Evi1 is predominantly expressed in LT-HSCs in adult BM
To elucidate Evi1 expression within the hematopoietic system, we have generated gene-targeted mice in which an internal ribosome entry site (IRES)-GFP cassette is knocked-in to the Evi1 locus by homologous recombination (). This knock-in allele functions in a bicistronic manner in that expression of both Evi1 and GFP is under the endogenous transcriptional regulatory elements of the Evi1 gene, thus enabling us to track Evi1 expression on an individual cell basis. Appropriately targeted TT2 embryonic stem (ES) cell clones were identified by Southern blotting (). Mice heterozygous for the Evi1-IRES-GFP allele (Evi1+/GFP) were distinguished from WT mice by genotyping PCR (). Western blot analysis showed the presence of GFP protein and comparable expression of Evi1 protein in embryonic fibroblast cells from Evi1+/GFP mice compared with WT mice (). Evi1+/GFP mice were phenotypically indistinguishable in survival, hematopoietic cellularity, and lineage composition from WT controls (unpublished data). Initial flow cytometric analysis of adult Evi1+/GFP mice revealed a small, but discrete, population of GFP+ cells (0.15 ± 0.6%; ), confirming the expression of the Evi1-IRES-GFP allele. To examine whether GFP expression levels correlated with those of endogenous Evi1 mRNA expression, Evi1 expression of sorted GFP− and GFP+ cells from BM of Evi1+/GFP mice was analyzed by real-time quantitative PCR (RQ-PCR). Evi1 mRNA was exclusively expressed in the GFP+ cells, and almost no expression was found in the GFP− cells (), indicating that GFP expression in this mouse model faithfully marks cells with active Evi1 expression.
Figure 1. Generation of Evi1-IRES-GFP knock-in mice. (A) The structure of murine Evi1 and the targeted Evi1-IRES-GFP locus is shown. RV, EcoRV; X, XbaI. (B) Southern blot analysis of genomic DNA isolated from WT ES cells (Evi1+/+) and two independent clones of (more ...)
Figure 2. Evi1 is predominantly expressed in LT-HSCs in adult BM. (A) FACS analysis of expression of lineage markers (Lin), c-kit, and Sca-1 on GFP+ cells in adult BM from Evi1+/GFP mice. Data are representative of three independent experiments. PI, propidium iodide; (more ...) Evi1
mRNA has been shown to be expressed at significantly higher levels in HSPCs (Lin−
[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–specific expression pattern, the distribution of GFP+
cells was examined in adult BM from Evi1+/GFP
mice. Beyond expectation, GFP expression was highly restricted to the LSK fraction (). To confirm stem/progenitor-specific expression of Evi1, we analyzed the GFP fluorescence of various hematopoietic cell populations from BM and spleen of Evi1+/GFP
mice. We found a heterogeneous expression of GFP in the LSK fraction, in which about half of the cells were GFP+
(). Conversely, only 2.5% of common myeloid progenitors (CMPs) expressed GFP, and almost no expression was found in granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs; ). In contrast to the previous study (Chen et al., 2008
), GFP was not expressed in CLPs (). In addition, no GFP expression was observed in mature hematopoietic lineages or nonhematopoietic cells in BM (). Together, these results suggest that Evi1 is uniquely expressed in HSPCs, but its expression is sharply down-regulated along with differentiation.
Because LSK cells, a population which contains multipotent progenitors (MPPs), short-term HSCs (ST-HSCs), and long-term HSCs (LT-HSCs), include both a GFP+
fraction and a GFP−
fraction, we next resolved GFP expression within the LSK compartment for other markers characteristic of LT-HSCs. When LSK cells were subdivided according to CD34 and Flk-2 expression (Orford and Scadden, 2008
), the Flk-2−
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 (). 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−
LSK cells, in which LT-HSCs are highly enriched. In contrast, GFP expression was substantially down-regulated in CD48+
LSK cells, irrespective of CD150 expression (). When we examined how GFP+
cells were distributed within the LSK fraction, GFP expression was highly enriched in the Flk-2−
LSK or CD48−
LSK fractions (). Therefore, these results indicate that Evi1 is dynamically regulated within HSPCs; its expression is predominantly enriched in LT-HSCs and rapidly extinguished during early stages of lineage commitment.
