Pharyngeal arch size is reduced in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutants.

<p>(A-C) Immunostaining of 36hpf <i>fli1a</i>:<i>egfp</i> labeled control, <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutant embryos with Zn-8 (red), which labels the endodermal pouches, revealed comparatively normal pharyngeal arch and pharyngeal pouch patterning. Pharyngeal arches 1–5 are indicated. (D-F) <i>fli1a</i>:<i>egfp</i> labeling of post-migratory NCC illustrates an overall reduction in pharyngeal arch size in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutants. Pharyngeal arches 1 and 2 are outlined in red. (G-H) Quantification of pharyngeal arches 1 & 2 (red arrows, D-F) volume revealed a reduction in <i>polr1c</i>^-/- (G) and <i>polr1d</i>^-/- (H) mutants. Scale bar = 100 μm. * = p < 0.05 and error bars represent 95% confidence intervals.</p

Analysis of NCC development in <i>polr1c</i> and <i>polr1d</i> mutant embryos.

<p>(A-H) <i>sox10</i> expression at 12 hpf and (I-P) <i>foxd3</i> expression at 14 hpf reveal relatively normal patterns of early cranial NCC specification and migration in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- embryos (black arrows). (Q-X) In contrast, <i>dlx2</i> expression at 36 hpf reveals slightly diminished domains of activity in mutant embryos, particularly with respect to the posterior pharyngeal arches, which is suggestive of fewer mature NCC colonizing the pharyngeal arches. White arrows indicate pharyngeal arches 1 and 2. Scale bar = 200 μm.</p

Craniofacial cartilage development is disrupted in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutant embryos.

<p>(A-C) Alcian blue staining reveals cranial cartilage in 5 dpf <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutant embryos is hypoplastic compared to controls. (D-F) The jaws of mutant embryos are smaller overall, with noticeable differences in the size of Meckel’s cartilage, the palatoquadrate, and ceratohyal elements. (G-I) Staining of the viscerocranium reveals smaller cartilage elements derived from each of the pharyngeal arches in mutant embryos, most notably the ceratobranchials, as well as altered polarity of the ceratohyal. (J-L) Staining of the neurocranium reveals hypoplasia of the ethmoid plate. Abbreviations: M, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; cb, ceratobranchial; ep, ethmoid plate; pch, parachordal. Scale bar = 200 μm.</p

<i>tp53</i> inhibition ameliorates cartilage anomalies in <i>polr1d</i>^-/-mutant embryos in a dosage-dependent manner.

<p>(A-D) Alcian blue staining of cartilage in an allelic series of <i>polr1d</i> and <i>tp53</i> mutant embryos. Dosage-dependent improvement in cartilage development is particularly noticeable in the jaw (E-H), elements of the viscerocranium (I-L), and more specifically the ceratohyal (G-L). Abbreviations: M, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; cb, ceratobranchial. Scale bar = 200 μm.</p

<i>polr1c</i> and <i>polr1d</i> are dynamically expressed during zebrafish embryogenesis.

<p><i>polr1c</i> and <i>polr1d</i> are maternally expressed at early stages (A,B, arrows) and ubiquitously expressed at 6 hpf (C,D) and 11 hpf (E,F) when the embryo surrounds the yolk (dashed lines). At 24 hpf, expression becomes enriched in regions such as the eye and midbrain-hindbrain boundary (G,H). Elevated levels of expression are evident in the pharyngeal arches (adjacent to curved line) at 36 hpf (I,J) whereas lower levels are observed throughout the embryo at 48 hpf and beyond (K,L) and beyond. Abbreviations: e, eye; mbhb, midbrain-hindbrain boundary; pa, pharyngeal arches; l, lens; t, tectum. Scale bar = 200 μm.</p

Ribosome biogenesis is disrupted in <i>polr1c</i> and <i>polr1d</i> mutant embryos.

<p>(A, B) qPCR quantification of 47S rRNA production. (A) <i>polr1c</i>^-/- mutants exhibit reduced levels of 5’ETS (39%), ITS2 (23%) and 18S rRNA (58%) compared to controls. (B) <i>polr1d</i>^-/- mutants similarly exhibit reduced levels of 5’ETS (25%), ITS2 (39%), and 18S rRNA (32%) compared to controls. (C, D) Polysome profiling shows decreased 80S and polysome peaks in <i>polr1c</i>^-/- (C) and <i>polr1d</i>^-/- (D) mutant embryos.</p

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

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