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. 2005 Nov 7;202(9):1261–1269. doi: 10.1084/jem.20051150

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Copyright © 2005, The Rockefeller University Press

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Figure 1. — In vivo evidence for the essential role of NFATc1 in osteoclast differentiation. (A) Radiographic analysis of Fos ^−/− mice transferred with NFATc1 ^+/− or NFATc1 ^−/− FLCs. Severe osteopetrosis persists after NFATc1 ^−/− FLC transfer. (B) Microradiographic analysis of femur (left: microcomputed tomography; right: microradiograph). Bone marrow cavity is filled with unresorbed bone in NFATc1 ^−/− FLC chimera. (C) Histologic examination of tibia (top: toluidine blue staining; middle: tartrate-resistant acid phosphatase [TRAP] staining for detection of osteoclasts and lumbar vertebra; bottom: toluidine blue staining). No osteoclasts are observed in NFATc1 ^−/− FLC chimera. (D) Histomorphometric evaluation of osteoclasts in NFATc1 ^+/− and NFATc1 ^−/− FLC chimera. (E) Reconstitution of the hematopoietic system of chimeric mice by the donor cells. PCR primers specific for joint region between the NFATc1 locus and the neomycin-resistant gene (NFATc1 neo) and for wild-type (WT) NFATc1 gene are used. The similar chimeric ratios were confirmed by quantitative PCR. (F) Complete blockade of in vitro osteoclastogenesis in osteoclast precursor cells from NFATc1 ^−/− FLC chimera. Splenocyte-derived osteoclast precursor cells were cultured in RANKL (50 ng ml⁻¹) and M-CSF (10 ng ml⁻¹). We counted multinucleated (>3 nuclei) cells (MNCs) positive for TRAP staining. (G) Radiographic analysis of neonates generated by Fos ⁻ ^/ ⁻ blastocyst complementation. Note the difference in radio-opacity at the femur (arrowheads). Right panels show the magnified view of the femur of neonates and tooth eruption of 6-wk-old mice. All of the Fos ^−/−/NFATc1 ^−/− chimeric mice exhibited osteopetrosis, but osteoclastogenesis is restored in Fos ⁻ ^/ ⁻ /NFATc1 ⁺ ^/ ⁺ chimeric mice when ES cells are transmitted to the hematopoietic system of recipient mice. **P < 0.001 versus control.