scale bars = 20 μm.</p

Stabilized β-catenin alters proliferation in anterior and posterior taste fields. A, GOF 2 days.

<p>After 2 days of dox, the proportion of Ki67^<b>+</b> basal keratinocytes in mutant CVP epithelium did not differ from controls (77.9 ± 2.3% vs. 76.2 ± 2.2% in controls vs. mutants; n = 3, p = 0.583, Student’s t-test). <b>A, GOF 4 days.</b> By day 4, proliferation in the mutant CVP was virtually abolished. <b>B</b>. In the FFP, the number of Ki67^<b>+</b> cells within the FFP epithelium are comparable in mutants and controls, but in mutants proliferation appears increased in epithelia at the FFP base (GOF 7 days vs control, white arrowheads). <b>C</b>. In controls, only cells in the basal layer of epithelium are Ki67^<b>+</b>, while in mutants, multiple layers or clusters of cells were actively proliferating (GOF 7d, arrowhead). <b>D</b>. The number of Ki67^<b>+</b> basal cells per 400μm of epithelium is significantly increased in mutants. <b>E,F</b>. Mutant and control mice were injected with BrdU 48 hours before tongues were harvested after 4 days on dox chow. Postmitotic BrdU^<b>+</b> cells were detected in and around taste buds (Krt8^<b>+</b>) in control CVP epithelium (white arrowheads), whereas β-catenin stabilization resulted in increased BrdU^<b>+</b> cells only within the expanded Krt8^<b>+</b> taste field. Nuclei were counterstained with Sytox Green. Stack images comprising 14 compressed 0.75 μm-thick optical sections are representative samples from 3 control and 3 mutant mice. Six CVP trenches and 15–16 sections of non-taste epithelium were analyzed per group. Data are represented as scatter plot (individual symbols), and mean ± SEM (blue bars). Student’s t-test. Dotted line delineates the basement membrane. Scale bars = 20 μm.</p

Stabilization of β-catenin in Krt5⁺ progenitors induces primarily Type I cell differentiation in CVP, FFP and anterior lingual epithelium.

<p><b>A</b>,<b>B.</b> SNAP25^<b>+</b> Type III taste cells (green) were present in comparable numbers in the CVP of control and mutant mice fed dox chow for 4 days. Note that CVP innervation is also SNAP25^<b>+</b> (green) (Yang et al., 2000). <b>C</b>,<b>D</b>. PLCβ2^<b>+</b> Type II cells (green) were slightly increased in mutant CVP compared to controls. <b>E</b>,<b>F</b>. NTPdase2^<b>+</b> Type I cells (green) cannot be counted (see text), and thus were quantified via measurement of NTPdase2 corrected fluorescence intensity, revealing a 2-fold increase in NTPdase2^<b>+</b> signal in mutant compared to control CVP, accounting for the majority of expanded Krt8^<b>+</b> cells (red in all panels). <b>G</b>. In the anterior tongue of mice fed doxycycline chow for 7 days, endogenous Krt8^<b>+</b> taste buds within FFP (red, white arrowhead) contained SNAP25^<b>+</b> Type III cells (green), while ectopic Krt8^<b>+</b> taste buds (red, white arrow) were devoid of SNAP25^<b>+</b> cells. <b>H</b>. Endogenous FF taste buds house numerous PLCβ2^<b>+</b> Type II taste cells (green, white arrowhead), while ectopic PLCβ2^<b>+</b> cells were not detected in ectopic taste buds (* indicates a PLCβ2-negative ectopic taste bud). <b>I</b>. Ectopic taste buds within FFP of mutants (white arrows), like endogenous FF taste buds (white arrowhead) were always NTPdase2^<b>+</b> (green). Nuclei were counterstained with DRAQ5 in blue. Representative stack images are made of 14 compressed 0.75 μm-thick optical sections. N = 3 control and 3 mutant mice. <b>B</b>,<b>D</b>: Student’s t-test; <b>F</b>: Mann and Whitney test. Data are represented as a scatter plot (individual symbols), and mean ± SEM (blue bars), except <b>F</b> (median with interquartile range). Dotted line delineates the basement membrane. Scale bars = 20 μm.</p

Stabilization of β-catenin in Shh⁺ precursor cells biases FFP taste cell fate.

