Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid

<div><p>The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of <i>Cyp26a1</i> and <i>Cyp26c1</i>. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which <i>Wnt10b</i> expression normally ceases and <i>Shh</i> becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of <i>Shh</i> and <i>Wnt10b</i> in the basal layer, specifying <i>de novo</i> taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.</p></div

Upregulation of <i>RARb</i> and <i>RARg</i> expression in <i>ShhGFPCRE/Smo</i>^<i>f/f</i> and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant tongues, indicating enhanced retinoic acid signaling.

<p>(<b>A-H’</b>) <i>RARg in situ</i> hybridization with oligonucleotide probes (brown) in parasagittal tongue sections. The dotted lines highlight the junction between the lingual epithelium and lingual mesenchyme. (<b>A-D’</b>) Sections from E12.5 (A,B) and E13.5 (C,D) control (A,C) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant (B,D) embryos. (A'-D') are enlarged images of the boxed areas in (A-D). (<b>E-F’</b>) Sections from E13.5 control (E) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (F) embryos first exposed to tamoxifen (TAM) at E10.5. (E’) and (F’) are enlarged images of the boxed areas in (E) and (F), respectively. (<b>G-H’</b>). Sections from E14 control (G) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (H) embryos first exposed to TAM at E11.5. (G’) and (H’) are enlarged images of the boxed (red) areas in (G) and (H), respectively. The inset in (H’) is an enlarged image of the boxed (green) area in (H). The <i>RARg</i> hybridization signals in the lingual epithelium (LE) of the controls, to a large extent, appear as punctae, whereas in the LE of the mutants they appear as globular aggregates, indicating higher levels of <i>RARg</i> expression in the mutants relative to the controls. Epithelial foci in the mutants with weaker <i>RARg</i> signals than the rest of the LE (arrow in H’). (<b>I-K</b>) RT-qPCR analysis for <i>RARb</i> and <i>RARg</i> relative to <i>Actb</i> (β-actin). (I) Upregulation of <i>RARb</i> (<i>P</i> = 0.0000) and <i>RARg</i> (<i>P</i> = 0.0000) levels in tongues from E13.5 <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutants (n = 6) as compared to tongues from controls (n <b>=</b> 6) first exposed to TAM at E11.5. (J) Upregulated <i>RARb</i> (<i>P</i> = 0.0000) and <i>RARg</i> (<i>P</i> = 0.0003) levels in tongues from E13.5 <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutants (n = 6) as compared to tongues from controls (n = 6) first exposed to TAM at E10.5. (K) Upregulation of <i>RARb</i> (<i>P</i> = 0.0015) and <i>RARg</i> (<i>P</i> = 0.0000) levels in tongues from E12.5 <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutants (n = 6) as compared to tongues from controls (n = 6). The data are mean values ± SD. CvP, circumvallate papilla; T, tongue. Scale bars: 500 μm (A-H) and 50 μm (A’-H’).</p

Ectopic and anachronic expression of <i>Shh</i> and <i>Wnt10b</i> in the lingual epithelium of <i>ShhGFPCRE/Smo</i>^<i>f/f</i> and tamoxifen-induced <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutants specify <i>de novo</i> taste placode formation.

<p>(<b>A-D</b>) Anti-Sonic hedgehog-stained (SHH; dark purple) parasagittal tongue sections from controls (A,C) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutants (B,D) at E16.5 (A,B) and postnatal day P0 (C,D). (<b>E-H</b>) <i>Shh</i> (E,F) and <i>Ptch1</i> (G,H) <i>in situ</i> hybridization in parasagittal tongue sections from E18.5 control (E,G) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> (F,H) embryos. The signals appear as black or shiny dots in bright-field (E,F) or dark-field (G,H) images, respectively. SHH protein and <i>Shh</i> mRNA (SHH/<i>Shh</i>) are confined to taste buds (TB) of fungiform papillae (FuP) in the control tongues (A,C,E). The <i>ShhGFPCRE/Smo</i>^<i>f/f</i> tongues display SHH+/<i>Shh+</i> placode-like entities (arrows in B,D and F) underlain by a <i>Ptch1</i>+ mesenchyme (arrows in H). (<b>I-J’</b>) Anti-SHH-stained parasagittal tongue sections from E16 control (I) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (J) embryos first exposed to tamoxifen at E11.5. (I'), (J'), and insets in (I) and (J) are enlarged images of the boxed areas in (I) and (J), respectively. TBs within fungiform papillae of the mutant tongue (inset in J) exhibit weaker SHH staining than those of the control tongue (inset in I). The mutant tongue shows SHH staining in placode-like structures in the epithelial basal layer (arrows in J’). (<b>K-P</b>) <i>Wnt10b in situ</i> hybridization (black) in parasagittal tongue sections. Sections from 17.5 control (K) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant (L) embryos. Sections from 17.5 control (M) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (N) embryos first exposed to tamoxifen (TAM) at E11.5. Sections from E15.5 control (O) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (P) embryos first exposed to TAM at E10.5. All the mutant tongues show abnormal expression of <i>Wnt10b</i> in the epithelial basal layer (arrows) separated by <i>Wnt10b</i>-negative gaps. Ic, incisor tooth. Scale bars: 250 μm (I,J), 200 μm (A,B,E-H,K-P), 100 μm (I';J'), and 50 μm (C,D).</p

