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Genes to Cells (2006) 11, 859-870. doi:10.1111/j.1365-2443.2006.00985.x
© 2006 Blackwell Publishing or its licensors
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Over-expression of c-Myb increases the frequency of hemogenic precursors in the endothelial cell population

Guoyou Dai1,2, Hiroshi Sakamoto2, Yuri Shimoda2, Tetsuhiro Fujimoto1,2,3, Shin-Ichi Nishikawa1,4 and Minetaro Ogawa1,2,3,*

1 Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan
2 Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
3 21st Century COE, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
4 Stem Cell Biology Laboratory, Riken Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Definitive hematopoiesis has been proposed to arise from hemogenic endothelial cells during mouse embryogenesis. The c-myb proto-oncogene is essential for the development of definitive hematopoiesis and was reported to be activated in hemogenic endothelial cells. To investigate whether c-Myb is involved in regulating the development of hemogenic endothelial cells, we conditionally induced c-myb over-expression during the in vitro differentiation of embryonic stem cells. VE-cadherin+ CD45– cells inducibly expressing c-Myb showed an increase in multilineage colony formation as well as an augmented capacity of the colony forming cells to self-renew in vitro under the condition that only the endogenous c-myb gene was expressed during differentiation of hematopoietic cells. Over-expression of c-Myb in the endothelial population led to activation of genes associated with definitive hematopoiesis such as Runx1, Hoxb4, Mll and Etv6. Our data provide evidence that c-Myb is able to exert an effect in endothelial cells which fosters the establishment of their hemogenic potential.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
c-Myb is a DNA binding transcription factor that plays a major role in development of erythroid, myeloid and lymphoid lineages of definitive hematopoiesis via its transcriptional regulation of proliferation, differentiation and apoptosis in hematopoietic progenitor cells (Oh & Reddy 1999). The c-myb gene is abundantly expressed in immature proliferating hematopoietic progenitor cells but not in mature non-proliferating cells (Gonda & Metcalf 1984; Sitzmann et al. 1995). c-Myb regulates self-renewal of hematopoietic progenitors by using FDCP-mix cell line as a model (White & Weston 2000). Similarly, analyses of a Friend murine erythroleukemia cell line indicated that the maintenance of the immature and proliferating status depended on c-Myb (Chen et al. 2002). Down-regulation of c-myb is a prerequisite for normal terminal differentiation of erythroid and myelomonocytic progenitors (Gonda & Metcalf 1984; Todokoro et al. 1988). While constitutive expression of c-myb blocked the differentiation of immature hematopoietic cells (Todokoro et al. 1988; Chen et al. 2002), anti-sense oligonucleotides that target c-myb, as well as the dominant negative form of c-Myb, inhibited proliferation and induced differentiation of normal myelomonocytes and myeloid leukemia cell lines (Gewirtz & Calabretta 1988; Lyon & Watson 1995; White & Weston 2000).

Two waves of hematopoiesis take place during mammalian embryogenesis. The first wave, primitive hematopoiesis, occurs in the yolk sac (Moore & Metcalf 1970); the second wave, definitive hematopoiesis, begins in the intraembryonic para-aortic splanchnopleure (P-Sp) and aorta-gonad-mesonephros (AGM) region followed by colonization and initiation of hematopoiesis in the fetal liver and then in the bone marrow (Muller et al. 1994; Ogawa et al. 2001). High levels of c-myb expression can be detected at sites of definitive hematopoiesis (Sitzmann et al. 1995). Targeted gene disruption has demonstrated an essential role of c-myb in establishment of the definitive hematopoietic system (Mucenski et al. 1991). c-myb null mutant mouse embryos showed an abrupt failure of fetal liver hematopoiesis, and die around 15 dpc due to severe anemia. While primitive hematopoiesis was not disturbed, myeloid, erythroid and lymphoid lineages of the definitive hematopoiesis were impaired. Subsequent analyses of c-myb–/– chimeric mice and c-myb knockdown studies showed that, even though definitive hematopoiesis can be initiated in the absence of or with lower levels of c-Myb, hematopoietic progenitors cannot self-renew or proliferate, thereby ultimately resulting in exhaustion during hematopoietic development (Allen et al. 1999; Clarke et al. 2000; Sumner et al. 2000; Emambokus et al. 2003). Despite the evident requirement for c-Myb in the establishment of definitive hematopoiesis, the exact stage from which c-Myb exerts its role in the developmental process of hematopoietic progenitors remains unclear. Melotti & Calabretta (1996a,b) reported that embryonic stem (ES) cells constitutively expressing c-Myb (undifferentiated and at various days after induction of differentiation) gave rise to increased number of hematopoietic colonies in a semisolid culture. Since c-Myb was expressed constitutively, however, differentiation steps between ES cells and hematopoietic cells in which the forced expression of c-Myb had an impact are not clear.

