Mandibular dysmorphology due to abnormal embryonic osteogenesis in FGFR2-related craniosynostosis mice

Susan M Motch Perrine, Meng Wu, Nicholas B Stephens, Divya Kriti, Harm van Bakel, Ethylin Wang Jabs, Joan T Richtsmeier, Susan M Motch Perrine, Meng Wu, Nicholas B Stephens, Divya Kriti, Harm van Bakel, Ethylin Wang Jabs, Joan T Richtsmeier

Abstract

One diagnostic feature of craniosynostosis syndromes is mandibular dysgenesis. Using three mouse models of Apert, Crouzon and Pfeiffer craniosynostosis syndromes, we investigated how embryonic development of the mandible is affected by fibroblast growth factor receptor 2 (Fgfr2) mutations. Quantitative analysis of skeletal form at birth revealed differences in mandibular morphology between mice carrying Fgfr2 mutations and their littermates that do not carry the mutations. Murine embryos with the mutations associated with Apert syndrome in humans (Fgfr2+/S252W and Fgfr2+/P253R ) showed an increase in the size of the osteogenic anlagen and Meckel's cartilage (MC). Changes in the microarchitecture and mineralization of the developing mandible were visualized using histological staining. The mechanism for mandibular dysgenesis in the Apert Fgfr2+/S252W mouse resulting in the most severe phenotypic effects was further analyzed in detail and found to occur to a lesser degree in the other craniosynostosis mouse models. Laser capture microdissection and RNA-seq analysis revealed transcriptomic changes in mandibular bone at embryonic day 16.5 (E16.5), highlighting increased expression of genes related to osteoclast differentiation and dysregulated genes active in bone mineralization. Increased osteoclastic activity was corroborated by TRAP assay and in situ hybridization of Csf1r and Itgb3 Upregulated expression of Enpp1 and Ank was validated in the mandible of Fgfr2+/S252W embryos, and found to result in elevated inorganic pyrophosphate concentration. Increased proliferation of osteoblasts in the mandible and chondrocytes forming MC was identified in Fgfr2+/S252W embryos at E12.5. These findings provide evidence that FGFR2 gain-of-function mutations differentially affect cartilage formation and intramembranous ossification of dermal bone, contributing to mandibular dysmorphogenesis in craniosynostosis syndromes.This article has an associated First Person interview with the joint first authors of the paper.

Keywords: Apert syndrome; Cartilage; Crouzon syndrome; FGFR2; Osteoclast; Pfeiffer syndrome; Transcriptome.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

© 2019. Published by The Company of Biologists Ltd.