SHH signaling is required for maintenance/reinforcement but not for induction of <i>Cyp26a1</i> and <i>Cyp26c1</i> expression.

<p>(<b>A-R</b>) <i>In situ</i> hybridization for <i>Cyp26c1</i> and <i>Cyp26a1</i> in the developing tongue (dark blue/brown). (<b>A</b>,<b>B</b>) E11.5 control (A) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant (B) tongues showing <i>Cyp26c1</i> expression in the control tongue (arrow in A) and absence of <i>Cyp26c1</i> expression in the mutant tongue (arrow in B). (<b>C</b>,<b>D</b>) <i>Cyp26c1</i> expression in E12.5-E13 control (C) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant (D) tongues. (<b>E</b>,<b>F</b>) <i>Cyp26c1</i> expression in tongues from E14-E14.5 control (E) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (F) embryos first exposed to tamoxifen (TAM) at E10.5. The inset in (E) is an enlarged image of the specimen in (E). (<b>G,H</b>) <i>Cyp26a1</i> expression in E14 control (G) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant (H) tongues. (<b>I,J</b>) <i>Cyp26a1</i> expression in tongues from E14.5 control (I) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (J) embryos first exposed to TAM at E10.5. (<b>K-N</b>) <i>Cyp26a1</i> (K,L) and <i>Cyp26c1</i> (M,N) expression in tongues from E15 control (K,M) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (L,N) embryos first exposed to TAM at E11.5. Severe downregulation of <i>Cyp26a1/Cyp26c1</i> expression in all mutants, except in epithelial foci (ef). Fungiform placodes or taste buds, which appear as unstained spots (C,G, inset in E), and the circumvallate papilla (CvP) are <i>Cyp26a1/Cyp26c1</i>-negative. Artefact due to loss of the thin epithelium of the intermolar eminence during tissue processing (arrowheads in G, I and K). (<b>O-R</b>) Unaltered <i>Cyp26c1</i> (O,P) and <i>Cyp26a1</i> (Q,R) expression in tongues from E14.5 control (O,Q) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant (P,R) embryos first exposed to TAM at E12.5. (<b>S,T</b>) <i>Cyp26a1</i> expression in tongue explants from E12 control embryos after <i>in vitro</i> culture for 2 days with DMSO (S) and 200 nM SAG (T). SAG enhanced <i>Cyp26a1</i> hybridization signals in the anterior tongue (blue) but failed to induce ectopic <i>Cyp26a1</i> expression in the posterior tongue and mandibular structures. (<b>U</b>) RT-qPCR analysis for <i>Cyp26b1</i> relative to <i>Actb</i> (β-actin) in tongues from E12.5 controls (n = 6) and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutants (n = 6), in tongues from E13.5 controls (n = 6) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutants (n = 6) first exposed to TAM at E10.5, and in tongues from E13.5 controls (n = 8) and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutants (n = 8) first exposed to TAM at E11.5. <i>Cyp26b1</i> levels are upregulated in mutants relative to controls first exposed to TAM at E11.5 (<i>P</i> = 0.0000). The data are mean values ± SD. Scale bars: 500 μm (A-N,Q,R) and 300 μm (O,P,S,T).</p

Early loss of SHH signaling in the tongue impinges upon growth and morphogenesis but is conducive to taste bud differentiation.