It has been proposed that the endothelial cell (EC) lineage is one of the direct sources of the definitive hematopoietic cells (Nishikawa et al. 2000; Ogawa et al. 2001). The presence of hemogenic ECs has been documented in chick, mouse and human embryos mainly by lineage tracing experiments as well as functional analyses (Jaffredo et al. 1998; Nishikawa et al. 1998a,b; de Bruijn et al. 2002; North et al. 2002; Sugiyama et al. 2003). Identification of vascular endothelial cadherin (VE-cadherin) as an EC-specific surface marker made it possible to isolate ECs from mouse embryos by using fluorescence activated cell sorting (FACS) (Nishikawa et al. 1998b). VE-cadherin+ CD45– ECs sorted from mouse embryos as well as differentiating ES cells can give rise to erythroid, myeloid and lymphoid cells in vitro (Nishikawa et al. 1998a,b; Ogawa et al. 1999; North et al. 2002). We have also shown that VE-cadherin+ CD45– ECs sorted from mouse embryos were capable of reconstituting long-term lymphopoiesis in SCID mice (Fraser et al. 2002). These data strongly suggest that ECs serve as a developmental source of the definitive hematopoietic cell lineages (Nishikawa et al. 2000; Ogawa et al. 2001; Dzierzak 2002) although the molecular events that specifically regulate the commitment of hemogenic ECs from mesoderm and subsequent hematopoietic development from these ECs are still unclear. Our recent studies found that a subpopulation of VE-cadherin+ CD45– ECs enriched for hematopoietic activity expressed the c-myb gene suggesting a putative role of c-Myb in the development of hemogenic ECs (Ogawa et al. 1999; Hirai et al. 2003).

In this study, we hypothesized that c-Myb may have a functional role in hemogenic ECs to regulate establishment of their potential and endothelial/hematopoietic cell transition. We addressed this issue by using an in vitro differentiation system of ES cells combined with a tetracycline-regulated inducible gene expression. Rapid degradative nature of c-Myb protein allowed us to induce over-expression of c-Myb restricted to the developmental stages before the divergence of hematopoietic cell lineages from ECs (Bies et al. 2002). Our results indicate that c-Myb positively regulates not only the formation of hemogenic ECs but also the proliferative capacity of EC-derived hematopoietic progenitors in which the transgene has been already shut down, probably by activating multiple genes related to the development of definitive hematopoietic progenitor cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
c-Myb over-expression can be induced and canceled efficiently during ES cell differentiation

The Tet-Off gene expressing system used here is a sensitive, efficient and reversible means of inducing expression of a specific gene (Era et al. 2002). c-myb-IRES-EGFP cDNA was placed under the control of the Tet-Off specific promoter which, when activated by tetracycline transactivator (tTA), drives expression of c-myb/EGFP transcripts in the absence of tetracycline (Tet–) or suppresses it in the presence of tetracycline (Tet+) (Fig. 1A). The construct was introduced into a wild-type (c-myb+/+) mouse ES cell line which constitutively expresses tTA. After drug selection, positive clones were characterized for their abilities to controllably induce c-Myb/EGFP expression in both undifferentiated and differentiated conditions.


Figure 1
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  		Figure 1  Tet-regulated inducible over-expression of c-Myb in ES cells. (A) Expression of a bicistronic transcript encoding c-Myb and EGFP is driven by a Tet-regulated promoter which is activated in the absence of tetracycline (Tet–) or suppressed in the presence of tetracycline (Tet+). (B, C) Undifferentiated ES cells maintained in the presence of tetracycline were induced to express c-Myb/EGFP by removing tetracycline for indicated periods of time. The level of EGFP fluorescence and c-Myb protein was analyzed by FACS (B) and Western blotting (C), respectively. (D) ES cells maintained in the presence of tetracycline were allowed to differentiate by culturing with OP9 stromal cells. Tetracycline was removed from Day 3 to Day 5 to induce over-expression of c-Myb/EGFP (Tet–). Expression of the transgene was suppressed by re-exposure to tetracycline for indicated periods of time. The level of c-Myb protein was analyzed by Western bloting. Tet+ an endogenous level of c-Myb protein expressed by ES cells cultured in the presence of tetracycline. OP9; no detectable c-Myb expression in OP9 stromal cells.

 
Inducible expression of EGFP and c-Myb in undifferentiated ES cells were monitored by FACS analysis and by Western blotting analysis, respectively. Expression of EGFP was not observed in the presence of Tet indicating that transcription of the c-myb/EGFP transgene is completely suppressed under the Tet+ condition (Fig. 1B). We detected a small amount of c-Myb protein expressed in the same condition, which is considered as a product of the endogenous c-myb gene (Fig. 1C). Expression of c-Myb/EGFP was significantly induced by 6 h after removal of Tet and reached a plateau at around 24 h (Fig. 1B,C).

We next assessed differentiating capability of ES cells during the induction of c-Myb/EGFP. ES cells were induced to differentiate by plating on OP9 stromal cell layer, and from Day 3 on, Tet was removed to allow the expression of the c-myb/EGFP transgene. Expression of VE-cadherin, a specific marker of ECs, was analyzed by FACS at Day 5. Around 25% of the cells expressed VE-cadherin on their surface (data not shown), indicating the ability of the ES cell clones to differentiate into endothelial cell lineage.

