Fig 1: Modulation of WNT signaling controls AF closure. (A-D) Wholemount Alizarin Red and Alcian Blue staining at E18.5 and P5 showing that Scx-Cre;Ctnnb1GOF mice exhibit a wide-open AF that fails to close compared to littermate controls. n=3 littermate pairs per stage. (E-G) A WNT inhibitor cocktail composed of recombinant mouse WIF1 protein and vantictumab was administered at P3 through a single injection under the scalp in the area above the AF. (E) Wholemount Alizarin Red and Alcian Blue staining of a treated control collected at P7 showed that the treatment had no negative effects on calvarial bone formation and AF closure. (F) Wholemount skeletal stains of three treated Wnt1-Cre;Fgfr2−/− littermates showed that AF closure is partially restored. (G) Quantification of the AF area at P7 in control and Wnt1-Cre;Fgfr2−/− mice that were treated (Tx) with WIF1/vantictumab at P3 or untreated.
Fig 2: Bulk RNA-seq reveals transcriptional differences in the AF of Wnt1-Cre;Fgfr2−/− embryos. (A) Diagram of the E18.5 mouse calvarium showing the region (box) that was analyzed. Heatmap analysis revealing overall differences in gene expression between the AF of Wnt1-Cre;Fgfr2−/− (top rows) and littermate controls (bottom rows) (n=5 each for control and Wnt1-Cre;Fgfr2−/−). (B) GO analysis showing that differentially regulated genes were associated with focal adhesion, axon guidance, and WNT signaling. (C) Dot plots of differentially expressed genes associated with the WNT pathway showing that Sfrp4, Notum, Wif1, and Lgr6 were downregulated in Wnt1-Cre;Fgfr2−/− mutants compared to controls. By contrast, Lgr5, Wnt11, and Sfrp2 were upregulated in Wnt1-Cre;Fgfr2−/− mutants compared to controls. (D) String analysis inferred interactions between these differentially expressed WNT pathways members with Fgfr2 and Fgf18. n=5 for control and Wnt1-Cre;Fgfr2−/− littermate pairs.
Fig 3: Differentially expressed WNT pathway members correspond to distinct populations within the AF. (A,A′) Violin plots (A) of WNT pathway members showing their cluster-specific enrichment in the developing AF at E18.5, with corresponding UMAP (A′). Expression of genes encoding the WNT receptor Fzd1, WNT ligands Wnt9a and Wnt11, and the WNT signaling target Lgr5 were enriched in the AF1, AF2, and EM clusters, while expression of the WNT inhibitor Wif1 was enriched in osteogenic populations OF1, OF2, and OB. (B) Cell communication analysis using CellChat predicted that EM is a crucial source of WNT ligand for AF1 and AF2. (C) Co-expression analysis of Fgfr2 and Wif1 showed significant overlap in OF1 and OF2 clusters (white cells). (D,E) RNAScope in situ hybridization in the AF at E18.5 identified low level expression of Lgr5 throughout both ecto- and endocranial domains in the control. In the Wnt1-Cre;Fgfr2−/− mutant AF, the domain of Lgr5 was expanded laterally towards the osteogenic fronts (asterisks). (F,G) Lgr6 was expressed throughout the ecto- and endocranial domains of the AF in control, but expression within the endocranial domain was lost in the Wnt1-Cre;Fgfr2−/− mutant (arrowhead). (H,I) In control, Wif1 was expressed throughout the osteogenic fronts. In the Wnt1-Cre;Fgfr2−/− mutant, Wif1 expression was greatly decreased (arrowheads). Dashed lines delineate the frontal bones. n=3 littermate pairs.
Fig 4: Recruitment model for frontal suture formation within the AF. A gradient of WNT signaling maintains cells of the AF in an undifferentiated state. As FGFR2+ cells of the osteogenic fronts advance, activated by FGF from the ectocranial mesenchyme, they secret WIF1 to locally inhibit WNT signaling in the AF. Downregulation of WNT signaling induces SCX+ cells in the ectocranial domain to form intramembranous bone, and SCX+/SOX9+ cells in the endocranial domain to form cartilage. Recruited SCX+ cells form the frontal suture, while SCX+ cells that remain outside the domain of WNT inhibition give rise to suprasutural ligament. Upon loss of FGFR2 signaling, WNT signaling remains high and AF cells fail to form the frontal suture joint.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse WIF-1 Protein, CF