<p>(<b>A-N</b>) Tongues and parasagittal tongue sections from control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to tamoxifen at E10.5. (<b>A</b>,<b>B</b>) Anti-Sonic hedgehog-stained (SHH; dark purple) sections of E15.5 control (A) and mutant (B) tongues showing focal epithelial hyperplasia (arrow in B) and severely decreased SHH immunostaining in the mutant. (<b>C</b>,<b>D</b>) <i>Gli1 in situ</i> hybridization (brown) in sections from E13.5 control (C) and mutant (D) tongues showing severe downregulation of <i>Gli1</i> expression in the mutant. (<b>E,F</b>) E15 control (E) and mutant (F) tongues after <i>in situ</i> hybridization with a riboprobe targeting both deleted (exon2) and non-deleted (exon1) <i>Shh</i>-coding sequences (dark purple). Abnormally small mutant tongue with a bifid tip, and exhibiting oversized <i>Shh</i>+ spots (arrowheads in F). (<b>G-L</b>) Sections of E15.5 (G-I) and E17.5 (J-L) control (G,K) and mutant (H-J,L) tongues immunostained (dark purple) for keratin 8 (K8; G-I, K,L) and Rab3c (J). The insets in (K) and (L) are enlarged images of the boxed areas in (K) and (L), respectively. The control and mutant tongues show K8+ taste buds (TB) in fungiform papillae (FuP), and the mutant tongues exhibit K8+ and Rab3c+ ectopic Merkel cells (MC). (<b>M,N</b>) Tongue sections from E18 control (M) and mutant (N) embryos after K8 (green) and P2X2 (red) double staining showing innervated TBs. (<b>O</b>) RT-qPCR analysis for <i>Ptch1</i> relative to <i>Actb</i> (β-actin) in tongues from E13.5 controls (n = 6) and mutants (n = 6) first exposed to tamoxifen (TAM) at E10.5. Severely decreased <i>Ptch1</i> levels in the mutant tongues as compared to the controls (<i>P</i> = 0.0000; mean values ± SD). PD, periderm; CvP, circumvallate papilla; Ic, incisor; RP, rugae palatinae; T, tongue. Scale bars: 500 μm (E,F,K,L), 200 μm (A-D), 100 μm (G,H), 50 μm (I,J), and 25 μm (M,N).</p

Tamoxifen induction at E11.5 causes overproduction of oversized, innervated taste buds in <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant tongues.

<p>(<b>A-N</b>) Analyses of tongues from control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to tamoxifen at E11.5. (<b>A,B</b>) <i>In situ</i> hybridization with a riboprobe targeting <i>Shh</i> exon2 (the deleted allele) in E13.5 control (A) and mutant (B) tongues showing severely decreased <i>Shh</i> expression (purple) in the mutant tongue. (<b>C,D</b>) <i>Gli1 in situ</i> hybridization (brown) in parasagittal sections of E14.5 control (C) and mutant (D) tongues showing severe downregulation of <i>Gli1</i> expression in the mutant tongue, except in a small domain (arrows in D). (<b>E</b>) RT-qPCR analysis for <i>Ptch1</i> relative to <i>Actb</i> (β-actin) in tongues from E13.5 controls (n = 9) and mutants (n = 9). Severely decreased <i>Ptch1</i> levels in the mutant tongues as compared to the controls (<i>P</i> = 0.0000; mean values ± SD). (<b>F,G</b>) E15.5 control (F) and mutant (G) tongues after <i>in situ</i> hybridization with a riboprobe targeting both deleted (exon2) and non-deleted (exon1) <i>Shh</i>-coding sequences (brown) showing overproduction of oversized <i>Shh</i>-expressing taste buds in the mutant tongue. (<b>H-L</b>) Immunostaining (dark purple) for Keratin 8 (K8; H-J) and SOX2 (K,L) in parasagittal sections of E18.5 (H-J) and E16 (K,L) control (H,K) and mutant (I,J,L) tongues. The insets in (H) and (I) are enlarged images of the boxed areas in (H) and (I), respectively. The mutant tongues exhibit crowded and oversized taste buds (TB; I,L) and a fungiform papilla (FuP) abnormally harboring three TBs (J). Artefact (asterisk in I). (<b>M,N</b>) Parasagittal sections of E18 control (M) and mutant (N) tongues after K8 (green) and P2X2 (red) double staining showing innervated TBs. CvP, circumvallate papilla; T, tongue. Scale bars: 500 μm (A,B,F,G,H,I), 200 μm (C,D), 50 μm (J-L), and 25μm (M,N).</p

Dramatic downregulation of <i>Cyp26a1</i> and <i>Cyp26c1</i> expression in the lingual epithelium of <i>ShhGFPCRE/Smo</i>^<i>f/f</i> and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant tongues.