We further tested whether conditional expression of the c-myb transgene could be shut off reversibly and efficiently. Expression of c-Myb was induced during ES cell differentiation by removal of Tet from Day 3 to Day 5, followed by re-institution of Tet for different periods of time. Western blotting analysis indicated that the c-Myb protein had already decreased to endogenous levels after re-exposure to Tet for 6 h (Fig. 1D), suggesting that over-expression of c-Myb can be abolished rapidly in the Tet-off cell lines. Two independently derived clones were used in the following experiments and generated similar results.

c-Myb over-expression increases the frequency of hemogenic precursors in the endothelial cell population

In order to study the biological effect of c-Myb over-expression on the hemogenic potential of ECs, we restricted the transgene induction from Day 3 to Day 5 of ES cell differentiation, the stage at which mesodermal cells are generated and subsequent commitment to the EC lineage takes place (Endoh et al. 2002). Given that c-Myb expression reaches a plateau within 24 h of induction (Fig. 1C) and VE-cadherin+ cells start to appear from Day 4 (Endoh et al. 2002) the 2-day-induction is supposed to focus most on the generating ECs. EC development was not affected by over-expression of c-Myb. Hence, while the number of total cells recovered at Day 5 was slightly decreased in Tet– cultures (85% that of Tet+ control cultures; data not shown), frequency of VE-cadherin+ CD45– ECs in the total population was increased (Fig. 2A; 25% in Tet– vs. 20% in Tet+). Therefore, total number of ECs appeared to be comparable between Tet– and Tet+ cultures.


Figure 2
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  		Figure 2  Over-expression of c-Myb/EGFP in the endothelial cells differentiated from ES cells. (A) ES cells maintained in the presence of tetracycline were allowed to differentiate by culturing with OP9 stromal cells. Tetracycline was removed from Day 3 to Day 5 in some cultures (Tet–) to induce over-expression of c-Myb/EGFP. Other cultures were kept in the presence of tetracycline (Tet+). Expression of EGFP, VE-cadherin and a pan-hematopoietic marker CD45 was analyzed by FACS at Day 5. Sorting gates for VE-cadherin+ CD45– EGFP+/– endothelial cells are indicated. (B) Experimental design shows the stage specific induction of c-Myb over-expression during ES cell differentiation and subsequent hematopoietic assays of sorted endothelial cells.

 
VE-cadherin+ CD45– ECs cultured in the presence or absence of Tet (as assessed by EGFP expression) were sorted at Day 5 and plated on to OP9 stromal cells in the presence of growth factors that favor differentiation of multilineage hematopoietic progenitors. Tet was also added to shut off and suppress c-myb transgene expression from Day 5 on (Fig. 2B). FACS analyses on the cells harvested at Day 12 revealed that erythrocytes, granulocytes and monocytes/macrophages were abundantly generated from both the Tet– and Tet+ ECs (data not shown). In order to examine whether the frequency of cells which have hemogenic activities among EC population was influenced by the over-expression of c-Myb, we quantified the hematopoietic cell colonies generated from sorted VE-cadherin+ CD45– ECs by overlaying cultures with semisolid medium one day after plating ECs (Ogawa et al. 1999). The frequency of immature multipotential progenitors which gave rise to mixed colonies of erythroid and myeloid lineages was increased six-fold in Tet– c-Myb-activated ECs compared to Tet+ control ECs (Fig. 3A,B). The mixed colonies derived from Tet– ECs were larger than that from control ECs (Fig. 3B). We confirmed that these colonies contained more cells (Fig. 3C). Similar results were also observed in erythroid and granulocyte/macrophage progenitor activities but not remarkable on mono-potential macrophage progenitors (Fig. 3A,C). We performed the same experiments by using a control ES cell clone in which EGFP, instead of c-Myb/EGFP, is induced under the Tet– condition. We did not detect any significant effect of EGFP expression in our experimental system (Fig. 3D).


Figure 3
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  		Figure 3  Enhanced hemogenic potential of endothelial cells by over-expression of c-Myb. (A) Over-expression of c-Myb/EGFP was induced during ES cell differentiation as described in Fig. 2. VE-cadherin+ CD45– (either EGFP+ or EGFP– according to the presence or absence of c-Myb induction) cells were sorted from Day 5 cultures and assessed for hematopoietic colony formation in the presence of OP9 cells and cytokines. Tetracycline was added to suppress over-expression of c-Myb during the hematopoietic assay. A representative result obtained from six independent experiments is shown (**P < 0.01 in Student's t-test). (B) (a–c) Morphologies of mixed colonies of erythroid and myeloid lineages, (d–f) burst colonies of erythroid lineage (g–i) and granulocyte/macrophage colonies which were derived either from control (Tet++) or c-Myb-activated (Tet–+) endothelial cells are shown. Colonies were picked and stained with May-Grunwald-Giemsa solution (c,f,i). Scale bars: 500 µm (a,b); 50 µm (c); 200 µm (d, e); 10 µm (f); 300 µm (g, h); 20 µm (i). (C) Different types of hematopoietic cell colonies were picked and number of cells per colony was scored. (n = 12; *P < 0.05) (D) Instead of c-Myb/EGFP, EGFP alone was induced to express during ES cell differentiation. VE-cadherin+ CD45– endothelial cells were sorted and analyzed for hemogenic potentials as described above.

