The transcription factors Foxf1 and Foxf2 integrate the SHH, HGF and TGFβ signaling pathways to drive tongue organogenesis

Abstract

The tongue is a highly specialized muscular organ with diverse cellular origins, which provides an excellent model for understanding mechanisms controlling tissue-tissue interactions during organogenesis. Previous studies showed that SHH signaling is required for tongue morphogenesis and tongue muscle organization, but little is known about the underlying mechanisms. Here we demonstrate that the Foxf1/Foxf2 transcription factors act in the cranial neural crest cell (CNCC)-derived mandibular mesenchyme to control myoblast migration into the tongue primordium during tongue initiation, and thereafter continue to regulate intrinsic tongue muscle assembly and lingual tendon formation. We performed chromatin immunoprecipitation sequencing analysis and identified Hgf, Tgfb2 and Tgfb3 among the target genes of Foxf2 in the embryonic tongue. Through genetic analyses of mice with CNCC-specific inactivation of Smo or both Foxf1 and Foxf2, we show that Foxf1 and Foxf2 mediate hedgehog signaling-mediated regulation of myoblast migration during tongue initiation and intrinsic tongue muscle formation by regulating the activation of the HGF and TGFβ signaling pathways. These data uncover the molecular network integrating the SHH, HGF and TGFβ signaling pathways in regulating tongue organogenesis.

Keywords: Gene regulatory network; Myoblast migration; Neural crest; SHH; TGFβ signaling; Tongue development.

Fig. 1.

Expression of Foxf1 and Foxf2 during tongue development. (A-L′) Frontal sections through the developing tongue in E12.5 (A-F) and E13.5 (G-L′) mouse embryos showing immunofluorescence detection of Foxf1 (A-C,G-I′, magenta), Foxf2 (D-F,J-L′, magenta), muscle actin (C,F,I,I′,L,L′, green) and Scx-GFP (B,E,H,H′,K,K′, green). Panels H′ and K′ show higher-magnification views of the boxed areas in H and K, respectively, revealing the substantial overlap of Foxf1 and Foxf2 with Scx-GFP in the developing lingual septum. Panels I′ and L′ show higher magnification views of the boxed areas in I and L, respectively, showing that there was little Foxf1 and Foxf2 expression in the muscle actin-positive muscle bundles. Arrows point to the developing midline lingual septum. White arrowheads point to the transverse muscle. Yellow arrowheads point to the superior longitudinal muscle. Images are representative of three embryos. Scale bars: 200 μm (A-L); 100 μm (H′-L′).

Fig. 2.

Foxf1^c/cFoxf2^c/+Wnt1-Cre and Foxf1^c/+Foxf2^c/cWnt1-Cre mutant mice exhibit defects in the intrinsic tongue muscles. (A-C) Whole-mount view of E16.5 control (A) (n=17), Foxf1^c/cFoxf2^c/+Wnt1-Cre (B) (n=6) and Foxf1^c/+Foxf2^c/cWnt1-Cre (C) (n=7) embryonic tongues showing the patterns of green fluorescence from the Scx-GFP transgenic reporter. Yellow arrows point to the location of the lingual septum tendon in the tongue. (D-L) Sagittal (n=3 for each genotype) (D-F) and frontal (n=3 for each genotype) (G-L) sections through the tongues of E14.5 control (D,G,J), Foxf1^c/cFoxf2^c/+Wnt1-Cre (E,H,K) and Foxf1^c/+Foxf2^c/cWnt1-Cre (F,I,L) embryos showing immunofluorescence detection of muscle actin (red). cl, combined longitudinal muscle (including hg and sg); gg, genioglossus muscle; h, hyoid bone; hg, hyoglossus muscle; il, inferior longitudinal muscle; pg, palatoglossus muscle; sg, styloglossus muscle; sl, superior longitudinal muscle; t, transverse muscle; v, vertical muscle. Yellow arrowheads point to the location of the transverse and vertical muscles in the tongue. Scale bars: 1000 μm (A-C); 200 μm (D-L).

Fig. 3.

Foxf1^c/cFoxf2^c/+Wnt1-Cre and Foxf1^c/+Foxf2^c/cWnt1-Cre mutant mice exhibit defects in tongue myogenesis. (A-F) Frontal sections through the developing tongue in E12.5 control (A,D) (n=7), Foxf1^c/cFoxf2^c/+Wnt1-Cre (B,E) (n=4) and Foxf1^c/+Foxf2^c/cWnt1-Cre (C,F) (n=4) embryos showing patterns of immunofluorescence staining of muscle actin (green) and MyoD (magenta). Yellow arrowheads in D-F point to the myogenic progenitor cells of transverse and vertical muscles of the tongue. (G-I) Quantification of the percentage of MyoD-positive nuclei in the anterior versus posterior and medial versus lateral domains of tongue mesenchyme (L, lateral; M, medial), depicted by the schematic in G. Statistical analysis was performed on data from five control embryos (63 sections for the posterior tongue and 66 sections for the anterior tongue) and four Foxf1^c/cFoxf2^c/+Wnt1-Cre (49 sections for the posterior tongue and 49 sections for the anterior tongue) embryos (H), or on data from four control embryos (51 sections for the posterior tongue and 50 sections for the anterior tongue) and four Foxf1^c/+Foxf2^c/cWnt1-Cre embryos (42 sections for the posterior tongue and 42 sections for the anterior tongue) (I). Statistical significance was determined by a two-tailed unpaired Student's t-test using Excel. The results are presented as mean±s.d. AL, anterior lateral; AM, anterior medial; PL, posterior lateral; PM, posterior medial. *P<0.05. Scale bar: 200 μm.