<p>ShhCreERT2;Ctnnb^{<b>(Ex3)fl/+</b>};R26R-YFP mutant mice and their control counterparts (ShhCreERT2;R26R-YFP) were gavaged with tamoxifen daily for 8 days, and tongues harvested 14 days after the last dose. <b>A.</b> The number of Shh-descendant taste cells labeled with YFP did not differ between mutants and controls. <b>B,C</b>. In mutants, corrected NTPdase2^<b>+</b> immunofluorescence intensity of Type I cells was significantly increased in YFP^<b>+</b> taste buds, but not YFP^<b>-</b> taste buds. <b>D, E.</b> Overall the total number of Type II cells did not differ between mutants and controls, nor did the number of Type III cells (<b>F,G</b>). Representative stack images and data from 4–5 control and 4–6 mutant mice. <b>A</b>: 53 vs 72 YFP^<b>+</b> taste bud profiles from 6 control mice vs 6 mutant mice, respectively; <b>B</b>: 15 vs 11 YFP^<b>-</b> taste bud profiles and 20 vs 27 YFP^<b>+</b> taste bud profiles from 4 control mice vs 4 mutant mice, respectively; <b>D</b>: 34 vs 42 YFP^<b>-</b> taste bud profiles and 28 vs 30 YFP^<b>+</b> taste bud profiles from 4 control mice vs 4 mutant mice, respectively; <b>F</b>: 19 vs 27 YFP^<b>-</b> taste bud profiles and 25 vs 42 YFP^<b>+</b> taste bud profiles from 5 control mice vs 6 mutant mice, respectively. Mann & Whitney test, except <b>B</b> (Student’s t-test). Data are represented as scatter plots (individual symbols), and median with interquartile range (blue bars), except <b>B</b> (mean ± SEM). Nuclei are counterstained with DRAQ5 (blue). Scale bars = 20 μm.</p

Beta-catenin stabilization in Shh⁺ cells biases taste cell fate in the CVP, both taste bud autonomously and indirectly.

<p>Beta-catenin stabilization in Shh^<b>+</b> precursors significantly increased the number of Shh- descendant taste cells labeled with YFP in mutant mice compared with controls (<b>A</b>). Overall the total number of Type II (<b>D,E</b>) and Type III cells (<b>F,G</b>) did not differ between mutants and controls. NTPdase2 corrected fluorescence intensity of Type I cells was significantly increased in both YFP^<b>+</b> and YFP^<b>-</b> taste buds (<b>B,C</b>). Representative stack images and data from 4–5 control and 5–6 mutant mice. <b>A</b>: 178 vs 246 YFP^<b>+</b> taste bud profiles from 6 control mice vs 6 mutant mice, respectively; <b>B</b>: 85 vs 64 YFP^<b>-</b> taste bud profiles and 82 vs 100 YFP^<b>+</b> taste bud profiles from 4 control mice vs 4 mutant mice, respectively; <b>D</b>: 133 vs 122 YFP^<b>-</b> taste bud profiles and 72 vs 101 YFP^<b>+</b> taste bud profiles from 4 control mice vs 4 mutant mice, respectively; <b>F</b>: 120 vs 93 YFP^<b>-</b> taste bud profiles and 106 vs 145 YFP^<b>+</b> taste bud profiles from 5 control mice vs 6 mutant mice, respectively. Mann & Whitney test. Data are represented as scatter plot (individual symbols), and median with interquartile range (blue bars). Nuclei are counterstained with DRAQ5 (blue). Scale bars = 20 μm.</p

Stabilized β-catenin depletes progenitors (Krt14⁺) and causes lingual epithelial cells to differentiate as taste cells (Krt8⁺) at the expense of non-taste cells (Krt13⁺).