 
Over-expression of c-Myb in ECs appeared to promote proliferation of descendant hematopoietic progenitor cells as indicated by the increase of colony size. Thus one can argue that increased frequency of hematopoietic cell colonies may be a result of the enhanced proliferation of committed hematopoietic progenitor cells during the liquid culture period that preceded semisolid medium overlay. The liquid culture step was necessary for settlement of plated ECs on OP9 cell layer. We compared frequency of hematopoietic cell colonies for the various length of liquid culture period and found that the frequency was not influenced by the duration of liquid culture in the range 2–24 h, excluding a possible contribution of expansion of committed progenitor cells (Supplementary Fig. S1). Thus, the observed increase of hematopoietic cell colonies was suggested to be attributed to an increase of hemogenic precursors in the EC population per se, which is closely associated with effects of over-expression of c-Myb conditionally induced during EC development.

c-Myb over-expression in endothelial cells promotes self-renewal of descendant hematopoietic progenitors

The enhanced hematopoietic activity of c-Myb-activated ECs, especially the higher frequency of multipotent hematopoietic progenitor activity and their high proliferation capability, prompted us to investigate self-renewal potential of the progenitors derived from ECs using a re-plating assay. VE-cadherin+ CD45– ECs sorted from either Tet+ or Tet– cultures were cultured in two different systems; either on OP9 with semisolid medium for primary colony formation assay or on OP9 with liquid medium for a week (Fig. 2B). The derived hematopoietic cells were harvested and re-plated for secondary colony formation assay on OP9 with semisolid medium. Tet was included in the media throughout the cultures after the initial sorting of the ECs to suppress the c-myb transgene. Table 1 shows the recovery of secondary colony forming cells after the one-week expansion culture together with the ratio to the initial number of primary colony forming cells. The cultures initiated from the Tet– c-Myb-activated ECs had significantly higher number of multipotential and erythroid progenitors compared to that from control ECs. In contrast, the effect of c-Myb induction on granulocyte/macrophage progenitors was less prominent. These results suggest that c-Myb over-expression in ECs promoted survival or self-renewal capacities of descendant multipotent hematopoietic progenitors in short-term culture.


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  		Table 1 Self-renewal activity of hematopoietic progenitor cells derived from c-Myb-activated endothelial cells
 
B lymphogenic potential of endothelial cells is augmented by c-Myb over-expression

B lymphocytes can be generated from hemogenic ECs (Nishikawa et al. 1998b; Fraser et al. 2002). We next analyzed the impact of c-Myb over-expression on B lymphogenic development from VE-cadherin+ progenitors. To determine the frequency of B cell progenitors which are clonable in the presence of OP9 and cytokines, VE-cadherin+ CD45– cells sorted either from Tet+ or Tet– ES cell cultures were plated under limiting dilution conditions with Tet (Fig. 2B). Clonal proliferation of B lymphocytes was monitored by FACS analyses for the expression of B220 and CD19 on the expanded clones (data not shown). Figure 4 shows that B cell progenitor frequency in the c-Myb-activated EC population was trebled compared to that in control ECs (1/700 vs. 1/2000), suggesting that over-expression of c-Myb augmented not only erythroid and myeloid potentials but also B lymphogenic potential of ECs.


Figure 4
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  		Figure 4  Enhanced B lymphogenic potential of endothelial cells by over-expression of c-Myb. Over-expression of c-Myb/EGFP was induced during ES cell differentiation as described in Fig. 2. VE-cadherin+ CD45– (either EGFP+ or EGFP– according to the presence or absence of c-Myb induction) cells were sorted from Day 5 cultures and assessed for a potential to give rise to B lymphocyte colonies in a limiting dilution analysis with OP9 cell layer and cytokines. Tetracycline was added to suppress over-expression of c-Myb during the assay. Generation of B lymphocytes was assessed after 2 weeks by using FACS analyses on the B lymphocyte markers, B220 and CD19. The frequency of B lymphogenic precursors was determined from 37% negative fraction according to Poisson distribution. The result shown is a representative of three independent experiments.

 
c-Myb over-expression activates genetic programs for definitive hematopoietic development in endothelial cells

