Figure 5
Figure 5. BM failure in BAF53a-deficient mice is hemopoietic-specific and cell-autonomous. (A) Experimental strategy to generate BAF53a+/+Cre+ (control) and BAF53afl/flCre+ hemopoietic chimeras in congenic mice. pIpC injections (n = 4) were performed 2 months after transplantation to allow complete reconstitution of the hemopoietic system (homeostasis). Analyses were performed on days 7, 10, 14, 17, and 21 after pIpC administration. (B) PCR analyses to determine the efficiency of BAF53a deletion in BAF53afl/flCre+ hemopoietic chimeras at different time points after pIpC-induced deletion. Twenty-one days after pIpC treatment, < 4% of donor (Ly5.2+) BM cells remained in BAF53afl/flCre+ chimeras (see also Figure 5G top panel). (C) Kinetics of loss of donor (Ly5.2+) myeloid cells (Mac1+) in the peripheral blood of BAF53afl/flCre+ chimeras after pIpC-induced deletion. Values represent fractions of the donor-derived (Ly5.2+) cell populations in BAF53afl/flCre+ chimeras relative to controls. n > 3 mice per genotype. (D) Representative FACS profiles of donor-derived (Ly5.2+) myeloid cells (Mac1+) in BAF53afl/flCre+ chimeras and controls (chimerism, 85%; donor Ly5.2+, 15%; recipient Ly5.1+, pIpC day 10). Note that similar results were obtained with mixed BAF53afl/flCre+ FL chimeras (steady state, 20%; donor Ly5.2+, 80%; recipient Ly5.1+ chimerism, data not shown). (E) Absolute numbers of donor-derived (Ly5.2+) BM cells in pIpC-treated BAF53afl/flCre+ chimeras on days 14-21 after pIpC treatment. Data are mean ± SD; n > 6 per genotype. (F) Absolute numbers of BM donor (Ly5.2+) and recipient (Ly5.1+) myeloid CFCs in pIpC-treated BAF53afl/flCre+ chimeras (days 14-21). Data are mean ± SD; n = 5 chimeras per genotype. (G) Kinetics of loss of donor-derived BM cells (top panel), B-lymphoid (Ly5.2+B220+; middle panel), and myeloid (Ly5.2+Mac1+) cells (bottom panel) in pIpC-treated mice. Data are mean ± SD; n > 3 mice per genotype. (H) Representative FACS profiles of donor-derived BM myeloid progenitors (Ly5.2+/Mac1+Gr1+) in pIpC-treated mice on days 7 and 10 after pIpC treatment as indicated. (I) Evaluation of apoptosis in donor-derived (Ly5.2+) myeloid progenitors in BAF53afl/flCre+ mice. TUNEL assays were performed on day 6 after pIpC treatment. Data are mean ± SD; n = 4 per genotype. Representative FACS profiles are shown. (J) Absolute numbers of donor-derived (Ly5.2+) BM LT-HSCs (Sca1+ Lin− CD150+ CD48−) in pIpC-treated mice. Data are mean ± SD; n = 5 per genotype. (K) Absolute numbers of cycling, Hoechst 333342+ Ly5.2+ donor-derived (Ly5.2+) BM LT-HSCs (Sca1+ Lin− CD150+ CD48−) in pIpC-treated BAF53afl/flCre+ chimeras. Data are mean ± SD; n = 5 per genotype.

BM failure in BAF53a-deficient mice is hemopoietic-specific and cell-autonomous. (A) Experimental strategy to generate BAF53a+/+Cre+ (control) and BAF53afl/flCre+ hemopoietic chimeras in congenic mice. pIpC injections (n = 4) were performed 2 months after transplantation to allow complete reconstitution of the hemopoietic system (homeostasis). Analyses were performed on days 7, 10, 14, 17, and 21 after pIpC administration. (B) PCR analyses to determine the efficiency of BAF53a deletion in BAF53afl/flCre+ hemopoietic chimeras at different time points after pIpC-induced deletion. Twenty-one days after pIpC treatment, < 4% of donor (Ly5.2+) BM cells remained in BAF53afl/flCre+ chimeras (see also Figure 5G top panel). (C) Kinetics of loss of donor (Ly5.2+) myeloid cells (Mac1+) in the peripheral blood of BAF53afl/flCre+ chimeras after pIpC-induced deletion. Values represent fractions of the donor-derived (Ly5.2+) cell populations in BAF53afl/flCre+ chimeras relative to controls. n > 3 mice per genotype. (D) Representative FACS profiles of donor-derived (Ly5.2+) myeloid cells (Mac1+) in BAF53afl/flCre+ chimeras and controls (chimerism, 85%; donor Ly5.2+, 15%; recipient Ly5.1+, pIpC day 10). Note that similar results were obtained with mixed BAF53afl/flCre+ FL chimeras (steady state, 20%; donor Ly5.2+, 80%; recipient Ly5.1+ chimerism, data not shown). (E) Absolute numbers of donor-derived (Ly5.2+) BM cells in pIpC-treated BAF53afl/flCre+ chimeras on days 14-21 after pIpC treatment. Data are mean ± SD; n > 6 per genotype. (F) Absolute numbers of BM donor (Ly5.2+) and recipient (Ly5.1+) myeloid CFCs in pIpC-treated BAF53afl/flCre+ chimeras (days 14-21). Data are mean ± SD; n = 5 chimeras per genotype. (G) Kinetics of loss of donor-derived BM cells (top panel), B-lymphoid (Ly5.2+B220+; middle panel), and myeloid (Ly5.2+Mac1+) cells (bottom panel) in pIpC-treated mice. Data are mean ± SD; n > 3 mice per genotype. (H) Representative FACS profiles of donor-derived BM myeloid progenitors (Ly5.2+/Mac1+Gr1+) in pIpC-treated mice on days 7 and 10 after pIpC treatment as indicated. (I) Evaluation of apoptosis in donor-derived (Ly5.2+) myeloid progenitors in BAF53afl/flCre+ mice. TUNEL assays were performed on day 6 after pIpC treatment. Data are mean ± SD; n = 4 per genotype. Representative FACS profiles are shown. (J) Absolute numbers of donor-derived (Ly5.2+) BM LT-HSCs (Sca1+ Lin− CD150+ CD48−) in pIpC-treated mice. Data are mean ± SD; n = 5 per genotype. (K) Absolute numbers of cycling, Hoechst 333342+ Ly5.2+ donor-derived (Ly5.2+) BM LT-HSCs (Sca1+ Lin− CD150+ CD48−) in pIpC-treated BAF53afl/flCre+ chimeras. Data are mean ± SD; n = 5 per genotype.

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