Volume 6, Issue 4, Pages (April 1997)

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Volume 6, Issue 4, Pages 437-447 (April 1997) PU.1 Functions in a Cell-Autonomous Manner to Control the Differentiation of Multipotential Lymphoid–Myeloid Progenitors Edward W Scott, Robert C Fisher, Marilyn C Olson, Eli W Kehrli, M.Celeste Simon, Harinder Singh Immunity Volume 6, Issue 4, Pages 437-447 (April 1997) DOI: 10.1016/S1074-7613(00)80287-3

Figure 1 Expression of PU.1 Protein in PU.1−/− Embryos Protein extracts from the fetal liver of PU.1+/+, PU.1+/−, and PU.1−/− littermate embryos (lanes 6–8) were resolved by SDS-PAGE and probed by Western blotting using an affinity purified anti-PU.1 antiserum. Lanes 1 and 2 contained full-length PU.1 and a truncated PU.1 protein, respectively, generated by in vitro translation. Lanes 3–5 contained protein extracts from indicated tissue of PU.1+/+ and PU.1+/− animals. Molecular weight markers are designated at left. Immunity 1997 6, 437-447DOI: (10.1016/S1074-7613(00)80287-3)

Figure 2 Analysis of PU.1+/+ and PU.1−/− Fetal Chimeras Wild-type or PU.1−/− ES cells were injected into C57BL/6 blastocysts to produce control and experimental chimeras. (A) GPI isozyme analysis of fetal liver protein from a wild-type and four PU.1−/− mutant chimeras (lanes 1–4). Extracts from C57BL/6 tissue (GPIb) and the parental ES cell line CCE.1 (GPIc) were used as GPI standards. (B) Hbb isoform analysis of fetal blood from chimeric embryos in (A). Blood samples from C57BL/6 and 129SV (ES cell genotype) mouse strains were used as Hbb standards. (C) FACS analysis of lymphoid and myeloid cells in wild-type and PU.1−/− mutant embryonic chimeras. Shown are analyses of the wild-type chimera (left) and the mutant chimera (#2) (right) described in (A). Ly 9.1 staining detects ES cell–derived progeny. Fetal thymocytes were analyzed using αCD4 (top). Fetal liver B lymphoid and myeloid cells were analyzed using αB220 (middle), and αCD11b (bottom), respectively. Immunity 1997 6, 437-447DOI: (10.1016/S1074-7613(00)80287-3)

Figure 3 Analysis of PU.1+/+ and PU.1−/− Adult Chimeras Wild-type or PU.1−/− chimeras were generated as described in Figure 2 but analyzed at 4–6 weeks after birth. (A) GPI isozyme analysis of tissue from representative wild-type and mutant chimeras. Tissues examined were blood (Bld), bone marrow (BnM), spleen (Spl), thymus (Thy), heart (Hrt), lung (Lng), brain (Brn), muscle (Mus), and kidney (Kid). Arrows indicate positions of GPIb and GPIc isozymes. (B) Hbb analysis of peripheral blood from chimeras in (A). Peripheral blood samples from C57BL/6 and 129SV mouse strains were used as Hbb standards. (C) FACS analysis of lymphoid and myeloid cells in wild-type and PU.1−/− mutant adult chimeras. Shown are analyses of the wild-type chimera (left) and the mutant chimera (right) described in (A). Ly 9.1 staining detects ES cell–derived progeny. Thymocytes were analyzed using αCD4 (top). Bone marrow B lymphoid and myeloid cells were analyzed using αB220 (middle) and αCD11b (bottom), respectively. Immunity 1997 6, 437-447DOI: (10.1016/S1074-7613(00)80287-3)

Figure 4 Analysis of Transplanted PU.1−/− Fetal Liver Cells in Lethally Irradiated Recipients Recipient animals were examined 3 weeks after transplanting wild-type or PU.1−/− fetal liver cells. (A) Hbb isoform analysis of peripheral blood from irradiated animals transplanted with the indicated donor fetal liver cells. Peripheral blood samples from C57BL/6 and 129SV mouse strains were used as Hbb standards. (B) FACS analysis of recipient bone marrow reconstituted with either PU.1+/+ or PU.1−/− fetal liver cells. Ly 5.2 staining indicates donor-derived cells. B lymphoid and myeloid cells were analyzed using αB220 and αCD11b antibodies, respectively. Immunity 1997 6, 437-447DOI: (10.1016/S1074-7613(00)80287-3)

Figure 5 Frequency Analysis of B Lineage Progenitors in the AA4.1+, Lin− Population of PU.1+/+ and PU.1+/− or PU.1−/− Embryos Fetal liver cells from day 14.5 littermates were sorted to obtain the AA4.1+, Lin− populations as described in Experimental Procedures. Purified progenitors were plated at 5, 20, 60, 100, or 200 cells/well in 96-well plates containing irradiated S17 cells and IL-7; 48 wells were used for each cell density. Wells were scored for proliferating cells after 9 days in culture. Results for PU.1+/+ and PU.1+/− (squares) or PU.1−/− (diamonds) AA4.1+, Lin− progenitors are represented as the percentage of nonresponding wells at each cell density. Immunity 1997 6, 437-447DOI: (10.1016/S1074-7613(00)80287-3)

Figure 6 Analysis of Ikaros and Pax-5 Expression in PU.1−/− Embryos Fetal liver RNA from PU.1+/+, PU.1+/−, or PU.1−/− littermate embryos (day 16.5) was analyzed by RT-PCR using primers specific for Ikaros, Pax-5, and hprt transcripts. RNA from the pre–B cell line 38B9 was used as a positive control. Lane M contains DNA molecular weight markers. Immunity 1997 6, 437-447DOI: (10.1016/S1074-7613(00)80287-3)