In order to get an insight into the mechanism underlying the enhanced hemogenic activities of ECs induced by c-Myb over-expression, we analyzed expression levels of genes critical for definitive hematopoiesis. Tal1 and Lmo2 are essential for commitment to hematopoietic lineages from mesoderm and endothelium (Yamada et al. 1998; Endoh et al. 2002). Gata2 is indispensable for hematopoietic progenitor cells to maintain their immaturity (Minegishi et al. 2003). Runx1 is essential for the transition of EC to definitive hematopoietic cell lineages (Yokomizo et al. 2001). Hoxb4, Mll, Etv6/Tel are genes involved in definitive hematopoiesis and the maintenance of hematopoietic stem cells (Wang et al. 1998; Kyba et al. 2002; Ernst et al. 2004). Bcl2, an identified target gene of c-Myb, modulates apoptosis in various types of cells (Frampton et al. 1996). VE-cadherin+ CD45– ECs, either EGFP+ (Tet–) or EGFP– (Tet+), were sorted at Day 5 of ES cell differentiation and subjected to semiquantitative RT-PCR analyses on the transcripts of these genes.

As shown in Fig. 5, all of the above genes were up-regulated for mRNA expression in c-Myb-activated ECs, except that the expression of Gata2 appeared to be suppressed. Consistent with the identity of VE-cadherin+ CD45– ECs, sorted cells expressed high levels of VE-cadherin while no detectable expression of CD45 was observed. Therefore, elevation of the hematopoiesis-related genes is not likely to be attributable to possible contamination of committed hematopoietic cells. The increased frequency of hemogenic precursors in the EC population may simply account for the elevation of gene expression. Alternatively, a cell-autonomous hemogenic program was suggested to be activated more positively in c-Myb induced ECs, which might partially contribute not only to the direct effect of c-Myb on generation of hemogenic ECs but also to indirect postc-Myb effect on the proliferative/survival activity of hematopoietic progenitor cells derived from c-Myb-activated ECs.


Figure 5
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  		Figure 5  Enhanced expression of hematopoiesis-related genes in c-Myb-activated endothelial cells. Over-expression of c-Myb/EGFP was induced during ES cell differentiation as described in Fig. 2. VE-cadherin+ CD45– (either EGFP+ or EGFP– according to the presence or absence of c-Myb induction) cells were sorted from Day 5 cultures and subjected to RT-PCR analyses for the expression of several hematopoiesis-related genes. The result shown is a representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The developmental origin of the definitive hematopoietic system has long been a controversial issue. We and others have proposed that the hemogenic endothelium is one of the developmental sources of the definitive hematopoietic cell lineages in the mouse embryos (Nishikawa et al. 2000; Ogawa et al. 2001; Dzierzak 2002). Hemogenic ECs are known to express c-myb which is crucial in definitive hematopoiesis (Ogawa et al. 1999; Hirai et al. 2003). In order to gain an insight into a possible role for c-Myb in hemogenic ECs, we assessed whether over-expression of c-Myb in the ECs has any effects on development of definitive hematopoietic cells from ECs by taking advantage of an in vitro differentiation system of murine ES cells combined with a Tet-Off inducible transgene expression (Endoh et al. 2002; Era et al. 2002). As the half-life of c-Myb proteins is very short and the suppression of transgenes in the presence of Tet is highly effective in the Tet-Off system (Bies et al. 2002; Era et al. 2002), over-expression of c-Myb was abolished rapidly by the re-introduction of Tet to the differentiation cultures.

VE-cadherin+ CD45– cells generated from ES cells give rise to VE-cadherin+ Flk-1+ CD31+ sheet-like structures which are capable of incorporating Ac-LDL when cultured on stromal cells (Hirashima et al. 1999). Therefore, VE-cadherin+ CD45– cells differentiated from ES cells are most likely to represent the EC lineage. We induced over-expression of c-Myb to coincide with development of VE-cadherin+ CD45– ECs from mesoderm during the course of differentiation of ES cells (Day 4 to Day 5 taking account of the delay of induction) (Endoh et al. 2002). ECs sorted from Day 5 cultures were analyzed for hemogenic potentials in the suppression of c-Myb over-expression by re-exposure to Tet. Our data showed that c-Myb over-expression augmented the hemogenic activity of ECs. Although over-expression of c-Myb was timed with the emergence of ECs in the ES cell cultures, immature mesodermal progenitors toward the EC lineage could also be induced with c-Myb, which may raise a possibility that the enhancement of hemogenic activity of ECs is attributed to ectopic expression of c-Myb in the mesodermal cell stage. However, we observed essentially the same extent of augmentation of hemogenic activity of ECs sorted from cultures in which c-Myb has been induced from Day 3 to Day 5 (again taking account of the delay), which is more likely to target the emergence of mesodermal cells. Therefore, we would prefer to conclude that over-expression of c-Myb in the ECs is responsible for the enhancement of hemogenic activity.

