Midface and upper airway dysgenesis in FGFR2-related craniosynostosis involves multiple tissue-specific and cell cycle effects

Abstract

Midface dysgenesis is a feature of more than 200 genetic conditions in which upper airway anomalies frequently cause respiratory distress, but its etiology is poorly understood. Mouse models of Apert and Crouzon craniosynostosis syndromes exhibit midface dysgenesis similar to the human conditions. They carry activating mutations of Fgfr2, which is expressed in multiple craniofacial tissues during development. Magnetic resonance microscopy of three mouse models of Apert and Crouzon syndromes revealed decreased nasal passage volume in all models at birth. Histological analysis suggested overgrowth of the nasal cartilage in the two Apert syndrome mouse models. We used tissue-specific gene expression and transcriptome analysis to further dissect the structural, cellular and molecular alterations underlying midface and upper airway dysgenesis in Apert Fgfr2^+/S252W mutants. Cartilage thickened progressively during embryogenesis because of increased chondrocyte proliferation in the presence of Fgf2 Oral epithelium expression of mutant Fgfr2, which resulted in a distinctive nasal septal fusion defect, and premature facial suture fusion contributed to the overall dysmorphology. Midface dysgenesis in Fgfr2-related craniosynostosis is a complex phenotype arising from the combined effects of aberrant signaling in multiple craniofacial tissues.

Keywords: Apert syndrome; Crouzon syndrome; Fibroblast growth factor; Midface dysgenesis; Mouse; Nasal cartilage; Suture.

Fig. 1.

Nasal airspace and three regions of investigation in representative Fgfr2 mutant mice. (A) Lateral view of 3D reconstructions of MRM images of P0 Fgfr2^+/+ and Apert Fgfr2^+/S252W heads showing soft tissues (orange) and an endocast of the segmented nasal passages (green). Dashed lines indicate the three locations shown in (B) and in Movies 1 and 2. Scale bars: 1 mm. (B) Alcian Blue/Eosin staining of sections in the pars anterior (anterior), pars intermedia (midnasal) and pars posterior (posterior) regions of P0 Fgfr2^+/+ and Apert Fgfr2^+/S252W mice. These locations were used for quantitative analysis. Asterisks in the anterior mutant panel indicate flaring of the paraseptal region and pronounced narrowing of the adjacent nasal space. Single asterisks in the midnasal mutant panel indicate ectopic cartilage projecting from the paries nasi; double asterisks indicate lack of fusion between septum and anterior secondary palate. Scale bars: 500 µm.

Fig. 2.

Nasal airspace and cartilage changes in Fgfr2 mutant mice. (A) Anterior, (B) midnasal and (C) posterior nasal space area measurements. (D) Anterior, (E) midnasal and (F) posterior total cartilage area measurements. S252W, Apert Fgfr2^+/S252W; P253R, Apert Fgfr2^+/P253R; CZ, Crouzon Fgfr2^C342Y/+. Blue boxes, unaffected littermates (Fgfr2^+/+; WT); red boxes, mutant. n=4-15. Data are presented as the median, 25th and 75th percentiles; whiskers indicate minimum and maximum values. *P<0.05 (Student's t-test).

Fig. 3.

Increased chondrocyte proliferation occurs early in the Apert Fgfr2^+/S252W anterior nasal septum. (A) Fgfr2^+/+ and Apert Fgfr2^+/S252W anterior nasal septa co-stained for SOX9 (red, to visualize chondrocytes) and BrdU (green) at E12.5, pHH3 (red) and BrdU (green) at E14.5, E16.5, and E17.5, and pHH3 (red) and MKI67 (green) at P0. Dashed lines indicate boundaries of nasal septum (NS). Scale bars: 100 µm. (B-D) BrdU incorporation (B), pHH3 expression (C) and chondrocyte density (D) in the E14.5 Fgfr2^+/+ and Apert Fgfr2^+/S252W nasal septum. (E-G) BrdU incorporation (E), pHH3 expression (F) and chondrocyte density (G) in the E14.5 Fgfr2^+/+ and Apert Fgfr2^+/S252W paries nasi. Blue boxes, WT; red boxes, mutant. n=4-17. Data are presented as the median, 25th and 75th percentiles; whiskers indicate minimum and maximum values. *P<0.05 (Student's t-test).

