Increased FGF8 signaling promotes chondrogenic rather than osteogenic development in the embryonic skull

ABSTRACT The bones of the cranial vault are formed directly from mesenchymal cells through intramembranous ossification rather than via a cartilage intermediate. Formation and growth of the skull bones involves the interaction of multiple cell-cell signaling pathways, with fibroblast growth factors (FGFs) and their receptors exerting a prominent influence. Mutations within the FGF signaling pathway are the most frequent cause of craniosynostosis, which is a common human craniofacial developmental abnormality characterized by the premature fusion of the cranial sutures. Here, we have developed new mouse models to investigate how different levels of increased FGF signaling can affect the formation of the calvarial bones and associated sutures. Whereas moderate Fgf8 overexpression resulted in delayed ossification followed by craniosynostosis of the coronal suture, higher Fgf8 levels promoted a loss of ossification and favored cartilage over bone formation across the skull. By contrast, endochondral bones were still able to form and ossify in the presence of increased levels of Fgf8, although the growth and mineralization of these bones were affected to varying extents. Expression analysis demonstrated that abnormal skull chondrogenesis was accompanied by changes in the genes required for Wnt signaling. Moreover, further analysis indicated that the pathology was associated with decreased Wnt signaling, as the reduction in ossification could be partially rescued by halving Axin2 gene dosage. Taken together, these findings indicate that mesenchymal cells of the skull are not fated to form bone, but can be forced into a chondrogenic fate through the manipulation of FGF8 signaling. These results have implications for evolution of the different methods of ossification as well as for therapeutic intervention in craniosynostosis.


Supplemental Figure 2. Embryonic expression of Msx2-Cre during craniofacial development.
Msx2-Cre mediated recombination was visualized using β-galactosidase staining (blue/green) of ROSA26 LacZ Reporter embryos. (A-C) Lateral views of whole mount β-galactosidase staining on E10.5 (A) and E11.5 (B) embryos. (C) At E16.5, almost the entire dorsal aspect of the head showed β-galactosidase staining (lateral view on the left, dorsal view on the right). Red and blue triangles denote regions over part of the parietal and intraparietal bones, respectively, where Msx2-Cre mediated recombination did not occur as efficiently. Black lines in B and C indicate plane of section in D, F and E, G respectively. (D-G) β-galactosidase staining of frozen sections at E11.5 in a frontal plane (D, F) and E16.5 in a sagittal plane (E, G), counterstained with nuclear fast red. F and G are magnifications of the regions outlined by black boxes in D and E, Disease Models & Mechanisms 11: doi:10.1242/dmm.031526: Supplementary information respectively. Sections are shown at both 10x (D, E) and 40x (F, G) magnification. Note in E, bone (arrow) and cartilage (ct), do not show β-galactosidase staining, whereas staining occurs in both ectoderm (boxed) and nasal epithelium ( Low magnification images in (A, B) show the approximate position of the lambdoid suture (Lb) from P12 sagittal sections. Sagittal sections from skulls of P0 (C-F) and P12 (G-J) control (C, E, G, I) and MR26F8 (D, F, H, J) pups. Sections (A-D, G-H) were stained with von Kossa, such that mineralized bone appears dark red/black. Sections (E-F, I-J) were stained with Goldner's Trichome stain such that mature bone matrix appears green whereas immature bone matrix stains red. In the P0 sections, blue arrowheads mark the limits of mineralization (C-D); red arrowheads show the extent of the unmineralized mature osteoid (E-F). In F, only one side of the mature osteoid is shown, with a mixture of mature and immature bone matrix occurring between the mature osteoid in the mutant as shown in greater detail in the inset (red arrow). The bar graph (K) shows the width of the lambdoid sutures at P0 and P12. Comparison of the width between the controls and MR26F8s is significantly different at both P0 (p<0.0001) and P12 (p<0.0003). Bars show average suture width (µM); error bars denote standard error within the group. Scale Bars: A-B: 1mm; C-J: 100µM. Aged-matched controls and mutants are at the same scale. mutants. In addition, the skulls of the mutants were more fragile than the controls and so it was only possible to clear the embryos for a limited time before they were too damaged for photodocumentation. This leads to greater trapping of alcian blue, especially in (H), than would normally occur. Scale bars= 1mm.
(A-C): Sagittal sections of E16.5 control (A) or OCAGF8 (B, C) skulls stained with toluidine blue and methyl green. Red lines superimposed on E16.5 embryo heads show plane of dissection. Note the cartilage in the dorsal (C), but not lateral (B) OCAGF8 E16.5 skull. In contrast, both the dorsal (A) and lateral skull sections (not shown) were similar in the controls. In C, the cartilage/lacunae morphology can be visualized in the lighter stained cartilage (top), whereas the characteristic reddish purple toluidine cartilage stain can be seen in the darker stained cartilage (bottom). Abbreviations: Ct, cartilage; SE, surface epithelium. Scale Bars = 80 µM.  Table 1. Gene comparison of Wildtype Cranial Vault vs. MCAGF8 Unless otherwise specified, all data are derived from comparing wildtype (WT, Control) to MCAGF8 (Mutant, Treatment) cranial vault. Cranial vault tissue was collected from 9 E14.5 control and mutant embryos as outlined in Fig. 7.

Supplemental
Tab1: All genes reported from RNA-seq sorted by ENSEMBL ID. Means are expressed as RPKM.
Tab3: Genes listed in text, sorted by order of appearance. Left: wildtype cranial vault vs mutant cranial vault; Right: wildtype cranial base vs mutant. Blue highlighted cells are genes downregulated <-1.5; yellow highlighted cells are genes upregulated >1.5. Significant P-values (p<0.05) highlighted in green.

Tab6
: Relevant values and histogram plot of normalized RPKM values of genes associated with BMP signaling and significantly dysregulated in MCAGF8 cranial vault samples (orange) as compared to controls (blue). Error bars represent relative standard error calculated from 3 replicates.

Tab7
: Relevant values and histogram plot of normalized RPKM values of genes associated with Hedgehog signaling and significantly dysregulated in MCAGF8 cranial vault samples (orange) as compared to controls (blue). Error bars represent relative standard error calculated from 3 replicates.
Tab8: Top functional annotation clusters as calculated by DAVID using all significant genes (p<0.05).
Tab9: Top functional annotation charting as calculated by DAVID using significant genes (p<0.05) that have a fold change of >1.5 or <-1.5. Unless otherwise specified, all data are derived from comparing wildtype cranial base (WT, Control) and MCAGF8 (Mutant, Treatment) cranial vault tissue. Cranial tissue was collected from 9 E14.5 control and MCAGF8 embryos as outlined in Fig. 7.

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Tab1: All genes reported from RNA-seq sorted by ENSEMBL ID. Means are expressed as RPKM.

Tab5
: Top functional annotation clusters as calculated by DAVID using all significant genes (p<0.05).
Tab6: Top functional annotation charting as calculated by DAVID using significant genes (p<0.05) that have a fold change of >1.5 or <-1.5.