To reinforce Evi1-IRES-GFP knock-in mice as a faithful tool for investigating HSCs, we assessed the number and function of LT-HSCs in BM from Evi1+/GFP mice. Flow cytometric analysis revealed that the frequencies of Flk-2− CD34− LSK or CD48− CD150+ LSK cells were comparable between Evi1+/+ and Evi1+/GFP mice (). In addition, a competitive repopulation assay (CRA) showed that Evi1+/GFP BM cells exhibited slightly less, but not significantly different, long-term reconstitution capacity (), indicating that the number and function of HSCs in Evi1+/GFP mice are similar to WT controls.
Evi1 expression represents a functionally distinct population that remains in an undifferentiated and quiescent state within HSPCs
As only a subset of LSK cells expressed GFP in Evi1+/GFP mice, we hypothesized that Evi1 expression functionally divides the LSK population and marks a more undifferentiated and quiescent state with multipotent differentiation properties in this population. To test this idea, we separated the LSK population into LSK GFP− and LSK GFP+ cells and compared their biological functions. Initially, we confirmed that LSK GFP+ cells had a much higher level of Evi1 transcripts than LSK GFP− cells by RQ-PCR analysis (). Interestingly, despite the negative GFP expression, LSK GFP− cells expressed Evi1 mRNA at a higher level compared with CMPs and GMPs (), which also suggests that Evi1 expression is inversely proportional to the differentiation status. To achieve an estimate of the differentiation stage of these two populations, LSK GFP− and LSK GFP+ cells were cultured in serum-free medium containing stem cell factor (SCF) and thrombopoietin (TPO). After 3 d of culture, the proportion that remained in the LSK fraction was significantly higher in LSK GFP+ cells than in LSK GFP− cells (), suggesting that LSK GFP+ cells are more primitive HSCs. Next, to evaluate the differentiation potential of LSK GFP− and LSK GFP+ cells, we performed colony-forming assays in vitro. Although both populations generated an equivalent number of myeloid colonies CFU-granulocyte/macrophage [CFU-GM]), LSK GFP+ cells gave rise to greater numbers of erythroid (burst-forming unit-erythrocyte [BFU-E]) and multipotential (CFU-granulocyte/erythrocyte/macrophage/megakaryocyte [CFU-GEMM]) colonies than LSK GFP− cells (). These data suggest that Evi1 expression correlates with multipotent differentiation capacity. In addition, to assess the colony-forming capacity at the clonal level, single LSK GFP− and LSK GFP+ cells were cultured in serum-free medium. LSK GFP− cells formed detectable colonies at a frequency comparable to LSK GFP+ cells, but generated smaller numbers of highly proliferative colonies (>300 cells; ), indicating that the LSK GFP+ fraction comprises a higher proportion of HSPCs with enhanced proliferative capacity.
Figure 3. Evi1 expression represents a functionally distinct population that remains in an undifferentiated and quiescent state within HSPCs. (A) RQ-PCR analysis of the expression of Evi1 mRNA in sorted GMPs, CMPs, LSK GFP− cells, and LSK GFP+ cells from (more ...)
Our observations suggested that Evi1 reporter activity is down-regulated as HSCs differentiate. To examine this issue, we forced LSK GFP+ cells to differentiate in vitro in response to SCF, TPO, IL-3, and IL-6. These LSK GFP+ cells predominantly generated GFP− cells (). After culture, the majority of cells that had become GFP− lost the LSK phenotype, whereas most cells that remained in GFP+ continued to express the LSK phenotype (), indicating that loss of GFP correlates with phenotypic differentiation. To confirm the differential phenotype of the GFP− and GFP+ cells after culture reflected their functional status, we compared their ability to form colonies in methylcellulose. GFP+ cells yielded significantly more colonies than GFP− cells (), suggesting that functionally primitive HSCs predominantly reside in the GFP+ fraction. Collectively, these data indicate that in vitro culture of LSK GFP+ cells leads to generation of GFP− cells that are more differentiated, and lend credence to the use of GFP as a fluorescent sensor for the differentiation state of hematopoietic cells.