<p>(<b>A-N’</b>) Parasagittal tongue (T) sections from controls and mutants after <i>in situ</i> hybridization with a ³⁵S-UTP-labelled riboprobe for <i>Cyp26c1</i> (A-F’) and oligonucleotide probes for <i>Cyp26a1</i> (G-N’; black). Genotypes are indicated in the panels. (<b>A-D</b>) Dark-field images showing <i>Cyp26c1</i> expression (shiny dots) in E12.5 (A,B) and E14 (C,D) control and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant tongues. (<b>A’-D’</b>) Enlarged bright-field images (hybridization signals appear as black dots) of the boxed areas in (A-D). (<b>E,F</b>) Dark-field images showing <i>Cyp26c1</i> expression in tongue sections from E14 control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to tamoxifen (TAM) at E10.5. (E’) and (F’) are enlarged, bright-field images of the boxed areas in (E) and (F), respectively. All mutants show virtual abrogation of <i>Cyp26c1</i> expression (B,D,F), except in epithelial foci (arrows in D,D’,F and F’). Absence of <i>Cyp26c1</i> transcripts in fungiform placodes (A’,C’) and pharyngeal tongue (A,C,E) in controls. (<b>G-J’</b>) <i>Cyp26a1</i> expression in tongue sections from E12.5 (G,H) and E13.5 (I,J) control and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant embryos. (G’-J’) Enlarged images of the boxed areas in (G-J). (<b>K</b>-<b>L’</b>) <i>Cyp26a1</i> expression in tongue sections from E13.5 control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to TAM at E10.5. (K’) and (L’) are enlarged images of the boxed areas in (K) and (L), respectively. (<b>M-N’</b>) <i>Cyp26a1</i> expression in tongue sections from E14 control and <i>ShhCreER</i>^<i>T2</i><i>/Shh</i>^<i>f</i> mutant embryos first exposed to TAM at E11.5. (M’) and (N’) are enlarged images of the boxed areas in (M) and (N), respectively. All mutants show virtual absence of <i>Cyp26a1</i> expression, except in epithelial foci (arrows in H’,J,J’,L’ and N’). <i>Cyp26a1</i> transcripts are excluded from fungiform placodes (G’,I’,K’ and M’) and pharyngeal tongue (G,I,K,M) in the controls. FP, fungiform placode; PT, pharyngeal tongue. Scale bars: 500 μm (A-F,I-N), 200 μm (G,H), and 50 μm (A’-N’).</p

The <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutant tongues show expansion of <i>Shh</i> expression in the lingual epithelium, and develop innervated taste buds.

<p>(<b>A-D</b>) Anti-Sonic hedgehog-stained (SHH; purple) parasagittal tongue sections from controls (A,C) and mutants (B,D) at E12.5 (A,B) and E14 (C,D) showing abnormally expanded SHH staining in the lingual epithelium (LE) of the E14 mutant. (<b>E,F</b>) Dark-field images of frontal tongue sections from E14.5 control (E) and mutant (F) embryos after <i>Shh in situ</i> hybridization showing abnormally expanded <i>Shh</i> expression in the LE of the mutant. (<b>G-L</b>) E14.5 (G-J) and E15.5 (K,L) control (G,I,K) and mutant (H,J,L) tongues after <i>in situ</i> hybridization (dark blue/brown; G,H,K,L) and green fluorescent protein (GFP) imaging (I,J) showing <i>Shh</i> expression. The mutant tongues exhibited abnormally enlarged <i>Shh</i>+ spots, some of which developed ectopically along the tongue midline (H,J,L). (<b>M,N</b>) Parasagittal tongue sections from control (M) and mutant (N) newborns (P0) after double staining for Keratin 8 (K8; green) and P2X2 (red) showing innervated TBs. CvP, circumvallate papilla; FP, fungiform placode; T, tongue; TB, taste bud; To, tooth bud. Scale bars: 500 μm (E-J), 300 μm (K,L), 200 μm (A-D), and 25 μm (M,N).</p

The <i>ShhGFPCRE/Smo</i>^<i>f/f</i> lingual epithelium undergoes Merkel cell metaplasia.