In this study, we showed that the frequency of hemogenic precursors in the EC population was increased by over-expression of c-Myb. Those hemogenic precursors encompassed multiple lineages including erythroid, myeloid and B lymphoid potentials. Augmentation was most prominent in CFU-Mix (six-fold) and least in CFU-M (1.5-fold). These results suggest that over-expression of c-Myb increased population size of the hemogenic ECs. A possible mechanism for this is that hemogenic ECs gained higher proliferative capacity against non-hemogenic ECs in response to the increase of c-Myb protein. This effect may be attributed simply to the increase of c-Myb protein beyond a threshold level, which may happen preferentially in the hemogenic ECs which express endogenous c-myb gene, or may be specific to the hemogenic EC population. The latter is reminiscent of the specific role of c-Myb in proliferation/survival of hematopoietic-committed progenitor cells. Alternatively, ectopic expression of c-Myb in non-hemogenic ECs could convert them into hemogenic ECs. This "differentiation" mechanism should rely on a pre-existing hematopoietic program since c-Myb alone is not capable of inducing hematopoietic commitment. Therefore only a subset of ECs which is already compatible with or competent for hemogenic differentiation might respond to the increment of c-Myb protein.

Hemogenic ECs derived from ES cells do not give rise to hematopoietic stem cells. Production of hematopoietic stem cells in vitro is generally rare, if not impossible, in ES cell differentiation systems (Kyba et al. 2002). Therefore, hematopoietic cell colonies generated from sorted ECs in this report more likely followed a direct differentiation process which skipped the stem cell stage to become multipotent or monopotent hematopoietic progenitor cells (Nishikawa et al. 2000). Differential effects of c-Myb over-expression on the frequency of precursor cells of different lineages (e.g. CFU-Mix vs. CFU-M) may support this notion. Hemogenic ECs might represent either a homogeneous population which has multiple programs for hematopoietic development in individual cells or heterogenous populations consisted of cells bearing different programs which prefer different lineages. Nevertheless, our results suggest that c-Myb is able to function in ECs to exert a positive influence on the hemogenic programs in this cell type.

c-Myb is a critical player in the development of the definitive erythropoiesis. Erythroid progenitors (BFU-E) are absent in fetal liver of c-myb–/– mouse embryos and significantly reduced in c-myb knockdown mouse embryos (Mucenski et al. 1991; Emambokus et al. 2003). Recently, it was also reported that c-Myb-dependent trans-activation of p300 was required for erythroid differentiation of megakaryocyte/erythrocyte progenitors (Sandberg et al. 2005). Our observation that the frequency of BFU-E activity was augmented in c-Myb-activated ECs is in agreement with above reports and may suggest that c-Myb promotes a process of erythroid commitment in the hemogenic ECs as in the case of early hematopoietic progenitor cells. Alternatively, c-Myb may modulate erythroid potential by rescinding the immature state of ECs as a hemogenic precursor cell. It was suggested that GATA-2 preserves immaturity of hemogenic precursors as a diminution of GATA-2 expression appears to be required for differentiation of the precursor cells (Minegishi et al. 2003). Our result showing that Gata2 expression was reduced in c-Myb-activated ECs may support this notion, although a direct link between c-Myb and GATA-2 remains to be an open issue. Furthermore, such a mechanism might also account for the augmentation of progenitor activity of other hematopoietic lineages.

Mice homozygous for the knockdown allele of the c-myb gene have reduced number of B220+ B lymphoid cells (Emambokus et al. 2003). B cell differentiation was arrested at the B220+ CD43+ pro-B cell stage in the c-myb knockdown bone marrow cells. Similarly, a mutation in the trans-activation domain of c-Myb which disrupts its interaction with the transcriptional coactivator p300 resulted in decrease of intramarrow pro-B and pre-B cells in number (Sandberg et al. 2005) suggesting that B cell differentiation or proliferation is dependent on c-Myb function. As the most immature B lymphoid precursors were unaffected in c-myb mutant mice, c-Myb is not essential for the commitment of early progenitors to the B cell lineage. Initiation of B cell development, however, is controlled by a combined dosage of a number of transcription factors such as E2A and PU.1 (Warren & Rothenberg 2003) thus it may be possible that the commitment process which putatively took place in ECs was modulated by over-expression of c-Myb thereby resulting in an augmentation of B cell potential as we observed. Alternatively, c-Myb may have increased precursors with multilineage potentials in ECs as discussed below, and the augmentation of B cell potential is a consequence of this effect.

It is well established that c-myb is predominantly expressed in immature hematopoietic cells (Gonda & Metcalf 1984; Sitzmann et al. 1995). The expression pattern suggests a role of c-Myb in maintaining the undifferentiated state of immature hematopoietic cells as early studies have demonstrated that down-regulation of c-myb is essential for terminal differentiation of hematopoietic cells (Gonda & Metcalf 1984; Todokoro et al. 1988). However, a role of c-Myb in earlier hematopoietic progenitors or hematopoietic stem cells is not well understood. An increased proportion of immature hematopoietic cells was observed in the fetal liver of c-myb knockdown embryos (Emambokus et al. 2003). Consistent with this notion, mice with a mutation in the trans-activation domain of c-Myb showed an increase in the number of hematopoietic stem cells in the bone marrow while subsequent differentiation was compromised (Sandberg et al. 2005). These reports suggested that c-Myb trans-activation may repress the proliferation of hematopoietic stem cells or couple the proliferation with subsequent differentiation. Our results showing that c-Myb over-expression enhanced the multilineage potential of ECs might be consistent with the latter possibility and suggest that c-Myb drives derivation of substantive multilineage precursor cells from a putative stem cell potential inherited in the hemogenic ECs.

