Tmprss6^–/– mice show increased Fgf23 expression in the BM. (A) Fgf23 mRNA expression relative to Actb in the total BM mRNA of Tmprss6^+/+, Tmprss6^+/−, and Tmprss6^–/– male mice (8-9 weeks old, C57BL/6 genetic background). The mean mRNA ratio of the Tmprss6^+/+ mice was normalized to 1. n = 7 per group; 1 outlier per genotype was identified using the Robust regression followed by OUTlier identification (ROUT) method (Q = 1%) and removed from subsequent analysis. Data represent the mean ± standard deviation. ∗P < .05 using one-way ANOVA with Tukey post hoc test. (B) Confirmation that the Fgf23^eGFP reporter allele yields green fluorescence in Tmprss6^–/– BM. Shown are confocal images of briefly fixed BM core biopsies from Tmprss6^–/–Fgf23^+/eGFP and Tmprss6^–/–Fgf23^+/+ mice (19 weeks old). Original magnification ×20. (C) Localization of green fluorescence and endomucin expression in the BM of mice carrying the Fgf23^eGFP reporter allele. Whole femurs from 6-month-male mice were fixed, decalcified, embedded, and cryosectioned before antiendomucin immunostaining. Pseudocolored merged images (GFP and endomucin) as well as individual channels in grayscale (GFP or endomucin) are presented in the same column; scale bar, 20 μm. Regions boxed in blue (Tmprss6^–/–Fgf23^+/eGFP) and yellow (Tmprss6^+/−Fgf23^+/eGFP) are presented at higher magnification in the adjacent column. The dashed line indicates the border between the bone (left) and BM (right).