<p>(<b>A-O</b>) Parasagittal tongue sections from newborn (P0) controls and <i>ShhGFPCRE/Smo</i>^<i>f/f</i> mutants. (<b>A-D</b>) Sections of control (A,C) and mutant (B,D) tongues immunostained (purple) for Keratin 8 (K8; A,B) and SOX2 (C,D). The inset in (B) is an enlarged image of the boxed area in (B). The mutant tongues exhibit K8-positive (+) and SOX2+ ectopic Merkel cells (MC) in the epithelial basal layer. Taste buds (TB) are also K8+ and SOX2+. (<b>E,F</b>) Sections of control (E) and mutant (F) tongues after double staining for K8 (green) and Sonic Hedgehog (SHH; red). TBs are K8+/SHH+. The K8+ ectopic Merkel cells in the mutant are SHH-negative (–). (<b>G,H</b>) Anti-Homer1-stained (purple) sections of control (G) and mutant (H) tongues showing Homer1(+) TBs. (<b>I-K</b>) Sections of control (I) and mutant (J,K) tongues after double staining for K8 (red) and Homer1 (green), showing K8+/Homer1+ TBs (I,J) and K8+/Homer1(–) ectopic Merkel cells (K). (<b>L,M</b>) Sections of control (L) and mutant (M) tongues after double staining for K8 (red) and Rab3c (green). The ectopic Merkel cells (arrowheads in M) are K8+/Rab3c+, whereas TBs are K8+/Rab3c(–). (<b>N</b>) Section across a mutant tongue after double staining for K8 (green) and NF-200 (NF; red) showing ectopic Merkel cells wrapped by NF-200+ neurites (arrowheads). The insets in (N) are non-merged, enlarged images of the boxed area in (N). (<b>O</b>) Section of a mutant tongue after double staining for K8 (green) and P2X2 (red) showing that the ectopic Merkel cells are not associated with P2X2+ gustatory neurites. (<b>P-R</b>) Sections across the whisker pad of control newborns after double staining for K8/SHH (P), K8/Homer1(Q) and K8/Rab3c (R). Merkel cells of the whisker follicles and adjacent epidermis are K8+/SHH(–), K8+/Homer1(–), and K8+/Rab3c+. FuP, fungiform papillae; HF, hair follicle; SG, sebaceous glands; Wh, whisker follicle. Scale bars: 200 μm (A,B), 50 μm (C-J, L,M, O-R), and 20 μm (K,N).</p

CA immunohistochemistry in postnatal molars at the maturation stage of enamel formation.

<p>Immunohistochemistry showing the distribution patterns of CARP XI, CA IX, CA XIII and CA XIV in sections of molars at 12 dpp as indicated on the panels. The latter three isoforms were detected with the Goat anti-CA IX, rabbit anti-mouse CA XIII antiserum and goat anti-CA XIV. CA IX (B) and CARP XI (F) are enriched in the ruffled border of ruffle-ended maturation-stage ameloblasts (RMA). Smooth-ended maturation-stage ameloblasts (SMA) exhibit a homogeneous staining portraying CA IX (C) and CARP XI (G). CA XIV immunostaining is homogeneous in RMA (N) and SMA (O). Odontoblasts (Od) express CA IX (D), CARP XI (H) and CA XIV (P). CA XIII immunostaining is strong in intracytoplasmic punctae/vesicles in the papillary layer (PL), RMA and SMA (J, K) as well as in odontoblasts, including the site of emergence of their processes (L). Because not all molar sections include SMA, the images in C and O were taken from other sections of the molars that have been processed for IHC under the same conditions. Images in B, D, F–H, J–L, N and P are high magnification views of the indicated areas in A, E, I and M, respectively. Additional abbreviations: DP, dental pulp mesenchyme, PD, predentin/dentin; TA, transition-stage ameloblasts. Scale bars: 200 µm (A, E, I, M), 20 µm (B–D, F–H, J–L, N–P).</p