Figures

Fig. 1.
Fig. 1.
Morphological differences in newborn (P0) mice carrying mutations associated with three FGFR2-related craniosynostosis syndromes and their unaffected littermates. (A-F) Results of PCA of mandibles based on unique linear distances among 3D landmarks (A-C) and EDMA of landmark coordinates (D-F). Scatter plots of individual scores on first and second PC axes (PC1 and PC2) of linear distance-based PCAs of the hemimandibles of mutant and unaffected littermates of Fgfr2+/S252W and Fgfr2+/P253R Apert syndrome mouse models (A,B, respectively) and Fgfr2cC342Y/+ Crouzon/Pfeiffer syndrome mouse model (C). Results of EDMA of each craniosynostosis mouse model and unaffected littermates showing linear distances within each model that are significantly different by at least 5% between mutant and unaffected littermates (D-F). Blue lines are significantly larger in mutant mice relative to unaffected littermates; fuchsia lines are significantly smaller in mutant mice. Thin lines indicate linear distances that are increased/decreased by 5-10% in mice carrying one of the Fgfr2 mutations whereas thick lines indicate linear distances that differ by >10% between mutant and unaffected mice. The buccal aspects of the left hemimandibles of the models were used for illustration. Hemimandibles were segmented into an anterior portion (anterior body, blue) and posterior portion (ramus, red) to indicate functional areas. Scale bars: 1 mm.
Fig. 2.
Fig. 2.
Histological analysis of mandible of Fgfr2+/S252W embryos at E16.5. (A) Schematic embryonic mouse head at E16.5 modified from the e-Mouse Atlas Project (http://www.emouseatlas.org/emap/eHistology). The red line indicates the location of sections used for B-O. (B) Cryosections of Fgfr2+/+ and Fgfr2+/S252W embryos were stained with the ALP assay (red) and Alcian Blue. MB, mandibular bone; MC, Meckel's cartilage; T, tongue. (C) The ALP-positive regions (red) were selected to quantify the areas and numbers of nuclei stained with Hoechst 33258 (blue). (D-I) The areas (D,G), cell numbers (E,H) and cell density (F,I) in the ALP-positive regions for Fgfr2+/+ (n=6) and Fgfr2+/S252W (n=6) embryos and MC of Fgfr2+/+ (n=6) and Fgfr2+/S252W (n=6) embryos. (J,M) Alizarin Red S staining (J) and von Kossa staining (M) showing ossification in the mandible of Fgfr2+/+ and Fgfr2+/S252W embryos. The areas and the percentages of the stained area in osteogenic tissue were measured for Alizarin Red S (K,L) and von Kossa (N,O) staining. Data are mean±s.e.m. *P<0.05, two-tailed Welch's t-test. Scale bars: 100 µm.
Fig. 3.
Fig. 3.
Laser capture microdissection and RNA-seq analysis of mandibular bone of Fgfr2+/S252W embryos at E16.5. (A) A representative mandibular region in cryosection was dissected by laser and collected for RNA-seq (left, before LCM; right, after LCM). (B) Hierarchical clustering of 122 genes significantly differentially expressed in the mandibular bone between Fgfr2+/S252W and Fgfr2+/+ littermate embryos. Three biological replicates were used for each genotype. (C) Volcano plot shows P-values and fold changes of DEGs in the mandibular bone between Fgfr2+/S252W and Fgfr2+/+ littermate embryos. Some of the most significantly differentially expressed genes [−log10(P-value)>4.5] implicated in mandibular dysmorphology are shown in blue. Scale bars: 400 µm.
Fig. 4.
Fig. 4.
Increased osteoclastogenesis in the mandibular bone of Fgfr2+/S252W embryos at E16.5. (A-H) The differential expression of Csf1r and Itgb3 in the mandible of Fgfr2+/+ (A,B,E,F) and Fgfr2+/S252W (C,D,G,H) embryos were validated by in situ hybridization (ISH). B,D,F and H show higher magnification of the boxed areas in A,C,E and G, respectively. (I-J) TRAP assay stained osteoclasts (purple) in the mandible of Fgfr2+/+ (I) and Fgfr2+/S252W (J) embryos. (K-L) Quantitative measurements of the density (K) and percentage (L) of osteoclasts in the bone area of Fgfr2+/+ (n=3) and Fgfr2+/S252W (n=3) embryos. Data are mean±s.e.m. *P<0.05, two-tailed Welch's t-test. Scale bars: 100 µm.
Fig. 5.
Fig. 5.
Increased expression of Enpp1 and Ank and elevated PPi concentration in the mandible of Fgfr2+/S252W embryos at E16.5. (A-H) RNA expression of Enpp1 and Ank in the mandible of Fgfr2+/+ littermate (A,B,E,F) and Fgfr2+/S252W embryos (C,D,G,H) was validated using ISH. B,D,F and H show higher magnification of the boxed areas in A,C,E and G, respectively. The weight of the mandible (I) and PPi concentration in the mandible (J) were measured for Fgfr2+/+ littermates (n=7) and Fgfr2+/S252W (n=11) embryos at E16.5. Data are mean±s.e.m. *P<0.05, two-tailed Welch's t-test. Scale bars: 100 µm.
Fig. 6.
Fig. 6.
Increased cell proliferation of osteoblasts and chondrocytes in the mandible of Fgfr2+/S252W embryos. (A) The osteoblasts in the mandibular bone at E16.5 were visualized using IHC for RUNX2. Boxed areas are shown at higher magnification on the right, respectively. (B) Double staining with EdU assay (green) and IHC for RUNX2 (red) at E12.5. (C) The percentage of proliferating osteoblasts (EdU-positive) in the total osteoblasts (RUNX2-positive) is shown for Fgfr2+/+ (n=4) and Fgfr2+/S252W (n=4) embryos. (D) EdU assay (green) with IHC for SOX9 (red) in MC at E12.5. (E) The percentage of proliferating cells (EdU-positive) in MC (SOX9-positive) is shown for Fgfr2+/+ (n=4) and Fgfr2+/S252W (n=4) embryos. Data are mean±s.e.m. *P<0.05, two-tailed Welch's t-test. Scale bars: 100 µm.
Fig. 7.
Fig. 7.
Proposed model of mandibular dysmorphogenesis in prenatal development of Fgfr2+/S252W mice. In the mandible of Fgfr2+/+ mice, FGFR2 signaling is activated by specific FGF binding, forming a complex of FGFs, heparan sulfate and FGFRs. The linker region between the immunoglobulin-like domains II and III regulates the ligand binding specificity and affinity. Dimerization and transphosphorylation by kinases in the intracellular domain of FGFR2 cause activation of downstream signaling cascades. These activated signaling pathways can regulate gene expression, controlling osteogenesis, osteoclastogenesis and chondrogenesis. Transcriptional level: The FGFR2 S252W mutation alters ligand specificity and affinity, resulting in abnormal FGFR2 signaling, which dysregulates the transcriptome in different cell types. Tissue level: Osteoblast proliferation is activated, enlarging the osteogenic tissue. Increased PPi inhibits mineralization, and bone resorption is promoted through osteoclastic activity, causing a change in the microarchitecture. Meckel’s cartilage is affected by increased proliferation of chondrocytes, resulting in an increase in size. Morphological level: Abnormal osteogenic activities contribute to changes in mandibular shape.

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