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. 2020 Jul 14;9:e55829. doi: 10.7554/eLife.55829

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© 2020, Hasan et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (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.

Figure 2—figure supplement 1. — (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.