_:b477750766 . _:b477750767 . _:b477750693 . _:b477750544 . _:b477750545 . _:b477750546 . _:b477750547 . _:b477750548 . . _:b477750549 . _:b477750550 . _:b477750551 . _:b477750662 . . _:b477750552 . _:b477750553 . _:b477750554 . _:b477750555 . _:b477750556 . _:b477750557 . _:b477750558 . _:b477750559 . _:b477750528 . . _:b477750529 . _:b477750530 . _:b477750531 . _:b477750532 . _:b477750689 . _:b477750533 . _:b477750534 . _:b477750535 . _:b477750536 . _:b477750537 . _:b477750538 . . _:b477750539 . _:b477750540 . _:b477750541 . _:b477750542 . _:b477750543 . _:b477750576 . _:b477750577 . _:b477750578 . _:b477750579 . _:b477750580 . . _:b477750581 . _:b13364261 "atlas and prior studies examining the expression pattern of Evi1 in various tissues have reported Evi1 expression in the kidney, ovary, uterus, intestine, stomach, lung, trachea, and nasal cavity in the adult mouse (Morishita et al., >>1990<<; Perkins et al., 1991; Su et al., 2004). It will be interesting to determine, using Evi1-IRES-GFP knock-in mice, whether Evi1-expressing cells in these organs are enriched with tissue stem cells." . _:b477750582 . _:b477750583 . _:b477750584 . _:b477750585 . _:b477750586 . _:b477750587 . _:b477750580 . _:b477750588 . _:b477750589 . _:b477750590 . _:b477750591 . _:b477750560 . _:b477750561 . _:b477750562 . _:b477750563 . _:b477750564 . _:b477750565 . _:b477750566 . _:b477750567 . _:b477750568 . _:b477750569 . _:b477750570 . _:b477750571 . _:b477750572 . _:b477750573 . _:b477750574 . _:b477750585 . _:b477750575 . _:b477750608 . . _:b477750609 . _:b477750641 . . _:b477750610 . _:b477750611 . _:b477750612 . _:b477750613 . _:b477750614 . _:b477750615 . _:b477750616 . _:b477750617 . _:b477750618 . _:b477750619 . _:b477750620 . _:b477750621 . _:b477750622 . _:b477750623 . _:b477750592 . _:b477750653 . _:b477750593 . _:b477750594 . _:b477750595 . _:b477750596 . _:b477750636 . _:b477750597 . _:b13364260 . _:b477750598 . _:b477750599 . . _:b477750600 . _:b477750601 . _:b477750664 . _:b477750602 . _:b477750603 . _:b477750604 . _:b477750605 . _:b477750606 . _:b477750607 . . _:b477750640 . _:b477750641 . . _:b477750642 . _:b477750643 . _:b477750644 . _:b477750645 . . _:b477750646 . _:b477750721 . _:b477750647 . . _:b477750648 . _:b477750649 . . _:b477750650 . _:b477750651 . _:b477750652 . _:b477750653 . _:b477750654 . _:b477750655 . . _:b477750624 . _:b477750625 . _:b477750626 . _:b477750627 . _:b477750628 . _:b477750629 . _:b477750630 . _:b477750631 . _:b477750632 . _:b477750633 . _:b477750634 . _:b477750635 . _:b477750636 . _:b477750637 . . _:b477750638 . _:b477750639 . _:b13364236 . . _:b477750556 . . _:b13364238 . _:b477750728 . . . . _:b477750624 . _:b13364235 . . _:b477750618 . . _:b13364264 . . _:b477750549 . _:b477750565 . . _:b13364239 . _:b13364235 "Heterozygous Evi1 KO mice (Evi1+/\u2212 mice) were previously described (Goyama et al., >>2008<<). C57BL/6-Ly5.1 mice were crossed with Ly5.2 mice to obtain Ly5.1/Ly5.2 mice. Littermates were used as controls in all experiments. All animal experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments" . _:b477750661 . _:b477750731 . . . . _:b477750592 . _:b477750623 . _:b477750691 . _:b477750744 . _:b477750751 . _:b13364257 . _:b477750597 . _:b477750591 . _:b13364258 . . . _:b477750733 . _:b13364240 . _:b477750734 . _:b477750567 . "10.1084%2Fjem.20110447" . . _:b477750655 . . _:b477750714 . . _:b477750589 . _:b13364252 "Fetal HSCs, in particular, divide rapidly and undergo massive expansion, whereas adult HSCs are mostly quiescent (Bowie et al., >>2006<<). It is known that Evi1 is highly expressed in the yolk sac, paraaortic splanchnopleura, and HSPCs (CD45+ CD34+ c-kit+) in early embryo (Yuasa et al., 2005). Therefore, we sought to determine whether Evi1 expression can mark fetal HSCs" . _:b477750644 . . . . _:b13364249 "LSK cells are transiently reconstituting MPPs/ST-HSCs or lymphoid-biased LT-HSCs with limited long-term engraftment and strong predominance of lymphoid reconstitution (Kiel et al., 2005; Weksberg et al., 2008; Grassinger