In this study, we demonstrated that hematopoietic progenitors derived from c-Myb-activated ECs possessed higher proliferating capability even after suppression of c-Myb over-expression. Multi-lineage mixed colonies derived from c-Myb-activated ECs contained more cells than those from control ECs. More interestingly, CFU-Mix and BFU-E which were generated from c-Myb-activated ECs showed sustained proliferation when cultured on OP9 stromal cells. These data suggested that high levels of c-Myb in ECs not only led to an increase in population size of hemogenic ECs but also conferred a significant self-renewal capability on descendant EC-derived hematopoietic progenitors. Possible molecular mechanisms underlying the enhancing effects of c-Myb may be suggested by the elevated expression of several genes which regulate the definitive hematopoiesis in the c-Myb-activated ECs. These genes included Bcl2, Hoxb4, Mll and Etv6. For instance, Bcl2 is one of the c-Myb-regulated genes so far identified and has been shown to confer hematopoietic progenitor cells with survival advantage by inhibiting apoptosis (Frampton et al. 1996). Hoxb4, a homeotic selector gene expressed in the hematopoietic stem cells, has been shown to regulate self-renewal capacity of the stem cells (Kyba et al. 2002). The Mll gene encodes a Trithorax-related chromatin-modifying transcriptional regulator which positively regulates Hox genes. Analyses of Mll-deficient embryos showed that the development of hematopoietic stem cells in the AGM region was severely impaired in the absence of the Mll gene (Yagi et al. 1998). Furthermore, an Ets-related transcription factor Tel/Etv6 is known to be unique in that it is selectively required for survival of adult hematopoietic stem cells in the bone marrow (Wang et al. 1998). Some of these molecules probably persisted even after c-Myb had receded and continuously exerted a function to regulate self-renewal or survival capability of EC-derived CFU-Mix. In that sense, c-Myb is a potential regulator that is able to influence a competency of definitive hematopoietic progenitor cells diverged from hemogenic ECs.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
ES cell lines

A parental ES cell clone transfected with pCAG-tTA and pUHD10-3-puro was provided by Dr T. Era in RIKEN CDB, Kobe (Era et al. 2002). ES cells were maintained on gelatin-coated culture dishes in the presence of leukemia inhibitory factor (LIF) as previously described (Endoh et al. 2002). pUHD-c-myb-IRES-EGFP was constructed by inserting a coding sequence of murine c-myb cDNA between PhCMV*-1 promoter and IRES-EGFP reporter cassette of pUHD10-3-IRES-EGFP (Era et al. 2002). Tet-OFF-regulated c-myb transgenic ES cell lines were established by introducing pUHD-c-myb-IRES-EGFP into the parental cell clone.

In vitro differentiation of ES cells

Thirty thousands ES cells were inoculated into a 25-cm2 culture flask preseeded with OP9 stromal cells and cultured for 5 days in MEM-alpha (Invitrogen) supplemented with 10% FCS and 5 x 10–5 mol/L 2-mercaptoethanol (induction medium) in the absence of LIF. Tetracycline (Tet) was added to the medium at the concentration of 1 µg/mL from Day 0 to Day 3 to prevent the c-myb/EGFP transgene expression. From Day 3 to Day 5, Tet was removed from some cultures to induce c-myb/EGFP transgene expression. Cultures were dissociated at Day 5 with cell dissociation buffer (Invitrogen), blocked with normal mouse serum and stained with phycoerythrin-conjugated anti-CD45 monoclonal antibody (mAb) (BD Pharmingen) and allophycocyanin-conjugated anti-VE-cadherin mAb (VECD1) (Nishikawa et al. 1998a). CD45– VE-cadherin+, either EGFP+ or EGFP–, endothelial cells were sorted using a FACS-Vantage cell sorter (BD Biosciences).

Hematopoietic colony formation assay

Hematopoietic colony formation assay was performed as previously described (Ogawa et al. 1999). FACS-purified VE-cadherin+ CD45– ECs were put into a 6-well plate (1000 cells per well) preseeded with OP9 stromal cells and incubated in the induction medium supplemented with 20 ng/mL rmSCF (R&D Systems), 2 IU/mL rhEPO (Kirin Brewery), 20 ng/mL rmIL-3 (R&D Systems) and 20 ng/mL rhG-CSF (Kyowa Hakko). Tet was also added in the medium to shut off the c-myb/EGFP transgene expression. After 24 h, medium was replaced with fresh semisolid medium that was consisted of the induction medium, mixture of growth factors, 1.2% methylcellulose and Tet. Cells were further cultured for 6 days and hematopoietic cell colonies generated were scored. Colonies were picked and morphologically examined by Giemsa staining. In some cultures, proliferation of hematopoietic cells were induced on OP9 for 7 days in the liquid induction medium without methylcellulose, followed by re-plating on to fresh OP9 cells to score secondary hematopoietic cell colonies.