To determine the cell-cycle distribution of LSK GFP− and LSK GFP+ cells, we performed Hoechst 33342 and pyronin Y staining, which revealed that the majority of LSK GFP+ cells were in G0 phase, whereas a significant proportion of LSK GFP− cells were actively cycling (). These data indicate that, within HSPCs, Evi1 expression represents a functionally distinct population that remains in an undifferentiated and quiescent state.
Evi1 expression marks in vivo long-term multilineage repopulating HSCs in adult BM
Based on the aforementioned data, we hypothesized that Evi1 expression would have the potential to effectively mark long-term multilineage repopulating HSCs. To examine this issue, we performed a CRA, in which 500 purified LSK GFP−
or LSK GFP+
cells were transplanted with 2 × 105
competitor BM cells into lethally irradiated recipients (Fig. S1 A
). At 16 wk after transplantation, flow cytometric analysis of donor-derived cells revealed long-term reconstitution in all recipients transplanted with LSK GFP+
cells (). Moreover, LSK GFP+
cells displayed multilineage potential with robust contribution to myeloid, B, and T cells in peripheral blood (PB) as well as the LSK fraction in BM (). In contrast, LSK GFP−
cells yielded an almost total inability to generate long-term chimerism (), which suggests that this population is devoid of self-renewal activity. To confirm the in vivo repopulating capacity of LSK GFP+
cells, we performed secondary transplantation. Similarly, LSK GFP+
cells showed remarkable long-term reconstitution, whereas LSK GFP−
cells consistently failed to produce detectable donor-derived cells (), demonstrating that in vivo long-term multilineage repopulating cells are exclusively enriched in the LSK GFP+
fraction in adult BM.
Figure 4. Evi1 expression marks in vivo long-term multilineage repopulating HSCs in adult BM. (A and B) PB donor chimerism in CRAs, in which 500 LSK GFP− or LSK GFP+ cells sorted from Evi1+/GFP mice (Ly5.2) were transplanted into lethally irradiated recipients (more ...)
To further refine our analysis designating Evi1 expression as a robust and reliable HSC marker, we compared the repopulating capacity of GFP−
cells within the CD48−
LSK fraction, which is enriched for LT-HSCs (Fig. S1 B). Intriguingly, CD48−
cells exhibited long-term multilineage reconstitution, whereas no engraftment was observed in recipients of CD48−
cells (), suggesting that long-term repopulating HSCs predominantly reside in the GFP+
fraction even within the highly subfractionated LT-HSC fraction. We then examined whether Evi1 expression is associated with repopulating capacity in the CD48+
LSK fraction, which is enriched for ST-HSCs/MPPs with limited self-renewal activity (Fig. S1 B). Although CD48+
cells provided only a transient reconstitution, CD48+
cells showed declining, but sustained engraftment 16 wk after transplantation (). In contrast to CD48−
cells mediated faint myeloid but superior lymphoid reconstitution (). Although it is controversial whether CD48+
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. When we subfractionated the LSK fraction according to CD34 and Flk-2 expression, and compared the repopulating capacity of GFP−
cells within these subsets (Fig. S1 C), we obtained similar results to the aforementioned findings using SLAM markers (). These data reveal that, irrespective of the combination of HSC surface markers used, GFP+
cells are the exclusive reservoir of HSC activity, with no reconstitution ability being observed in GFP−
cells within the LT-HSC compartment. Altogether, our results demonstrate that Evi1 expression can further augment the conventional HSC purification strategy, and suggest that Evi1-IRES-GFP knock-in mice allow us to functionally identify HSCs on the ground of self-renewal capacity.