et al., >>2010<<), Evi1-expressing cells possess higher repopulating capacity within this fraction." . . . _:b477750629 . _:b477750666 . . . . _:b477750625 . . _:b477750752 . . _:b477750569 . . . . _:b477750681 . _:b477750738 . _:b477750690 . _:b477750581 . _:b477750594 . . _:b477750531 . . _:b477750563 . _:b13364272 "Our data fit with other studies showing that retroviral integration at the Evi1 locus can be associated with long-term in vivo clonal dominance, occasionally leading to leukemic transformation (Stein et al., >>2010<<). The genetic events underlying AML pathogenesis fall into two groups: (1) mutations that enhance proliferation and survival of hematopoietic progenitors, or (2) mutations that result in impaired differentiation or aberrant acquisition of" . _:b477750712 . . _:b477750526 . _:b13364248 "whether CD48+ CD150\u2212 LSK cells are transiently reconstituting MPPs/ST-HSCs or lymphoid-biased LT-HSCs with limited long-term engraftment and strong predominance of lymphoid reconstitution (Kiel et al., 2005; Weksberg et al., >>2008<<; Grassinger et al., 2010), Evi1-expressing cells possess higher repopulating capacity within this fraction." . . _:b477750729 . . _:b477750611 . . . . _:b477750590 . . . _:b477750566 . _:b13364243 "Evi1 mRNA has been shown to be expressed at significantly higher levels in HSPCs (Lin\u2212 Sca-1+ c-kit+ [LSK]) and common lymphoid progenitors (CLPs) than in other hematopoietic cells (Yuasa et al., 2005; Chen et al., >>2008<<). To gain insight into the biological function of Evi1 through its cell type\u2013specific expression pattern, the distribution of GFP+ cells was examined in adult BM from Evi1+/GFP mice." . _:b477750540 . _:b477750767 . . _:b477750769 . _:b13364242 "Evi1 mRNA has been shown to be expressed at significantly higher levels in HSPCs (Lin\u2212 Sca-1+ c-kit+ [LSK]) and common lymphoid progenitors (CLPs) than in other hematopoietic cells (Yuasa et al., >>2005<<; Chen et al., 2008). To gain insight into the biological function of Evi1 through its cell type\u2013specific expression pattern, the distribution of GFP+ cells was examined in adult BM from Evi1+/GFP mice." . _:b477750747 . _:b477750700 . _:b477750596 . . _:b477750657 . _:b477750602 . _:b477750523 . _:b477750614 . . . . _:b477750610 . . _:b13364252 . _:b477750740 . . _:b477750634 . . . _:b477750765 . _:b477750704 . _:b13364249 . _:b477750685 . . . . . . . _:b477750726 . . _:b477750711 . . . _:b477750656 . . _:b477750603 . _:b477750543 . . _:b477750762 . _:b13364256 "5, A-C; Takakura et al., 2000; Kim et al., 2006; McKinney-Freeman et al., >>2009<<). When the distribution of GFP+ cells in the fetal hematopoietic system was analyzed, most GFP+ cells exhibited the HSPC-specific marker profile in all embryonic tissues examined (Fig. 5, D\u2013F), indicating the predominant expression of" . _:b13364274 "These findings may explain the underlying mechanisms of the clinical observations that, irrespective of ME expression, aberrant EVI1 expression carries an adverse prognostic value in AML (Lugthart et al., >>2008<<, 2010). As it is becoming evident that leukemic stem cells share self-renewal machinery with normal HSCs, the elucidation of how Evi1 controls HSC self-renewal may provide biological insight into the pathogenesis of Evi1-related leukemia." . _:b477750536 . . . . _:b13364263 "expression pattern of Evi1 in various tissues have reported Evi1 expression in the kidney, ovary, uterus, intestine, stomach, lung, trachea, and nasal cavity in the adult mouse (Morishita et al., 1990; Perkins et al., 1991; Su et al., >>2004<<). It will be interesting to determine, using Evi1-IRES-GFP knock-in mice, whether Evi1-expressing cells in these organs are enriched with tissue stem cells." . _:b13364237 . _:b477750554 . . . . . . _:b477750717 . . . . _:b477750559 . . _:b477750727 . . . _:b13364271 . _:b13364234 "materials and methods" . _:b477750600 . . . . _:b13364240 "ECs were defined as CD31+ TER-119\u2212 CD45\u2212, OBs were defined as CD31\u2212 TER-119\u2212 