Augmentation of BMP Signaling in Cranial Neural Crest Cells Leads to Premature Cranial Sutures Fusion through Endochondral Ossification in Mice

Abstract

Craniosynostosis is a congenital anomaly characterized by the premature fusion of cranial sutures. Sutures are a critical connective tissue that regulates bone growth; their aberrant fusion results in abnormal shapes of the head and face. The molecular and cellular mechanisms have been investigated for a long time, but knowledge gaps remain between genetic mutations and mechanisms of pathogenesis for craniosynostosis. We previously demonstrated that the augmentation of bone morphogenetic protein (BMP) signaling through constitutively active BMP type 1A receptor (caBmpr1a) in neural crest cells (NCCs) caused the development of premature fusion of the anterior frontal suture, leading to craniosynostosis in mice. In this study, we demonstrated that ectopic cartilage forms in sutures prior to premature fusion in caBmpr1a mice. The ectopic cartilage is subsequently replaced by bone nodules leading to premature fusion with similar but unique fusion patterns between two neural crest-specific transgenic Cre mouse lines, P0-Cre and Wnt1-Cre mice, which coincides with patterns of premature fusion in each line. Histologic and molecular analyses suggest that endochondral ossification in the affected sutures. Both in vitro and in vivo observations suggest a greater chondrogenic capacity and reduced osteogenic capability of neural crest progenitor cells in mutant lines. These results suggest that the augmentation of BMP signaling alters the cell fate of cranial NCCs toward a chondrogenic lineage to prompt endochondral ossification to prematurely fuse cranial sutures. By comparing P0-Cre;caBmpr1a and Wnt1-Cre;caBmpr1a mice at the stage of neural crest formation, we found more cell death of cranial NCCs in P0-Cre;caBmpr1a than Wnt1-Cre;caBmpr1a mice at the developing facial primordia. These findings may provide a platform for understanding why mutations of broadly expressed genes result in the premature fusion of limited sutures. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: BMP SIGNALING; CELL FATE; CRANIOSYNOSTOSIS; P0‐Cre; Wnt1‐Cre.

Fig. 1

Skull deformities of Wnt1‐Cre;caBmpr1a and P0‐Cre;caBmpr1a mice. (A) Representative lateral and top views of head of Wnt1‐Cre;caBmpr1a and P0‐Cre;caBmpr1a mice at postnatal day 17 (P17) (n = 6). Wnt1‐Cre;caBmpr1a and P0‐Cre;caBmpr1a mice display short/broad snouts and hypertelorism. (B, C) Micro‐computed tomography (μCT) image at P17 showed suture fusion in Wnt1‐Cre;caBmpr1a (n = 11) and P0‐Cre;caBmpr1a (n = 6) mice. Representative lateral view, top view, and enlarged top view are shown (B). Two‐dimensional sections of μCT image at AF suture and naso‐premaxillary (naso‐premax) suture (C). Sutures that caused premature fusion in mutant mice are indicated by red arrows (AF suture) and red arrowheads (naso‐max suture), respectively. (D) Length of the nasal bone (a), the frontal bone (b), the parietal bone (c), and between eyes (d) is measured using μCT image. Relative length of the nasal bone divided by length of the frontal bone (a/b), length of the frontal bone divided by length of the parietal bone (b/c), and length of the nasal and frontal bone divided by length of the parietal bone ((a + b)/c) are quantified. White bars, control mice; blue bars, Wnt1‐Cre;caBmpr1a mice; red bars, P0‐Cre;caBmpr1a mice. Student's t test or one‐way ANOVA with Tukey's test were used for the statistical analysis. *p < 0.05, **p < 0.01. Scale bars: 5 mm (A), 2 mm (B), 1 mm (C).

Fig. 2

Histological observation of respective sutures in control, Wnt1‐Cre;caBmpr1a mice, and P0‐Cre;caBmpr1a mice. (A–F) Hematoxylin and eosin staining for nasal‐frontal suture (A), nasal‐posterior suture (B), naso‐premaxillary suture (C), anterior frontal suture (D), sagittal suture (E), and coronal suture (F) of control mice (n = 6), Wnt1‐Cre;caBmpr1a mice (n = 6), and P0‐Cre;caBmpr1a mice (n = 6), respectively. Frontal bone (f) and posterior bone (p) of coronal suture. (G) Schematic image of mouse skull indicating sutures. Arrowheads indicate patent sutures. Scale bars: 1 mm (A–F), 500 mm (A, B, boxed area), and 200 μm (C–F).