Evi1 expression marks in vivo long-term multilineage repopulating HSCs in embryo
The formation of blood cells begins in the yolk sac of the embryo, and then shifts to the aorta-gonad-mesonephros (AGM) region, and then sequentially to the placenta, fetal liver (FL), and adult BM. 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
). 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+
) 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 restricted to HSPCs in the embryonic tissues; CD45+
cells in embryonic day 10.5 (E10.5) AGM, CD34+
cells in E12.5 placenta, and Mac-1+
cells in E14.5 FL (; 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 (), indicating the predominant expression of Evi1 in HSPCs during fetal hematopoiesis.
Figure 5. Evi1 expression marks in vivo long-term multilineage repopulating HSCs in embryo. (A–C) Frequency of GFP+ cells in each subpopulation of E10.5 AGM (A; n = 5) or E12.5 placenta (B; n = 3) or E14.5 FL (C; n = 5) from Evi1+/GFP embryos. (D–F) (more ...)
To determine whether Evi1 expression is associated with hematopoietic activity in the embryonic tissues, we performed colony-forming assays in vitro using CD45+ GFP− and CD45+ GFP+ cells from E10.5 AGM, and found that CD45+ GFP+ cells contained almost all colony-forming cells, with few detectable hematopoietic colonies in CD45+ GFP− cells (). In the same manner, within the CD34+ c-kit+ CD48− fraction from E12.5 placenta of Evi1+/GFP embryos, colony-forming activity was exclusively present in GFP+ cells, regardless of colony type (). These data suggest that clonogenic hematopoietic progenitors predominantly reside in the Evi1-expressing fraction in fetal hematopoiesis.
To examine whether Evi1 expression would have the potential to effectively mark long-term repopulating HSCs in embryo, we performed a CRA using sorted CD34+
cells from E12.5 placenta of Evi1+/GFP
embryos (Fig. S2 A
). It was obvious that CD34+
cells contributed to the long-term reconstitution of irradiated recipients, whereas donor chimerism was almost undetectable in mice transplanted with CD34+
cells (), which is in agreement with the results obtained with their adult counterparts. To further assess whether Evi1 expression can enrich long-term repopulating HSCs in embryo, we performed a CRA using purified MSL GFP−
and MSL GFP+
cells from E14.5 FL (Fig. S2 B). Along with cells in E12.5 placenta, MSL GFP+
cells gave rise to long-term multilineage reconstitution, whereas no engraftment was observed in recipients of MSL GFP−
cells (). These results indicate that fetal HSCs with active Evi1
transcription exclusively harbor stem cell activity. Collectively, despite the functional differences between fetal and adult HSCs, Evi1 expression marks long-term multilineage repopulating HSCs throughout ontogeny, suggesting a specific relationship between Evi1 expression and HSC self-renewal capacity.
Evi1 heterozygosity leads to an almost complete loss of LT-HSCs in a cell-autonomous manner
The aforementioned observations led us to predict that Evi1 plays a functional role specifically in LT-HSCs. To clarify this issue, we analyzed heterozygous Evi1
KO mice (Evi1+/−
). We previously showed that heterozygosity of Evi1 leads to decreased numbers of LSK and CD34−
LSK cells, as well as impaired long-term repopulating activity (Goyama et al., 2008
). In the current study, although Flk-2+
LSK cells were moderately decreased, Flk-2−
LSK cells from Evi1+/−
mice exhibited a marked reduction in frequency compared with WT controls (). Likewise, when LSK cells were subdivided according to SLAM markers, we observed substantial decreases in CD48−
LSK subsets (). Therefore, the number of each subpopulation within the LSK fraction in Evi1+/−
mice was declined in proportion to their expression level of Evi1, indicating that Evi1 has a dominating effect on the maintenance of LT-HSCs. In contrast, there were no significant differences in BM cellularity and the frequencies of lymphoid and myeloid progenitors, and mature blood cells between Evi1+/+
mice ( and not depicted), indicating that the differentiation potential to all mature lineages and committed progenitors is normal in Evi1+/−
mice. Collectively, these observations suggest that Evi1 serves as a specific regulator in the earliest stage of adult hematopoietic development.