CD45\u2212 Sca-1\u2212 ALCAM+ cells (Nakamura et al., 2010), and MSCs were defined as CD31\u2212 TER-119\u2212 CD45\u2212 Sca-1+ PDGFR\u03B1+ cells (Morikawa et al., >>2009<<)." . _:b477750735 . . _:b477750660 . _:b13364259 "discussion" . _:b477750635 . . _:b477750671 . . _:b477750702 . . _:b13364267 "In fact, several molecules involved in the regulation of HSC self-renewal have been identified as downstream targets or interacting proteins of Evi1, including Gata2 (Sato et al., 2008; Yuasa et al., 2005), Pbx1 (Shimabe et al., >>2009<<), Runx1 (Senyuk et al., 2007), and TGF-\u03B2 (Kurokawa et al., 1998). Together with these findings, our data strongly support a model in which Evi1 gene dosage is a critical determinant of HSC self-renewal potential." . . . _:b477750571 . _:b13364254 "5, A-C; Takakura et al., >>2000<<; Kim et al., 2006; McKinney-Freeman et al., 2009)." . _:b477750759 . _:b477750534 . . _:b13364239 "ECs were defined as CD31+ TER-119\u2212 CD45\u2212, OBs were defined as CD31\u2212 TER-119\u2212 CD45\u2212 Sca-1\u2212 ALCAM+ cells (Nakamura et al., >>2010<<), and MSCs were defined as CD31\u2212 TER-119\u2212 CD45\u2212 Sca-1+ PDGFR\u03B1+ cells (Morikawa et al., 2009)." . . _:b477750665 . . . . _:b477750584 . _:b13364266 "In fact, several molecules involved in the regulation of HSC self-renewal have been identified as downstream targets or interacting proteins of Evi1, including Gata2 (Sato et al., 2008; Yuasa et al., >>2005<<), Pbx1 (Shimabe et al., 2009), Runx1 (Senyuk et al., 2007), and TGF-\u03B2 (Kurokawa et al., 1998)." . . _:b13364265 "In fact, several molecules involved in the regulation of HSC self-renewal have been identified as downstream targets or interacting proteins of Evi1, including Gata2 (Sato et al., >>2008<<; Yuasa et al., 2005), Pbx1 (Shimabe et al., 2009), Runx1 (Senyuk et al., 2007), and TGF-\u03B2 (Kurokawa et al., 1998)." . . _:b477750613 . _:b13364255 . . _:b13364251 . _:b13364255 "5, A-C; Takakura et al., 2000; Kim et al., >>2006<<; McKinney-Freeman et al., 2009)." . _:b477750570 . . _:b13364245 "When LSK cells were subdivided according to CD34 and Flk-2 expression (Orford and Scadden, >>2008<<), the Flk-2\u2212 CD34\u2212 LSK fraction, which is considered to contain most LT-HSC activity, had the highest expression of GFP, and its expression decreased with differentiation to hematopoietic progenitors (Fig." . . _:b13364267 . _:b477750639 . . _:b477750755 . _:b477750745 . . _:b477750525 . . _:b477750524 . _:b477750527 . . _:b477750526 . . . _:b477750763 . . _:b477750718 . _:b477750698 . _:b477750523 . _:b477750575 . _:b13364257 "We previously showed that heterozygosity of Evi1 leads to decreased numbers of LSK and CD34\u2212 LSK cells, as well as impaired long-term repopulating activity (Goyama et al., >>2008<<). In the current study, although Flk-2+ CD34+ and Flk-2\u2212 CD34+ LSK cells were moderately decreased, Flk-2\u2212 CD34\u2212 LSK cells from Evi1+/\u2212 mice exhibited a marked reduction in frequency compared with WT controls (Fig. 6, A and C). Likewise," . . _:b477750522 . . _:b477750552 . _:b477750526 "8"^^ . _:b477750527 "7"^^ . _:b477750694 . . _:b477750524 "9"^^ . _:b477750525 "8"^^ . _:b477750523 "10"^^ . _:b477750716 . _:b477750651 . _:b477750588 . _:b477750522 "13"^^ . _:b477750579 . . _:b477750686 . _:b477750533 "5"^^ . _:b477750532 "6"^^ . _:b13364268 . _:b477750535 "5"^^ . _:b477750534 "5"^^ . _:b477750530 "6"^^ . . _:b477750531 "6"^^ . _:b477750683 . _:b477750568 . _:b477750528 "7"^^ . . _:b477750529 "6"^^ . _:b477750541 "5"^^ . _:b13364269 . _:b477750540 "5"^^ . . _:b477750543 "4"^^ . _:b477750542 "4"^^ . _:b477750537 "5"^^ . . _:b477750536 "5"^^ . _:b477750646 . _:b477750654 . _:b477750546 . _:b477750539 "5"^^ . _:b477750538 "5"^^ . _:b477750628 . . _:b477750549 "4"^^ . . _:b477750548 "4"^^ . . _:b477750551 "4"^^ . . . . _:b477750550 "4"^^ . _:b13364261 . _:b477750708 . _:b477750545 "4"^^ . . _:b477750544 "4"^^ . _:b477750724 . . _:b477750547 "4"^^ . _:b477750722 . _:b477750546 "4"^^ . _:b477750557 "4"^^ . _:b477750593 . . _:b477750556 "4"^^ . _:b477750559 "4"^^ . _:b477750558 "4"^^ . . _:b477750553 "4"^^ . _:b477750552 "4"^^ . _:b477750605 . _:b477750555 "4"^^ . _:b477750554 "4"^^ . . _:b477750565 "3"^^ . . . _:b477750564 "3"^^ . _:b477750567 "3"^^ . _:b477750566 "3"^^ . . _:b477750561 "3"^^ . . _:b477750560 "4"^^ . _:b477750524 . _:b477750563 "3"^^ . _:b477750541 . . _:b477750562 "3"^^ . _:b477750598 . _:b477750573 "3"^^ . . . _:b477750572 "3"^^ . _:b477750753 . _:b477750575 "3"^^ . _:b477750742 . _:b477750574 "3"^^ . _:b477750768 . _:b477750569 "3"^^ . . _:b477750620 . _:b477750568 "3"^^ . _:b13364275 . _:b477750571 "3"^^ . _:b13364262 "examining the expression pattern of Evi1 in various tissues have reported Evi1 expression in the kidney, ovary, uterus, intestine, stomach, lung, trachea, and nasal cavity in the adult mouse (Morishita et al., 1990; Perkins et al., >>1991<<; Su et al., 2004). It will be interesting to determine, using Evi1-IRES-GFP knock-in mice, whether Evi1-expressing cells in these organs are enriched with tissue stem cells." . . _:b477750570 "3"^^ . _:b477750581 "3"^^ . . _:b477750580 "3"^^ . _:b477750583 "3"^^ . _:b477750582 "3"^^ . _:b477750577 "3"^^ . . _:b477750576 "3"^^ . _:b13364241 "results" . _:b13364246 "In addition, further enrichment for LT-HSCs within the LSK fraction using SLAM family receptors (CD48 and CD150; Kiel et al., >>2005<<) revealed that GFP+ cells were found in greatest abundance within CD48\u2212 CD150+ LSK cells, in which LT-HSCs are highly enriched." . _:b477750579 "3"^^ . . _:b477750578 "3"^^ . _:b477750589 "3"^^ . _:b13364270 . _:b477750527 . _:b477750588 "3"^^ . _:b477750642 . _:b477750591 "3"^^ . _:b477750590 "3"^^ . _:b477750599 . _:b477750585 "3"^^ . _:b13364243 . _:b477750584 "3"^^ . _:b13364275 "These findings may explain the underlying mechanisms of the clinical observations that, irrespective of ME expression, aberrant EVI1 expression carries an adverse prognostic value in AML (Lugthart et al., 2008, >>2010<<). As it is becoming evident that leukemic stem cells share self-renewal machinery with normal HSCs, the elucidation of how Evi1 controls HSC self-renewal may provide biological insight into the pathogenesis of Evi1-related leukemia." . . _:b477750758 . _:b477750695 . _:b477750587 "3"^^ . _:b477750709 . _:b477750586 "3"^^ . _:b477750684 . _:b477750597 "2"^^ . _:b477750596 "3"^^ . _:b477750599 "2"^^ . _:b477750598 "2"^^ . . _:b13364244 . _:b477750558 . _:b477750593 "3"^^ . _:b477750592 "3"^^ . _:b477750663 . _:b477750595 "3"^^ . . _:b477750594 "3"^^ . _:b13364271 "Inappropriate expression of EVI1 confers poor prognosis in patients with AML (Lugthart et al., 2008; Gr\u00F6schel et al., >>2010<<), and therefore improvement of the therapeutic outcome of leukemia with high EVI1 expression is needed." . _:b477750605 "2"^^ . _:b477750622 . _:b477750604 "2"^^ . _:b477750561 . _:b477750607 "2"^^ . . _:b477750548 . _:b477750606 "2"^^ . . _:b477750601 "2"^^ . _:b477750600 "2"^^ . _:b13364260 "We show that mesenchymal stem cells (MSCs), one of the few tissue stem cell types that have been established to self-renew in vivo (Morikawa et al., >>2009<<), do not express Evi1." . _:b13364268 "involved in the regulation of HSC self-renewal have been identified as downstream targets or interacting proteins of Evi1, including Gata2 (Sato et al., 2008; Yuasa et al., 2005), Pbx1 (Shimabe et al., 2009), Runx1 (Senyuk et al., >>2007<<), and TGF-\u03B2 (Kurokawa et al., 1998). Together with these findings, our data strongly support a model in which Evi1 gene dosage is a critical determinant of HSC self-renewal potential." . _:b13364264 "In addition, a recent gene expression profile analysis showed that Evi1 binding sites are enriched in the upstream region of genes expressed selectively in LT-HSCs (Forsberg et al., >>2010<<). In fact, several molecules involved in the regulation of HSC self-renewal have been identified as downstream targets or interacting proteins of Evi1, including Gata2 (Sato et al., 2008; Yuasa et al., 2005), Pbx1 (Shimabe et al., 2009)," . _:b477750603 "2"^^ . _:b477750602 "2"^^ . _:b13364251 "major phenotypic and functional differences between fetal and adult HSCs in surface marker profile, cell cycle status, self-renewal potential, gene expression profile, and regulatory mechanism (Mikkola and Orkin, 2006; Orkin and Zon, >>2008<<). Fetal HSCs, in particular, divide rapidly and undergo massive expansion, whereas adult HSCs are mostly quiescent (Bowie et al., 2006)." . _:b477750613 "2"^^ . . _:b477750612 "2"^^ . . _:b477750615 "2"^^ . . _:b477750614 "2"^^ . _:b477750609 "2"^^ . _:b477750756 . _:b13364274 . _:b477750608 "2"^^ . _:b477750611 "2"^^ . _:b477750640 . _:b477750610 "2"^^ . _:b477750621 "2"^^ . _:b477750620 "2"^^ . . _:b477750623 "2"^^ . _:b477750719 . _:b477750622 "2"^^ . _:b477750576 . _:b477750617 "2"^^ . _:b477750616 "2"^^ . _:b477750533 . _:b477750529 . _:b477750748 . . _:b477750619 "2"^^ . _:b477750532 . . . _:b477750618 "2"^^ . _:b477750535 . . . _:b477750629 "2"^^ . _:b477750534 . . _:b477750628 "2"^^ . _:b477750529 . _:b477750601 . _:b477750631 "2"^^ . _:b477750528 . _:b477750630 "2"^^ . _:b477750531 . . _:b477750749 . _:b477750625 "2"^^ . _:b477750530 . _:b477750616 . _:b13364256 . _:b477750624 "2"^^ . _:b477750541 . _:b477750675 . _:b477750764 . _:b477750741 . _:b477750627 "2"^^ . _:b477750540 . . . _:b477750626 "2"^^ . _:b477750543 . _:b477750619 . _:b477750637 "2"^^ . _:b477750542 . _:b477750636 "2"^^ . _:b477750537 . _:b477750578 . _:b13364262 . _:b477750639 "2"^^ . _:b477750536 . _:b477750701 . _:b477750525 . _:b477750638 "2"^^ . _:b477750539 . . . . _:b477750633 "2"^^ . _:b477750538 . _:b477750615 . _:b477750632 "2"^^ . _:b477750549 . _:b477750635 "2"^^ . _:b477750548 . _:b477750634 "2"^^ . _:b477750551 . . . . _:b477750645 "2"^^ . _:b477750550 . . . _:b477750644 "2"^^ . _:b477750545 . . . _:b477750647 "2"^^ . _:b477750544 . _:b477750646 "2"^^ . _:b477750547 . _:b477750535 . _:b477750551 . _:b477750641 "2"^^ . _:b477750546 . . _:b13364263 . _:b477750669 . _:b477750640 "2"^^ . _:b477750557 . _:b477750643 "2"^^ . _:b477750556 . _:b477750643 . _:b477750642 "2"^^ . _:b477750559 . . _:b477750653 "2"^^ . _:b477750558 . _:b477750761 . _:b477750574 . . . _:b477750652 "2"^^ . _:b477750553 . . . _:b477750655 "2"^^ . _:b477750552 . _:b477750654 "2"^^ . _:b477750555 . . . . _:b477750649 "2"^^ . _:b477750554 . _:b477750687 . _:b477750648 "2"^^ . _:b477750565 . _:b477750651 "2"^^ . _:b477750564 . _:b477750650 "2"^^ . _:b477750678 . _:b477750567 . _:b13364236 . . . _:b477750661 "2"^^ . _:b477750566 . _:b477750537 . _:b13364237 . _:b477750660 "2"^^ . _:b477750561 . _:b13364238 . _:b477750663 "2"^^ . _:b477750560 . _:b13364239 . _:b477750662 "2"^^ . _:b477750577 . _:b477750563 . _:b477750657 "2"^^ . _:b477750562 . _:b477750656 "2"^^ . _:b477750573 . _:b477750538 . _:b13364234 . _:b477750659 "2"^^ . _:b477750572 . _:b13364235 . _:b477750633 . _:b477750575 . _:b13364244 . _:b477750658 "2"^^ . _:b477750669 "2"^^ . _:b477750574 . _:b13364245 . _:b477750668 "2"^^ . _:b477750569 . _:b13364246 . . _:b477750671 "2"^^ . _:b477750568 . _:b13364247 . . _:b477750670 "2"^^ . _:b477750571 . _:b13364240 . _:b477750665 "2"^^ . _:b477750570 . _:b13364241 . _:b477750664 "2"^^ . _:b477750581 . _:b13364242 . _:b477750627 . _:b477750667 "2"^^ . _:b477750580 . _:b13364243 . _:b477750647 . _:b477750666 "2"^^ . _:b477750583 . _:b13364252 . . _:b477750677 "2"^^ . _:b477750582 . _:b13364253 . _:b477750723 . _:b13364273 "two groups: (1) mutations that enhance proliferation and survival of hematopoietic progenitors, or (2) mutations that result in impaired differentiation or aberrant acquisition of self-renewal properties of HSPCs (Fr\u00F6hling et al., >>2005<<). Our data indicate that Evi1 activation can function as the latter mutation and confer enhanced self-renewal capacity in myeloid neoplasms." . _:b477750676 "2"^^ . _:b477750577 . _:b13364254 . . _:b477750679 "2"^^ . _:b477750576 . _:b13364255 . _:b477750678 "2"^^ . _:b477750579 . _:b13364248 . _:b477750673 "2"^^ . _:b477750578 . _:b13364249 . . _:b477750676 . _:b477750672 "2"^^ . _:b477750589 . _:b13364250 . _:b477750607 . _:b477750675 "2"^^ . _:b477750588 . . _:b13364251 . _:b477750674 "2"^^ . _:b477750591 . _:b13364260 . _:b477750685 "2"^^ . _:b477750590 . _:b13364261 . . _:b477750684 "2"^^ . _:b477750585 . _:b13364262 . . _:b477750687 "2"^^ . _:b477750584 . _:b13364263 . . _:b477750686 "2"^^ . _:b477750587 . _:b13364256 . _:b477750681 "2"^^ . _:b477750586 . _:b13364257 . _:b477750680 "2"^^ . _:b477750597 . _:b13364258 . _:b477750683 "2"^^ . _:b477750596 . _:b13364259 . . _:b477750682 "2"^^ . _:b477750599 . _:b13364268 . _:b477750693 "2"^^ . _:b477750598 . _:b13364269 . _:b477750583 . _:b477750692 "2"^^ . _:b477750593 . _:b13364270 . _:b477750695 "2"^^ . _:b477750592 . _:b13364271 . . _:b477750694 "2"^^ . _:b477750595 . _:b13364264 . _:b477750689 "2"^^ . _:b477750594 . _:b13364265 . _:b477750688 "2"^^ . . _:b477750605 . _:b13364266 . _:b13364248 . _:b477750691 "2"^^ . _:b477750604 . _:b477750743 . _:b13364267 . _:b477750690 "2"^^ . _:b477750607 . . _:b477750701 "2"^^ . _:b477750606 . _:b477750631 . _:b477750700 "2"^^ . _:b477750601 . _:b477750649 . _:b477750703 "2"^^ . _:b477750600 . . _:b13364269 "self-renewal have been identified as downstream targets or interacting proteins of Evi1, including Gata2 (Sato et al., 2008; Yuasa et al., 2005), Pbx1 (Shimabe et al., 2009), Runx1 (Senyuk et al., 2007), and TGF-\u03B2 (Kurokawa et al., >>1998<<). Together with these findings, our data strongly support a model in which Evi1 gene dosage is a critical determinant of HSC self-renewal potential." . _:b477750702 "2"^^ . _:b13364272 . _:b477750603 . _:b477750697 "2"^^ . _:b13364273 . _:b477750602 . . _:b477750696 "2"^^ . _:b13364274 . _:b477750613 . _:b13364244 "In contrast to the previous study (Chen et al., >>2008<<), GFP was not expressed in CLPs (Fig." . _:b477750699 "2"^^ . _:b13364275 . _:b477750612 . . . "PMC0" . _:b477750698 "2"^^ . _:b477750615 . _:b477750709 "2"^^ . _:b477750614 . . _:b477750539 . _:b477750708 "2"^^ . _:b477750609 . . _:b477750650 . . _:b477750711 "2"^^ . _:b477750608 . _:b477750522 . _:b477750608 . _:b477750710 "2"^^ . _:b477750611 . . _:b477750705 "2"^^ . _:b477750610 . _:b477750704 "2"^^ . _:b477750621 . . _:b477750707 "2"^^ . _:b477750620 . . . _:b477750706 "2"^^ . _:b477750623 . _:b477750617 . _:b477750717 "2"^^ . _:b477750622 . _:b477750716 "2"^^ . _:b477750617 . . _:b477750719 "2"^^ . _:b477750616 . _:b477750718 "2"^^ . _:b477750619 . _:b477750677 . . _:b477750713 "2"^^ . _:b477750618 . . _:b477750712 "2"^^ . _:b477750629 . _:b477750630 . _:b477750757 . _:b477750715 "2"^^ . _:b477750628 . _:b477750714 "2"^^ . _:b477750631 . _:b477750725 "2"^^ . _:b477750630 . _:b477750724 "2"^^ . _:b477750625 . _:b13364266 . _:b477750727 "2"^^ . _:b477750624 . _:b477750726 "2"^^ . _:b477750627 . _:b477750721 "2"^^ . _:b477750626 . _:b477750720 "2"^^ . _:b477750637 . _:b13364234 . _:b477750604 . _:b477750723 "2"^^ . _:b477750636 . . . . _:b477750722 "2"^^ . _:b477750639 . _:b13364241 . . _:b477750699 . _:b477750638 . _:b477750733 "2"^^ . . _:b477750633 . _:b477750732 "2"^^ . _:b13364242 . _:b477750632 . _:b477750735 "2"^^ . . _:b477750635 . _:b477750734 "2"^^ . _:b13364259 . _:b477750634 . _:b477750729 "2"^^ . _:b477750645 . _:b477750728 "2"^^ . _:b477750644 . _:b477750731 "2"^^ . _:b477750647 . _:b477750730 "2"^^ . _:b477750646 . _:b477750741 "2"^^ . _:b13364253 . _:b477750641 . _:b477750740 "2"^^ . _:b477750640 . _:b477750743 "2"^^ . _:b477750643 . _:b477750742 "2"^^ . . _:b477750642 . _:b477750737 "2"^^ . _:b477750653 . _:b477750736 "2"^^ . . _:b477750652 . _:b477750739 "2"^^ . _:b477750655 . _:b477750738 "2"^^ . . _:b477750654 . _:b477750749 "2"^^ . _:b477750703 . _:b477750649 . _:b477750748 "2"^^ . . _:b477750648 . _:b477750751 "2"^^ . _:b477750545 . _:b477750651 . _:b477750750 "2"^^ . _:b477750658 . _:b477750564 . _:b477750650 . _:b477750745 "2"^^ . _:b477750661 . _:b477750744 "2"^^ . _:b477750660 . _:b477750747 "2"^^ . _:b477750663 . _:b477750746 "2"^^ . . _:b477750662 . _:b477750757 "2"^^ . _:b477750657 . _:b477750756 "2"^^ . _:b13364237 "When CD48+ CD150\u2212 LSK cells were transplanted, nonblocking anti-CD48 antibody (MRC OX78 clone) was used (Grassinger et al., >>2010<<)." . _:b477750673 . _:b477750705 . _:b477750656 . _:b477750759 "2"^^ . _:b477750659 . _:b477750758 "2"^^ . _:b477750659 . _:b477750658 . _:b477750753 "2"^^ . . _:b477750669 . _:b477750752 "2"^^ . . _:b477750668 . _:b477750755 "2"^^ . _:b477750555 . . _:b477750562 . _:b477750671 . _:b477750754 "2"^^ . _:b477750670 . _:b477750765 "2"^^ . _:b477750682 . . _:b477750560 . _:b477750665 . _:b477750764 "2"^^ . _:b477750725 . . . _:b477750767 "2"^^ . _:b477750664 . _:b477750667 . _:b477750766 "2"^^ . _:b13364258 "Because both ME and Evi1 are inactivated in our Evi1 KO model (Goyama et al., >>2008<<), we attempted to genetically dissect the relative roles of ME and Evi1 in maintaining LT-HSCs." . _:b477750666 . _:b477750761 "2"^^ . _:b13364259 _:b13364264 . _:b13364259 _:b13364265 . _:b13364259 _:b13364266 . . _:b13364259 _:b13364267 . _:b477750677 . _:b477750760 "2"^^ . _:b13364259 _:b13364268 . _:b13364259 _:b13364269 . _:b13364259 _:b13364270 . _:b13364259 _:b13364271 . _:b477750676 . _:b477750763 "2"^^ . . . _:b477750679 . _:b477750762 "2"^^ . _:b13364259 _:b13364260 . _:b13364259 _:b13364261 . _:b13364259 _:b13364262 . . _:b13364259 _:b13364263 . _:b477750678 . _:b477750673 . _:b477750648 . _:b477750672 . _:b13364259 _:b13364272 . _:b13364259 _:b13364273 . _:b13364259 _:b13364274 . _:b13364259 _:b13364275 . _:b477750675 . _:b477750532 . _:b477750674 . _:b477750769 "2"^^ . . _:b477750685 . _:b477750768 "2"^^ . . _:b477750684 . _:b477750544 . _:b477750687 . . _:b477750686 . _:b477750681 . _:b477750680 . . _:b477750683 . _:b13364247 "Although it is controversial whether CD48+ CD150\u2212 LSK cells are transiently reconstituting MPPs/ST-HSCs or lymphoid-biased LT-HSCs with limited long-term engraftment and strong predominance of lymphoid reconstitution (Kiel et al., >>2005<<; Weksberg et al., 2008; Grassinger et al., 2010), Evi1-expressing cells possess higher repopulating capacity within this fraction." . . _:b477750682 . _:b13364241 _:b13364256 . _:b13364241 _:b13364257 . _:b13364241 _:b13364258 . _:b477750693 . . _:b477750670 . . _:b477750736 . _:b477750732 . _:b477750754 . _:b477750692 . _:b477750730 . _:b13364253 "It is known that Evi1 is highly expressed in the yolk sac, paraaortic splanchnopleura, and HSPCs (CD45+ CD34+ c-kit+) in early embryo (Yuasa et al., >>2005<<). Therefore, we sought to determine whether Evi1 expression can mark fetal HSCs despite their distinct features from adult HSCs, and thus analyzed the expression pattern of GFP in Evi1+/GFP embryos. As expected, GFP expression was highly" . . _:b477750695 . . _:b477750694 . . _:b477750689 . _:b477750688 . . _:b477750691 . _:b477750750 . _:b477750713 . _:b477750690 . _:b477750701 . _:b477750638 . _:b477750700 . . _:b477750703 . _:b477750702 . _:b13364241 _:b13364242 . _:b13364241 _:b13364243 . _:b477750697 . _:b13364241 _:b13364244 . _:b13364241 _:b13364245 . _:b13364241 _:b13364246 . _:b13364241 _:b13364247 . _:b477750746 . _:b477750612 . _:b13364241 _:b13364248 . _:b477750696 . _:b13364241 _:b13364249 . _:b13364241 _:b13364250 . . _:b477750621 . _:b13364241 _:b13364251 . _:b477750699 . _:b13364241 _:b13364252 . _:b477750557 . _:b13364241 _:b13364253 . _:b13364241 _:b13364254 . _:b13364241 _:b13364255 . _:b477750698 . _:b13364234 _:b13364236 . _:b13364234 _:b13364237 . _:b13364234 _:b13364238 . _:b13364234 _:b13364239 . _:b477750709 . _:b13364234 _:b13364235 . . _:b477750708 . _:b477750711 . _:b477750737 . . _:b477750710 . . _:b477750760 . . _:b477750542 . _:b477750705 . _:b477750667 . _:b477750704 . . _:b477750707 . _:b13364234 _:b13364240 . . _:b477750706 . _:b477750717 . . _:b477750716 . _:b477750719 . . . _:b477750718 . _:b477750533 . . _:b477750713 . . _:b477750712 . _:b477750715 . _:b477750714 . _:b477750725 . . . . _:b477750724 . _:b477750727 . _:b477750726 . _:b477750522 . _:b13364270 "Inappropriate expression of