FGFR2c-mediated ERK-MAPK activity regulates coronal suture development - PubMed (original) (raw)

FGFR2c-mediated ERK-MAPK activity regulates coronal suture development

Miles J Pfaff et al. Dev Biol. 2016.

Abstract

Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis.

Keywords: Coronal suture; Craniosynostosis; Crouzon syndrome; ERK–MAPK; FGF; FGFR2.

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Conflict of interest statement

Financial Disclosure

The author and all involved parties have no commercial associations or financial disclosures that would create a conflict of interest with the information presented.

Figures

Figure 1. Effects of loss-of-function and gain-of-function mutations in Fgfr2c on coronal suture patency and craniofacial development

A) Representative photographs of 3-month-old WT, _Fgfr2c_−/−, and Fgfr2cC342Y/+ mice. Both _Fgfr2c_−/− and Fgfr2cC342Y/+ mice present with dome-shaped skulls and a retruded midface. Ocular proptosis is also present in Fgfr2cC342Y/+ mice. B) Representative microCT images reveal patent sutures in WT mice and fusion of the coronal sutures in Fgfr2c−/− (black arrow) and Fgfr2cC342Y/+ mice (black arrowhead), with additional sutures fused in the Fgfr2cC342Y/+ mice. 2D cross-sectional images of the coronal suture demonstrate fusion in _Fgfr2c_−/− (white arrow) and Fgfr2cC342Y/+ mice (white arrowhead). C) Morphometric analysis of 3-month-old mouse skulls demonstrates more severe dysmorphia in Fgfr2cC342Y/+ mice compared to _Fgfr2c_−/− and WT controls. _Fgfr2c_−/− differed from WT controls with respect to the frontal height:skull height ratio; no difference was detected between Fgfr2c_−/− and Fgfr2cC342Y/+ mice (WT, Fgfr2c−/−, and Fgfr2c_C342Y/+: n = 5, 7, and 9 mice, respectively). c, coronal suture; s, sagittal suture; pf, posterior interfrontal suture; and o, occipital suture; fb, frontal bone; pb, parietal bone. *_P_≤0.05; **_P_≤0.01. Scale bars: 3 mm (A) and 5 mm (C).

Figure 2. Effects of loss-of-function and gain-of-function mutations in Fgfr2c on craniofacial skeletal growth

Alizarin red‒stained WT, _Fgfr2c_−/−, and Fgfr2cC342Y/+ mouse skulls at 1, 6, and 12 weeks of age. The _Fgfr2c_−/− and Fgfr2cC342Y/+ mice have fusion of the coronal sutures (arrows) at 1 week, with near complete fusion by 6 and 12 weeks. Note the patent anterior fontanel in _Fgfr2c_−/− mice (n=3 mice/group/time point). c, coronal suture.

Figure 3. Coronal suture development in loss-of-function and gain-of-function Fgfr2c mice

Representative Goldner’s trichrome (top) and von Kossa (bottom) stained histological sections of the coronal suture in WT, _Fgfr2c_−/−, and Fgfr2cC342Y/+ mice at 1, 6, and 12 weeks of age. At 1 week, WT mice show normal overlap of the frontal and parietal bones (arrows). _Fgfr2c_−/− mice have reduced cellular proliferation within the suture mesenchyme, with wide spacing between the osteogenic fronts of the frontal and parietal bones (arrowheads); Fgfr2cC342Y/+ mice have increased proliferation and mineralization of the mesenchyme, with trabeculae formation and suture fusion (asterisks). At 6 weeks of age, the coronal sutures in the _Fgfr2c_−/− and Fgfr2cC342Y/+ mice are completely fused (n=3 mice/group/time point). fb, frontal bone; pb, parietal bone; me, mesenchyme; tb, trabeculae. Scale bar: 50 μm for all images (10× objective for all images).

Figure 4. In vivo ERK-MAPK activation within the coronal suture of loss-of-function and gain-of-function Fgfr2c mice

Representative immunoblot and densitometric analysis of phospho-ERK and total-ERK levels within the coronal sutures of E18.5 through P1 _Fgfr2c_−/− and Fgfr2cC342Y/+ mice. The sutures in the _Fgfr2c_−/− mice have decreased levels of phospho-ERK (relative to total-ERK) compared to WT controls, whereas Fgfr2cC342Y/+ mice have significantly increased levels of phospho-ERK:total-ERK (n=4–5/group).

Figure 5. Calvarial osteoblast function and ERK-MAPK activation in loss-of-function and gain-of-function Fgfr2c mice

A) ALP-stained WT, _Fgfr2c_−/−, and Fgfr2cC342Y/+ calvarial osteoblasts revealed increased ALP staining in Fgfr2cC342Y/+ osteoblasts compared to WT cells, but no significant difference between WT and _Fgfr2c_−/− cells. B) ALP activity was significantly higher in Fgfr2cC342Y/+ cells compared to WT controls, with no difference between WT and _Fgfr2c_−/− cells. C and D) Similarly, Fgfr2cC342Y/+ cells have significantly increased mineralized matrix production, whereas _Fgfr2c_−/− osteoblasts did not difference from WT cells (results from two independent experiments). E) Immunoblot and densitometric analyses of osteoblasts revealed increased ERK phosphorylation in Fgfr2cC342Y/+ cells compared to WT controls, but no difference between WT and _Fgfr2c_−/− osteoblasts. F) ALP activity was significantly lower in U0126-treated Fgfr2cC342Y/+ osteoblasts compared to WT cells. *_P_≤0.05; **_P_≤0.01.

Figure 6. Effects of heterozygous gain-of-function mutations in combination of the loss of the WT Fgfr2c allele on coronal suture patency and craniofacial development

A) Representative alizarin red (top, dorsal view) and microCT (bottom; dorsal and lateral views) images reveal patent sutures in 10-day-old WT mice (arrow) and fused coronal sutures in Fgfr2cC342Y/+ mice and Fgfr2cC342Y/− mice (arrowheads). Note the severe midface retrusion with resulting class III malocclusion and a domed-shaped skull in the Fgfr2cC342Y/− mice (bottom; lateral view). B) Representative Goldner’s trichrome (top) and von Kossa (bottom) stained histological sections of the coronal suture of 10-day-old WT, Fgfr2cC342Y/+, and Fgfr2cC342Y/− mice show normal overlap of the frontal and parietal bones (arrows); Fgfr2cC342Y/+ and Fgfr2cC342Y/− mice have advanced trabecule formation and coronal suture fusion (arrowheads). C) Morphometric analysis of 10-day-old mouse skulls revealed more severe dysmorphia in the Fgfr2cC342Y/+ (n=12 mice) and Fgfr2cC342Y/− mice (n=7 mice) compared to WT controls (n=5 mice). c, coronal suture; fb, frontal bone; pb, parietal bone. **_P_≤0.01. Scale bars: 5 mm (A, top), 2 mm (A, bottom; lateral), and 200 μm (B); 20× objective (B).

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FGFR2c-mediated ERK-MAPK activity regulates coronal suture development - PubMed (original) (raw)