<p>Mutant (Krt5rtTA;tetOCre;Ctnnb1^{<b>(Ex3)fl/+</b>}) and control mice were fed doxycycline-supplemented chow for 4 or 7 days, their tongues harvested, and CVP and anterior tongue sections immunostained for markers of taste cells (Krt8), non-taste squamous epithelium (Krt13), and basal progenitor cells (Krt14). <b>A</b>. In the CVP of control mice, distinct Krt8^<b>+</b> taste buds (red, * indicates a taste bud) are interspersed in the apical epithelium with Krt13^<b>+</b> postmitotic squamous keratinocytes (green, white arrowheads). Ovoid Krt14^<b>+</b> progenitor cells (cyan) are restricted to the basal epithelium (white bent arrows), although occasional elongate Krt8^<b>+</b> cells are also dimly Krt14^<b>+</b> (yellow arrowheads). <b>B</b>. In the CVP of Krt5rtTA;tetOCre;Ctnnb^{<b>(Ex3)fl/+</b>} mice, Krt8^<b>+</b> cells are greatly expanded, and Krt13^<b>+</b> cells typically found between taste buds are absent (compare white arrowheads in <b>A</b> and <b>B</b>). Ovoid Krt14^<b>+</b> basal cells are also slightly reduced (compare white arrows in <b>A</b> and <b>B</b>, and white arrows in <b>A</b> and <b>B</b> Merge’, while persistent Krt14 staining in the Krt8^<b>+</b> taste field is dramatically increased compared to controls (<b>A</b> and <b>B</b>, yellow arrowheads). <b>C</b>. In the anterior tongue of controls, each FFP houses a single Krt8^<b>+</b> taste bud (red, * indicates a taste bud) surrounded by Krt13^<b>+</b> squamous epithelium (green), while basal cells adjacent to taste buds and throughout the FFP epithelium are Krt14^<b>+</b> (cyan). <b>D</b>. In mutants with stabilized β-catenin, ectopic Krt8^<b>+</b> cell clusters occur in FFP (red), which are devoid of Krt13 (green; white asterisks), while many are Krt14^<b>+</b> (cyan, yellow arrowheads, white signal in merged panel). <b>E</b>. Krt8^<b>+</b> cells (red) are detected ectopically, interspersed among filiform papillae (“f”) of the non-taste lingual epithelium. These Krt8^<b>+</b> cells are Krt13-negative, and frequently Krt14^<b>+</b>. Stack images are representative samples from 3 control and 3 mutant mice. Dotted line delineates the basement membrane. Scale bars = 20 μm.</p

Prolonged stabilization of β-catenin in Krt5⁺ progenitors induces differentiation of a small number of Type II cells in ectopic taste bud-like structures in the anterior tongue.

<p>After induction with dox chow for 14 days, 5.8 ± 2% of anterior Krt8^<b>+</b> ectopic structures (total of 485 Krt8^<b>+</b> structures counted, 23 with PLCβ2, n = 3 mice) expressed PLCβ2 (green, white arrows). These ectopic structures never expressed the Type III cell marker SNAP25 (total of 356 Krt8^<b>+</b> structures counted, 0 with SNAP25^<b>+</b> cells, n = 3 mice). Nuclei were counterstained with DRAQ5 in blue. Representative stack images from 3 mutant mice. Dotted line delineates the basement membrane. Scale bars = 20 μm.</p

Krt5-β-catenin-LOF results in reduced basal keratinocytes and proliferation, and leads to a smaller CVP.

<p><b>(A)</b> Many proliferating Ki67⁺ cells (magenta) are evident at the basement membrane of a control CVP, while in Krt5-β-catenin LOF mutants at 4 days and 2 weeks, fewer Ki67⁺ cells are evident in each CVP trench. Representative images are compressed z-stacks. Nuclei were counterstained with Sytox Green (green). Dotted line delineates the basement membrane. Taste buds are marked with asterisks. Scale bars = 20 μm. <b>(B)</b> Progenitor proliferation was significantly decreased in LOF mutants compared to controls at both 4 days and 2 weeks. <b>(C)</b> The total number of basal progenitor cells residing along the basement membrane was significantly lower in mutants compared to controls after 4 days and 2 weeks of Krt5-β-catenin LOF (white arrow). CVP size was assessed <i>via</i> the cross sectional area between the two trenches (<b>D</b>, green area), and CVP trench depth (<b>D</b>, blue arrow). By 2 weeks of doxycycline chow, CVP size and trench depth were reduced in mutant mice compared with controls (<b>E</b>,<b>F</b>). Data are represented as scatter plots (individual symbols), and mean ± SEM (<b>B</b>,<b>E</b>, blue bars. Student’s t-test) or median with 1^st and 3^rd quartile (<b>C</b>,<b>F</b>, blue bars. Mann & Whitney test). Sample sizes: (<b>B</b>,<b>C</b>) 32 vs 34 CVP trench sections at 4 days and 34 vs 21 CVP trench sections at 2 weeks from 3 control mice vs 3 mutant mice, respectively. <b>(E)</b> 6–10 CVP profiles from 3–4 control mice and 3 mutant mice; <b>(F)</b> 12–20 CVP trench profiles from 3–4 control mice and 3 mutant mice.</p

β-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice

<div><p>Taste stimuli are transduced by taste buds and transmitted to the brain <i>via</i> afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional β-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and <i>Shh</i>⁺ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest β-catenin plays a greater role in renewal of anterior versus posterior taste buds.</p></div