Fig. 4.

Early fusion of the frontomaxillary suture in Apert Fgfr2^+/S252W mice. (A-D) The expression of RUNX2 protein (red) in the left and right frontomaxillary sutures of WT (A,B; n=3) and Apert Fgfr2^+/S252W (C,D; n=3) mice at E16.5 is shown. Sections are counterstained with Hoechst 33258. Scale bars: 100 µm. (E,G) Alcian Blue/Eosin-stained sections near-adjacent to the sections shown in A-D. Black boxes in E and G indicate the approximate locations of the frontomaxillary sutures of WT and Apert Fgfr2^+/S252W mice, respectively, shown in A-D. Red boxes indicate the areas enlarged in F and H. Scale bars: 500 µm. (F,H) Small cartilages external to the paries nasi in WT (F) and Apert Fgfr2^+/S252W (H) mice are indicated (dashed outlines). Scale bars: 100 µm. (I,K) The expression of SP7 protein in the frontomaxillary sutures of WT (I; n=7) and Apert Fgfr2^+/S252W (K; n=4) mice at E14.5 is shown. Dashed lines outline the frontal and maxillary bones in I and the fused bones in K. Scale bars: 100 µm. (J,L) Alcian Blue/Eosin-stained sections near-adjacent to the sections shown in I and K. Black boxes indicate the approximate locations of the frontomaxillary sutures of WT and Apert Fgfr2^+/S252W mice, respectively, shown in I and K. Scale bars: 500 µm. F, frontal bone; M, maxillary bone; PN, paries nasi.

Fig. 5.

Tissue-specific contributions to the P0 Apert Fgfr2^+/S252W midfacial phenotype. (A) Alcian Blue/Eosin staining of sections in the anterior, midnasal and posterior regions of WT and indicated Cre+;Fgfr2^+/S252W mice. Asterisks indicate ectopic cartilage projecting from the midnasal paries nasi in Col2a1-Cre+;Fgfr2^+/S252W mice. Scale bars: 500 µm. (B) Total cross-sectional area of cartilage in representative anterior, midnasal and posterior locations in the indicated tissue-specific Cre driver lines. (C) Total cross-sectional area of nasal space in representative anterior, midnasal, and posterior locations in the indicated tissue-specific Cre driver lines. Blue boxes, WT; red boxes, mutant. n=4-11. Data are presented as the median, 25th and 75th percentiles; whiskers indicate minimum and maximum values. *P<0.05 (Student's t-test).

Fig. 6.

Differential gene expression at E14.5 in Apert Fgfr2^+/S252W nasal cartilage and validation in both Apert mutants. (A) Anterior nasal septum cartilage was isolated from WT (n=3) and Apert Fgfr2^+/S252W (n=3) embryos by LCM to obtain RNA for RNA-seq analysis. Images show a representative section with nasal septum outlined (left) and a nearby Cresyl Violet-stained section after LCM (right). (B) Heat map (left) and GO analysis for significantly (P<0.05) enriched BP categories (right) of differentially expressed genes in Apert Fgfr2^+/S252W nasal cartilage compared with WT (FDR<0.01). (C,D) Fluorescent RNA ISH (green) for Top2a and Igfbp5 in E14.5 nasal septum cartilage (NS, within dashed lines) of Apert Fgfr2^+/S252W (n=3) (C) and Apert Fgfr2^+/P253R (n=3) (D) mice. White boxes in upper panels outline areas of enlargement shown in lower panels. Scale bars: 100 µm in upper panels; 50 µm in lower panels.