Fig. 4.

Foxf1^c/cFoxf2^c/+Wnt1-Cre and Foxf1^c/+Foxf2^c/cWnt1-Cre mutant mice exhibit defects in lingual septum tendon formation. (A-C) Whole-mount view of E13.5 control (A), Foxf1^c/cFoxf2^c/+Wnt1-Cre (B) and Foxf1^c/+Foxf2^c/cWnt1-Cre (C) embryonic tongues to visualize the patterns of green fluorescence from the Scx-GFP transgenic reporter (n=3 for each genotype). Arrows point to the location of the lingual septum tendon in the tongue. (D-I) Frontal sections through the developing tongue in E13.5 control (D,G), Foxf1^c/cFoxf2^c/+Wnt1-Cre (E,H) and Foxf1^c/+Foxf2^c/cWnt1-Cre (F,I) embryos showing the patterns of Scx-GFP (green) and immunostaining of muscle actin (magenta). Higher-magnification views of the boxed areas in D-F are shown in G-I, respectively. (J-O) Frontal sections through the developing tongue in E16.5 control (J,M), Foxf1^c/cFoxf2^c/+Wnt1-Cre (K,N) and Foxf1^c/+Foxf2^c/cWnt1-Cre (L,O) embryos showing immunofluorescence detection of muscle actin (magenta) and collagen I (green). Panels M-O show higher-magnification views of the boxed areas in J-L, respectively. Arrows in M-O point to the location of the lingual septum tendon. Scale bars: 500 μm (A-C); 200 μm (D-L).

Fig. 5.

Foxf2 directly binds to the promoter regions of the Tgfb3 and Tgfb2 genes in the developing tongue. (A) The sequence of the most enriched Foxf2-binding motif identified by ChIP-seq analysis of the E12.5 embryonic tongue is shown together with the statistical P-value and percentage of the ChIP-seq peaks containing the motif. (B,C) Gene Ontology analysis of the Foxf2 ChIP-seq peaks performed with GREAT (http://great.stanford.edu). The plot indicates the −log₁₀ of binomial P-values of each GO term. The most significant GO biological process categories are shown in B and the most significant GO molecular function terms are shown in C. (D,E) Genome Browser views of the genomic regions containing the Tgfb3 (D) and Tgfb2 (E) genes. Vertical arrows point to the Foxf2-bound peak regions. Asterisks mark the ChIP-seq peaks containing the canonical Foxf1/Foxf2-binding motif.

Fig. 6.

Loss of Foxf1 and Foxf2 function in the CNCC-derived tongue mesenchyme affects TGFβ signaling in tongue development. (A-F) Frontal sections through the developing tongue in E12.5 control (A,D), Foxf1^c/cFoxf2^c/+Wnt1-Cre (B,E) and Foxf1^c/+Foxf2^c/cWnt1-Cre (C,F) embryos showing patterns of expression of the Tgfb3 (A-C) and Tgfb2 (D-E) mRNAs (n=3 for each genotype). Black arrows point to the developing lingual septum. Red arrows point to the developing dorsal lingual aponeurosis. Black arrowheads point to the developing muscle. (G-O) Frontal sections through the developing tongue in E12.5 control (G,J,M), Foxf1^c/cFoxf2^c/+Wnt1-Cre (H,K,N) and Foxf1^c/+Foxf2^c/cWnt1-Cre (I,L,O) embryos showing patterns of immunofluorescence staining of pSmad2 (magenta) and muscle actin (green) (n=3 for each genotype). Panels J-O show higher-magnification views of the boxed areas in G-I, respectively. (P-U) Frontal sections through the developing tongue in E12.5 control (P,S), Foxf1^c/cFoxf2^c/+Wnt1-Cre (Q,T) and Foxf1^c/+Foxf2^c/cWnt1-Cre (R,U) embryos showing patterns of immunofluorescence staining of pSmad2 (magenta) and of the Scx-GFP reporter (green). Panels S-U show higher-magnification views of the boxed areas in P-R, respectively. (V) Western blot analysis of pSmad2/3, total Smad2/3 and β-actin in the E12.5 control, Foxf1^c/cFoxf2^c/+Wnt1-Cre and Foxf1^c/+Foxf2^c/cWnt1-Cre tongue tissues. Images are representative of three embryos for each genotype. (W,X) Quantitative comparison of the levels of pSmad2/3 between the tongue tissues of control and Foxf1^c/cFoxf2^c/+Wnt1-Cre littermates (W) (n=4 for each genotype) and between control and Foxf1^c/+Foxf2^c/cWnt1-Cre littermates (X) (n=3 for each genotype). Data are shown as mean±s.e.m. and compared by two-tailed unpaired t-test. N.S., not significant; *P<0.05. Scale bars: 200 μm.

