Deletion of Rac genes in Nestin⁺ cells leads to alterations in osteoblasts populations. (A) Photomicrographs of trabecular bone from mouse femurs after hematoxylin and eosin staining demonstrating osteoblasts (white arrow) recognized as basophilic cells with a cuboidal-columnar shape, arranged in rows along trabecular bone and the inner surface of BM space, and lining osteoblasts presenting a flat shape, lining the bone surface cells (*). The right panel shows expanded views of the boxed area on left. The scale bar represents 100μm; images were viewed at 200× original magnification using a Nikon Eclipse 80i microscope, and analyzed with NIS Elements Version 2BR imaging software. (B) The number of morphologically-defined osteoblasts was quantified from representative animals of each genotype. Data represent mean ± SD, N = 3 animals per group, **P < .01. (C) FACS gating strategy to define osteoblast progenitor cell populations by Sca-1 and ALCAM staining. (D) Percentages of ALCAM and Sca-1–expressing cells in live CD45/CD31/Ter119^– BM-associated cell fractions. Data represent mean ± SD, *P < .05, **P < .01, N = 4 individual experiments. (E) Comparison of mRNA levels of HSCs maintenance genes and osteopontin (Opn) from freshly sorted Nestin⁺ cells from TG WT and TGRac1^Δ/Δ/Rac3^−/− mice by real-time PCR. Data represent mean ± SD, N = 3 individual experiments, **P < .01.