Fig. 3

Spatial correlations of ectopic cartilage and premature fusion of cranial sutures in Wnt1‐Cre;caBmpr1a and P0‐Cre;caBmpr1a mice. (A) Ectopic cartilage in head stained with Alcian blue solution at newborn (NB), postnatal day 1 (P1), and P2 (n = 6). Ectopic cartilage in anterior frontal (AF) suture is indicated by red arrows. (B) Calvaria formation of control mice and P0‐Cre;caBmpr1a mice stained with Alizarin red solution at NB, P1, and P2. Ectopic bone nodules in the AF suture are indicated by red arrowheads. (C) Ectopic cartilage in AF suture of P0‐Cre;caBmpr1a mice at NB was histologically detected with Alcian blue solution. (D) Whole cartilage staining with Alcian blue of control, Wnt1‐Cre;caBmpr1a, and P0‐Cre;caBmpr1a mice at NB are shown. (E, F) Ectopic cartilage in naso‐premaxillary suture (E) and AF suture (F) were histologically stained with Alcian blue, SOX9, and pSMAD 1/5/9 (n = 6). The ectopic cartilage in the naso‐premaxillary suture is indicated by white arrowheads, and the ectopic cartilage in the AF suture is indicated by white arrows. Osteogenic front of both the naso‐premaxillary suture and the AF suture are indicated by dotted lines. (G) Histological assessments for presence of ectopic bone formation in relation to positions of ectopic cartilage by Alcian blue staining followed by hematoxylin staining. Black arrows indicate patent AF suture. Black arrowheads indicate ectopic cartilage. Boxed area in (G) is enlarged. (H, I) Double immunohistochemistry for COL1 (red) and COL2 (green) at NB, P3, and P6 (H), and immunohistochemistry for COL10 or CD31 (I) are shown. A yellow arrowhead in (H ) indicates a COL1‐positive cell in ectopic cartilage at NB. Nuclei were stained with DAPI. Dotted lines indicate AF suture in control mice. Scale bars: 1 mm (A, B, D), 100 μm (C, E, F, H, I), 500 μm (G).

Fig. 4

Cranial neural crest stem cells (NCSCs) with enhanced BMP signaling showed higher capacity for chondrogenic differentiation. (A) Cells isolated from control or P0‐Cre;ca‐Bmpr1a mutant embryos labeled with R26R ^mTmG reporter were visualized by fluorescent microscopy before and after FACS. (B) RT‐PCR results of neural crest and stem cell markers in NCSCs at passage 18 (n = 3). (C) Alkaline phosphatase staining and Safranin O staining of cranial NCSCs with or without osteogenic or chondrogenic induction, respectively (n = 3). (D, E) Chondrogenic induction of control and P0‐Cre;caBmpr1a cranial NCSCs after 14 days of culture with or without chondrogenic induction. Alcian blue staining and representative pellets are shown (D). The diameter of the pellets was measured. Relative gene expression of chondrogenic markers of control and P0‐Cre;ca‐Bmpr1a cranial NCSCs were quantified by quantitative RT‐PCR (E) (n = 6). Basal, cells cultured with basal medium; chondrogenic (Genic), cells cultured with chondrogenic medium. One‐way ANOVA with Tukey's test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 400 μm (A), 100 μm (C), 50 μm (D, sections), 1 mm (D, pellets).

Fig. 5

Cranial NCCs with enhanced BMP signaling showed a lower capacity for osteogenic differentiation. (A) Representative embryos at E11.5. Nasal process (NP) cells (green area) at E11.5 were isolated then induced chondrogenic and osteogenic differentiation. Capability of osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining and quantitative RT‐PCR for osteogenic marker genes. Capability of chondrogenic differentiation was evaluated by Alcian blue (AB) staining and quantitative RT‐PCR for chondrogenic marker genes. (C) The calvarial formation at E15.5 was assessed by ALP staining and AB staining. Student's t test was used for statistical analysis. Each p value is shown on the data. Scale bars: 1 mm (A, B), 500 μm (C).

Fig. 6

The distribution patterns of Wnt1‐Cre expressing and P0‐Cre expressing NCCs. Wnt1‐Cre and P0‐Cre mice were crossed with tdTomato and LacZ reporter mice to visualize NCCs, respectively. (A) NCCs derivatives at E16.5 were histologically revealed by LacZ staining (n = 6). Suture mesenchyme at nasal suture (nasal, left column), the premaxillary suture (premaxillary, center column), and anterior frontal (AF) suture (right column) are shown, respectively. (B) Quantification of LacZ‐negative area in suture mesenchyme. (C) NCCs at E12.5, E14.5, and E16.5 were visualized with tdTomato (n = 6). Representative sections of tdTomato staining in both P0‐Cre and Wnt1‐Cre mice at E12.5 are shown. Arrows indicate place showing higher tdTomato fluorescence in P0‐Cre mice than Wnt1‐Cre mice. Arrowheads indicate region where tdTomato signal is found in Wnt1‐Cre line but not in P0‐Cre line. Student's t test was used for statistical analysis. N.S., no significance. Scale bars: 500 μm (A, C).

Fig. 7

Effect of augmented BMP signaling in NCCs at early embryonic stage. (A) Whole LacZ staining and histological LacZ staining for medial nasal process (MNP) at E10.5 (n = 6). Counterstaining for nuclei was performed with nuclear fast red staining. (B) Activation of BMP‐SMAD signaling in MNP and lateral nasal process (LNP) was analyzed by phospho‐SMAD 1/5/9 staining (n = 6). (C) Cell death in MNP was revealed by TUNEL staining (n = 6). (D, E) Number of positive cells for p‐SMAD1/5/9 or TUNEL in each transgenic mouse was quantified. One‐way ANOVA with Tukey's test was used for statistical analysis. NS, no significance; * p < 0.05, ** p < 0.01. Scale bars: 100 μm.