Fig. 7.

Failure of myoblast migration into the developing tongue primordium in the Smo^c/cHand2-Cre embryos correlated with loss of HGF expression in the mandibular arch. (A) Genome Browser view of the genomic region containing the Hgf gene. Vertical arrows point to the Foxf2-bound peak regions in E12.5 tongue tissues overlapping with the accessible chromatin regions in E10.5 mandibular tissues detected by ATAC-seq (Minoux et al., 2017). Asterisks mark the peaks containing the canonical Foxf1/Foxf2-binding motif. (B,C) Frontal views of E10.5 Smo^c/c control (B) and Smo^c/cHand2-Cre (C) embryos showing failure of Myf5-expressing tongue myogenic progenitor cells to enter the mandibular arch in the Smo^c/cHand2-Cre embryo in comparison with control littermates (n=3 for each genotype). Black arrowheads point to the Myf5-expressing tongue muscle precursor cells. (D,E) Rostral views of the mandibular arches showing patterns of Hgf mRNA expression in E10.5 Smo^c/c control (D) and Smo^c/cHand2-Cre (E) embryos detected by whole-mount in situ hybridization. Black arrows point to the domain of Hgf mRNA expression in the oral side of the mandibular arch (n=3 for each genotype). (F-I) Sagittal sections through the developing mandibular arch of the Smo^c/c control (F,H) and Smo^c/cHand2-Cre (G,I) embryos at E10.5 (F,G) and E11.5 (H,I), showing patterns of immunofluorescence staining of HGF (n=3 for each genotype at each stage). White arrows point to the domain of HGF expression in the tongue primordium and white arrowheads points to the domain of HGF expression in the caudal mandibular arch. (J,K) Sagittal sections through the mandibular arch of E11.5 Smo^c/c control (J) and Smo^c/cHand2-Cre (K) embryos showing patterns of immunofluorescence staining of MyoD. Yellow arrowheads point to the MyoD-positive tongue muscle precursor cells. (L,M) Sagittal sections through the mandibular arch of E11.5 Smo^c/c control (L) and Smo^c/cHand2-Cre (M) embryos showing patterns of c-Met mRNA expression (blue). Yellow arrowheads point to the domain of c-Met mRNA expression. t, tongue primordium. Scale bars: 500 μm (B-E); 200 μm (F-M).

Fig. 8.

Disruption of tongue myogenesis in the Foxf1^c/cFoxf2^c/cWnt1-Cre embryos correlated with loss of HGF expression in the tongue primordium. (A,B) Sagittal sections through the tongue and mandible of E16.5 control (A) and Foxf1^c/cFoxf2^c/cWnt1-Cre (B) embryos showing patterns of immunofluorescence staining of muscle actin (red). Asterisks mark the tongue muscle in the pharyngeal region. (C,D) Frontal views of E10.5 control (C) and Foxf1^c/cFoxf2^c/cWnt1-Cre (D) embryos showing Myf5-expressing tongue muscle precursor cells migrating to the caudal mandibular arch (n=3 for each genotype). Black arrowheads point to the Myf5-positive (purple) tongue muscle precursor cells. (E,F) Sagittal sections through the developing tongue primordium of E11.5 control (E) and Foxf1^c/cFoxf2^c/cWnt1-Cre (F) embryos showing patterns of immunofluorescence staining of HGF (magenta) and MyoD (green) (n=3 for each genotype). White arrows point to the expression of HGF in the tongue primordium. Yellow arrowheads point to the expression of HGF in the caudal mandibular arch. White arrowheads point to the MyoD-positive tongue muscle precursor cells. (G,H) Sagittal sections through the developing tongue of E12.5 control (G) and Foxf1^c/cFoxf2^c/cWnt1-Cre (H) embryos showing patterns of immunofluorescence staining of muscle actin (magenta) and MyoD (green). The hash (#) indicates the agenesis of oral tongue tissue in Foxf1^c/cFoxf2^c/cWnt1-Cre (F,H) embryos. t, tongue. Scale bars: 500 μm (A-D); 200 μm (E-H).

Fig. 9.

The roles of hedgehog-Foxf1/Foxf2 signaling in tongue morphogenesis. (A) At the initiation stage of tongue myogenesis, SHH-Foxf1/Foxf2 signaling regulates the expression of HGF in the CNCC-derived mandibular mesenchyme to direct the migration of c-Met+ myoblasts into the tongue primordium. (B) After myoblast arrival in the tongue primordium, Foxf1/Foxf2 in the CNCC-derived tongue mesenchyme control lingual tendon formation and subsequently the morphogenesis of intrinsic tongue muscles by regulating the expression Tgfb2 and Tgfb3. The CNCC-derived mesenchyme is indicated in blue, myogenic cells are indicated in magenta, Scx+ tendon progenitors in fibrous lingual septum at the midline and the dorsal submucosal lingual aponeurosis are indicated in green. os, occipital somites.