Fig 2: Effects of FGF10 in organ culture. (A–H) Explants viewed at day 6. (A) Organ fed basal media only, with high power of the boxed area shown in (B). SM (sm, arrowed) and epithelial (e) zones are indicated. (C,D) Organ fed with basal media supplemented with 5 ng/ml TGFβ1 has a cocoon‐like appearance. Interstitial zone indicated by asterisk. (E,F) Organ fed with basal media supplemented with 500 ng/ml FGF10. Note the apparent increased length versus organ fed basal media alone. (G,H) Organ fed with basal media supplemented with both 5 ng/ml TGFβ1 and 500 ng/ml FGF10. Cocooning is still apparent with prominent interstitial tissue (*) but the length of the tube appears increased versus the organ exposed to TGFβ1 alone. (I,J) Quantification of increases in urothelial tube length (I) and area (J) show that FGF10 (n = 12) caused significant linear growth versus basal media alone (n = 13). Addition of TGFβ1 (n = 13) caused significant reduction in linear and area growth versus basal media alone. When FGF10 was added together with TGFβ1 (n = 13) the negative effect of the latter on linear growth was overcome.

Fig 3: Gene expression analysis in Wt and Rab23-/- lambdoid suture.(A) Wt and Rab23-/- calvaria at E15.5 (dotted ring, excluding skin) processed for Illumina based microarray (n = 3 for each age and genotype). (B) Analysis of mRNA based microarray data by Chipster, a bioinformatics tool, reveals that 223 genes are differentially expressed (t-test, p<0.05, 115 genes are upregulated, red and 108 genes are downregulated, green) in Rab23-/- calvaria, represented by volcano plot (n = 3 for each age and genotype). Fold change of genes is calculated by arithmetic mean in linear scale and shown in the volcano plot. Fold change >1 (up-regulated gene), fold change < 1 (down-regulated gene). The gene list is provided in Supplementary file 1. (C) Represents individual searches of genes based on their contribution of suture fusion reveals Fgf10 as a candidate gene and Pitx2 as another developmentally important gene that can regulate Fgf10 expression. Both genes are upregulated in Rab23-/- calvaria. (D, E) RT-qPCR analysis of Fgf10 (D) and Pitx2 (E) mRNA extracted from E15.5 Wt and Rab23-/- calvaria (excluding skin), cultured calvaria derived primary cells (CDC) and from lambdoid sutural tissue reveals that Fgf10 and Pitx2 are overexpressed in Rab23-/- samples (n = 3 calvaria and eight lambdoid sutures for each genotype). Gene expressions were normalized by 18S rRNA. Data are represented as mean ± SD, paired Student’s t-test was used and as relative gene expression is shown using ΔΔCт values. Statistical significance was defined as a p-value <0.05 (*), p-value <0.005 (**). (F) Pitx2 expression analysis by whole-mount ISH in Wt and Rab23-/- calvaria at E15.5. Arrows indicate Pitx2 overexpression in the lambdoid suture. pb: parietal bone, ipb: interparietal bone, ls: lambdoid suture. Scale bar: 500 µm. (G) RT-qPCR expression analysis of Fgfr1b, Fgfr2b and Fgfr2c mRNA from E15.5 Wt and Rab23-/- lambdoid suture reveals Fgfr1b overexpression in Rab23-/- sample (n = 8 for each genotype). Gene expressions were normalized by 18S rRNA. Data are represented as mean ± SD, paired Student’s t-test was used and relative gene expression is shown using ΔΔCт values. Statistical significance was defined as a P-value <0.05 (*). (H, I) Western blotting of proteins extracted from Wt and Rab23-/- lambdoid suture at E15.5 (H) and relative intensity measurement (I) reveals over expression of FGFR1 in the Rab23-/- lambdoid suture (n = 6 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a p-value <0.05 (*). (J) Exogenous FGF10 treatment for 3 hr on Wt calvaria derived (CD) cells and subsequent Fgfr1b and Fgfr2b mRNA analysis by q-PCR shows induction of Fgfr1b expression in FGF10 treated cells compare to untreated Wt CD cells (n = 3 for each genotype). Gene expressions were normalized by 18S rRNA. Data are represented as mean ± SD, paired Student’s t-test was used and relative gene expression is shown using ΔΔCт values. Statistical significance was defined as a P-value.

Fig 4: Model: regulation of suture patency by RAB23.In the developing calvaria RAB23 regulates FGFR signaling by repressing FGF10 expression. This regulates the delicate balance of osteoprogenitor proliferation and differentiation. RAB23 also known as a negative regulator of Hh signaling. The regulation of GLI1 through ERK1/2 and Hh in the mesenchymal stem cell niche is maintained along with RUNX2 during osteoprogenitor proliferation and differentiation. Thus, through a combination of FGF and Hh signaling, RAB23 tightly synchronizes suture morphogenesis and patency.

Fig 5: Analysis of MAPK signaling in Wt and Rab23-/- lambdoid suture.(A) Represents two downstream pathway subtypes p38 and ERK of MAPK signaling involve in RUNX2 activation and suture fusion. (B, C) Western blotting analysis of pERK44/42 and α-ERK44/42 protein levels extracted from Wt and Rab23-/- lambdoid suture at E15.5 (B) and relative intensity measurement (C) shows higher pERK44 and pERK42 levels in Rab23-/- lambdoid suture (n = 6 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a P-value <0.05 (*). (D, E) Western blotting analysis of phospho-p38 and p38 protein levels extracted from Wt and Rab23-/- lambdoid suture at E15.5 (D) and relative intensity measurement (E) shows lower phospho-p38 and p38 levels in Rab23-/- lambdoid suture (n = 6 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a p-value <0.05 (*), p-value <0.005 (**). (F, G) Western blotting analysis of RUNX2 protein level extracted from Wt and Rab23-/- lambdoid suture at E15.5 (F) and relative intensity measurement (G) shows higher RUNX2 level in Rab23-/- lambdoid suture (n = 6 for each genotype). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a p-value <0.05 (*). (H) Runx2 expression analysis by RT-qPCR in exogenous FGF10 treated (500 ng/ml) and untreated Rab23-/- calvaria derived cells at 2 and 4 hr. (n = 3 for each genotype). Gene expressions were normalized by 18S rRNA. Data are represented as mean ± SD, paired Student’s t-test was used and relative gene expression is shown using ΔΔCт values. Statistical significance was defined as a P-value <0.05 (*), P-value <0.005 (**). (I, J) RT-qPCR expression analysis of hedgehog signaling components Hh (H), Gli1, Gli2 and Gli3 (I) in the Wt and Rab23-/- lambdoid suture reveals overexpression of Hh and Gli1 in Rab23-/- lambdoid suture (n = 8 for each genotype). Gene expressions were normalized by 18S rRNA. Data are represented as mean ± SD, paired Student’s t-test was used and relative gene expression is shown using ΔΔCт values. Statistical significance was defined as a P-value <0.05 (*), P-value <0.005 (**). n.s: non-significant.