EVI1 confers poor prognosis in patients with AML (Lugthart et al., >>2008<<; Gr\u00F6schel et al., 2010), and therefore improvement of the therapeutic outcome of leukemia with high EVI1 expression is needed." . _:b477750721 . _:b477750550 . _:b477750523 . _:b477750524 . _:b477750525 . _:b477750526 . _:b477750720 . _:b477750527 . _:b477750723 . _:b477750722 . . _:b477750733 . _:b477750732 . . _:b477750735 . _:b477750734 . _:b477750729 . _:b477750688 . _:b477750728 . _:b477750572 . _:b477750731 . _:b477750606 . _:b477750730 . _:b477750692 . . _:b477750741 . _:b13364254 . _:b477750740 . _:b477750553 . _:b477750743 . _:b477750710 . _:b477750742 . _:b477750737 . . _:b477750637 . _:b477750736 . _:b477750739 . _:b477750680 . _:b477750738 . _:b477750749 . . _:b477750748 . _:b477750751 . _:b477750674 . _:b477750750 . _:b13364238 "Western blotting was performed as previously described (Goyama et al., >>2008<<). In brief, mouse embryo fibroblast cells were lysed in TNE buffer, subjected to 7% SDS-PAGE, and transferred to a PVDF membrane (Millipore)." . _:b477750745 . . _:b477750744 . . _:b477750747 . _:b477750746 . _:b477750645 . _:b477750757 . _:b477750756 . _:b477750672 . _:b477750706 . _:b477750759 . . _:b477750758 . _:b477750753 . . . _:b477750766 . _:b477750752 . _:b13364245 . _:b477750528 . . _:b477750755 . _:b477750587 . _:b13364265 . _:b477750754 . _:b477750697 . _:b477750765 . . _:b477750764 . _:b477750739 . _:b477750672 . _:b477750673 . _:b477750674 . _:b477750767 . _:b477750675 . _:b477750676 . _:b477750677 . . _:b477750678 . _:b477750766 . _:b477750679 . _:b477750680 . _:b13364247 . _:b477750681 . _:b477750682 . _:b477750761 . _:b477750683 . _:b477750684 . _:b13364246 . _:b477750685 . _:b13364250 . _:b477750686 . _:b477750760 . _:b477750687 . _:b477750656 . _:b477750530 . _:b477750657 . . _:b477750658 . _:b477750763 . _:b477750659 . . _:b477750660 . _:b477750661 . _:b477750662 . _:b477750762 . _:b477750663 . _:b477750664 . _:b477750665 . _:b477750666 . _:b477750667 . _:b477750668 . _:b477750669 . _:b477750670 . _:b477750671 . _:b13364250 "There are several major phenotypic and functional differences between fetal and adult HSCs in surface marker profile, cell cycle status, self-renewal potential, gene expression profile, and regulatory mechanism (Mikkola and Orkin, >>2006<<; Orkin and Zon, 2008)." . _:b477750704 . _:b477750705 . _:b477750706 . _:b477750707 . _:b477750708 . _:b477750709 . _:b477750710 . . _:b477750609 . _:b477750711 . _:b477750679 . _:b477750712 . _:b477750713 . _:b477750714 . _:b477750769 . . _:b477750715 . _:b477750716 . _:b477750717 . _:b477750718 . _:b477750768 . _:b477750719 . _:b477750688 . _:b477750689 . _:b477750690 . _:b477750582 . _:b477750691 . _:b477750692 . _:b477750693 . _:b477750694 . _:b13364273 . _:b477750695 . _:b477750547 . _:b477750696 . _:b477750697 . _:b477750698 . _:b477750699 . . _:b477750700 . _:b477750701 . _:b477750702 . _:b477750703 . _:b477750736 . _:b477750737 . . _:b477750738 . _:b477750739 . _:b13364236 "Evi1+/\u2212 mice were genotyped by PCR as previously described (Goyama et al., >>2008<<). Evi1+/GFP mice were genotyped using a multiplex PCR to detect both WT and Evi1-IRES-GFP alleles." . _:b477750740 . _:b477750741 . . _:b477750742 . _:b477750743 . _:b477750744 . _:b477750745 . _:b477750746 . _:b477750707 . _:b477750747 . _:b477750748 . _:b477750749 . _:b477750652 . _:b477750696 . _:b477750750 . _:b477750751 . _:b477750720 . _:b477750721 . _:b477750722 . _:b13364272 . _:b477750723 . _:b477750586 . _:b477750724 . _:b477750725 . _:b477750726 . . _:b477750727 . _:b477750728 . _:b477750729 . _:b477750730 . _:b477750731 . _:b477750732 . _:b477750733 . _:b477750734 . _:b477750735 . _:b477750768 . _:b477750769 . _:b477750626 . _:b477750715 . _:b477750668 . _:b477750720 . _:b477750752 . _:b477750753 . _:b477750754 . _:b477750595 . _:b477750632 . _:b477750755 . _:b477750756 . _:b477750757 . . _:b477750758 . _:b477750759 . _:b477750760 . _:b477750761 . _:b477750573 . _:b477750762 . _:b477750763 . . _:b477750764 . _:b477750765 .