Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development.

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning

Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning

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

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