Limiting dilution assay for B lymphogenic potential

For limiting dilution assay of B cell progenitor activity, FACS-purified VE-cadherin+ CD45– ECs were diluted in various concentrations and inoculated into 24-well plates preseeded with OP9 cells. Cells were cultured in the induction medium in the presence of 50 ng/mL rmSCF, 20 ng/mL rmFlt3L (R&D Systems), 20 ng/mL rmIL-7 (R&D Systems) and Tet. After 2 weeks of culture, harvested cells were stained with mAbs against B220 and CD19 (BD Pharmingen) and analyzed by FACS-Vantage. Positive wells with B220+ CD19+ cells were scored.

Western blotting

Cell lysate was subjected to SDS-PAGE, and proteins were transferred on to a PVDF membrane (Millipore). Membrane was incubated with a rabbit polyclonal antibody against murine c-Myb (Santa Cruz Biotechnology) in PBST buffer containing 5% skimmed milk. Blots were then probed with a HRP-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratory) and c-Myb band was visualized by enhanced chemiluminescence (ECL, Pierce).

RT-PCR

Total RNA was prepared from sorted cells and reverse-transcribed with Supercript II reverse transcriptase (Invitrogen) according to maker's instructions. PCR was performed by Ex Taq DNA polymerase (Takara Shuzo). Primers used were: Tal1(f) 5' ATT GCA CAC ACG GGA TTC TG 3'; Tal1(r) 5' CAT ACA GTA CGA CAC TGA CG 3'; Lmo2(f) 5' AGA ACA TAG GGG ACC GCT AC 3'; Lmo2(r) 5' GAT GAT CCC ATT GAT CTT GG 3'; Gata2(f) 5' TGC AAC ACA CCA CCC GAT ACC 3'; Gata2(r) CAA TTT GCA CAA CAG GTG CCC 3'; Hoxb4(f) 5' AGA ACC CCC TGC ATC CCA 3'; Hoxb4(r) 5' CCG AGC GGA TCT TGG TGT 3'; Mll(f) 5' CCA GCA GTG AGC ATG TAG AG 3'; Mll(r) 5' TGA AGG CGG AAG CAC TGC GT 3'; Etv6(f) 5' TCC TGC ATC AGA ACC ATG AC 3'; Etv6(r) 5' CTC TTC CGG TGG GGA CAT A 3'; Bcl2(f) 5' TCG CTA CCG TCG TGA CTT C 3'; Bcl2(r) 5' AAA CAG AGG TCG CAT GCT G 3'; Runx1(f) 5' CCT GCT TGG GTG TGA GGC CC 3'; Runx1(r) 5' GCC TCG CTC ATC TTG CCG GG 3'; c-myb(f) 5' AAG CTT GTC CAG AAA TAT GGT CCG AAG 3'; c-myb(r) 5'GGC TGC CGC AGC CGG CTG AGG GAC 3'; Cdh5(f) (VE-cadherin) 5' GGA TGC AGA GGC TCA CAG AG 3'; Cdh5(r) 5' CTG GCG GTT CAC GTT GGA CT 3'; Ptprc(f) (CD45) 5' GAC CAT GGG TTT GTG GCT CAA AC 3'; Ptprc(r) 5' CAC AGT AAT GTT CCC AAA CAT GGC 3'; Actb(f) (beta-actin) 5' CCT AAG GCC AAC CGT GAA AAG 3'; Actb(r) 5' TCT TCA TGG TGC TAG GAG CCA 3'. Cycle conditions were: one cycle at 94 °C for 5 min followed by 40 cycles at 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min and one additional cycle at 72 °C for 10 min. RT-PCR products were electrophoresed through 1% agarose gel and stained with ethidium bromide.


   Acknowledgements
 
We are grateful to Dr S. Ishii for the c-myb cDNA clone and Dr T. Era for the Tet-Off vectors and the parental ES cell line which constitutively expresses tTA. We would like to thank Kyowa Hakko Kogyo Co., Ltd. and Kirin Brewery Co., Ltd. for generous gifts of recombinant human G-CSF and erythropoietin, respectively. We also thank Dr S. Fraser for critical comments of the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 14081204) and Japan Society for the Promotion of Science (nos. 16390227, 15790495). T.F. was supported by a postdoctoral fellowship from the 21st Century COE, Kumamoto University.


   Footnotes
 
Communicated by: Tetsuya Taga

* Correspondence: E-mail: minetaro{at}kaiju.medic.kumamoto-u.ac.jp


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Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 3 November 2005
Accepted: 21 April 2006





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