Atf4 deficiency alters MSC function but not HSC maintenance. (A) qRT-PCR analysis of Atf4 expression in CD45^–Ter119^–LepR⁺ cells from Prx1-Cre;Atf4^fl/fl and Atf4^fl/fl mice (n = 3 mice). (B) Gross phenotype of Atf4^fl/fl and Prx1-Cre;Atf4^fl/fl mice at 7 weeks of age (left) and body, forelimb, and hindlimb length measurements (right). ∗∗∗P < .001; ∗∗P < .01; n = 5. (C) Microcomputed tomography (micro-CT) image showing the limb phenotype of Atf4-mutant vs WT mice. (D-E) Micro-CT analysis showing decreased bone volume fraction in Atf4 mutants; BV/TV, bone volume per total volume (n = 5 mice). (F) Representative calcein double-labeling images (left) with quantification of trabecular bone formation in the femur metaphysis (right) (n = 4-6 mice). (G) Representative femur sections from Prx1-Cre;Atf4^fl/fl;Col2.3-GFP and Atf4^fl/fl;Col2.3-GFP mice showing the decrease in the number of Col2.3-GFP⁺ osteoblasts (n = 3 mice). (H) Representative flow cytometry dot plots of CD45^–Ter119^–LepR⁺ cells (left) and corresponding quantitative data showing the decreased frequency of CD45^–Ter119^–LepR⁺ cells in Prx1-Cre;Atf4^fl/fl mice at 7 weeks of age (right) (n = 3-4 mice). (I) Representative images of CFU-F colonies formed when cells were cultured from enzymatically dissociated BM (left) and corresponding quantitative data (right); n = 3 mice from at least 3 independent experiments. (J) Representative images of CFU-F differentiating into osteoblasts after culture in differentiation medium for 2 to 3 weeks, showing the reduced osteogenic potential of Atf4-depleted MSCs. (K) BM cell numbers of Prx1-Cre;Atf4^fl/fl and Atf4^fl/fl mice (n = 3 mice). (L) Percentages of the indicated cell populations in Prx1-Cre;Atf4^fl/fl and Atf4^fl/fl mice BM (n = 3 mice). (M) Results from colony formation assays using 10⁴ BM cells cultured for 10 to 14 days in a complete methylcellulose-based medium (n = 3 mice). (N) Experimental workflow of competitive transplantation. (O) Percentage of CD45.2⁺ donor cells in the peripheral blood (PB) of recipient mice at the indicated time points after competitive BM transplantation (n = 4-8 mice). (P) Percentage of CD45.2⁺ donor-derived HSCs in the BM of recipients (n = 5 mice). (Q) Percentage of CD45.2⁺ donor cells in the PB of recipient mice at the indicated time points after secondary BM transplantation (n = 4-7 mice). (R) Percentage of CD45.2⁺ donor-derived HSCs in the BM of recipients after the secondary BM transplantation (n = 4 mice). (S) Experimental workflow of the assay in which 10⁶ BM cells were transplanted from donor mice (CD45.1) into irradiated Prx1-Cre;Atf4^fl/fl or Atf4^fl/fl mice. (T) Percentage of CD45.1⁺ donor cells in the PB of recipient mice at indicated time points after BM transplantation. The donor cells were CD45.1⁺ (n = 5 mice). (U) Percentage of CD45.1⁺ donor-derived HSCs in the BM of recipients (n = 4-5 mice). (V-W) Percentage of CD45.2⁺ donor cells in the PB of recipient mice at the indicated time points after competitive BM transplantation (n = 4-6 mice). Data represent mean ± standard deviation (SD). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; unpaired 2-tailed Student t test.