Figure 6. Evi1 heterozygosity leads to an almost complete loss of LT-HSCs in a cell-autonomous manner. (A) FACS analysis of expression of Flk-2 and CD34 or CD48 and CD150 on LSK cells in BM from Evi1+/+ and Evi1+/− mice. Data are representative of at least (more ...)
To exclude the possibility that a defect of BM microenvironment could be responsible for the observed hematopoietic abnormalities in Evi1+/− mice, we performed reciprocal transplantation experiments, in which WT BM cells were transplanted into lethally irradiated Evi1+/+ or Evi1+/− mice. At 16 wk after transplantation, flow cytometric analysis showed no differences in the percentages of Flk-2− CD34− LSK or CD48− CD150+ LSK cells in both groups of recipient mice (), demonstrating that the profound loss of LT-HSCs in Evi1+/− mice is attributed to cell-intrinsic mechanisms.
Evi1 heterozygosity causes specific abrogation of self-renewal capacity in ST- and LT-HSCs
To further characterize which subpopulation in HSPCs is most dependent on Evi1, we purified CD34+ and Flk-2− CD34− LSK cells from Evi1+/+ and Evi1+/− mice and compared their differentiation and self-renewal capacity in vitro and in vivo. First, to assess the effect of Evi1 heterozygosity on the biological functions of ST-HSCs/MPPs, we performed colony-forming assays in vitro using Evi1+/+ and Evi1+/− CD34+ LSK cells, which demonstrated no significant differences in the number and type of colonies (). Similarly, we found the capacity of Evi1+/− CD34+ LSK cells to form colonies in the spleen 11 d after transplantation (CFU-spleen [CFU-S]) was also equivalent to that of WT littermates (), indicating Evi1 is dispensable for the regulation of the differentiation and proliferation capacity in ST-HSCs/MPPs. Moreover, to investigate the self-renewal ability of ST-HSCs/MPPs in vivo, we evaluated the short-term repopulating capacity of purified CD34+ LSK cells using a CRA. At 2 wk after transplantation, we detected comparable frequencies of Evi1+/+ and Evi1+/− CD34+ LSK cell–derived myeloid and B cells (), suggesting that heterozygosity of Evi1 does not affect the engraftment and differentiation potential of ST-HSCs/MPPs in vivo. However, at later time points in the experiment, we found a moderate but significant decline in the percentage of donor-derived cells from Evi1+/− mice (). These data indicate that heterozygosity of Evi1 attenuates the self-renewal capacity of ST-HSCs/MPPs, but is not accompanied by any specific differentiation defects in them.
Figure 7. Evi1 heterozygosity causes specific abrogation of self-renewal capacity in ST- and LT-HSCs. (A) Numbers of CFU-GM, CFU-GEMM, and BFU-E colonies derived from 100 sorted Evi1+/+ and Evi1+/− CD34+ LSK cells (n = 3). (B) Appearance and number of CFU-S (more ...)
To assess whether Evi1 is required for the functions of LT-HSCs, we compared the self-renewal and proliferation capacity of Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells when cultured in serum-free medium. Evi1+/− Flk-2− CD34− LSK cells showed comparable proliferation with WT cells for the first week of culture, but thereafter they exhibited pronouncedly impaired growth (). After incubation, a significantly lower proportion of cultured Evi1+/− Flk-2− CD34− LSK cells remained in the LSK fraction than those from Evi1+/+ mice (). In addition, we observed a prominent reduction of hematopoietic colonies contained in cultured Evi1+/− Flk-2− CD34− LSK cells (). Besides, most of the colonies generated from cultured Evi1+/− Flk-2− CD34− LSK cells consisted of only CFU-GM. These data indicate that heterozygosity of Evi1 results in accelerated loss of HSPCs, leading to the inefficient expansion of their progeny. To evaluate the colony-forming capacity at the single cell level, Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells were clonally sorted and cultured in serum-free medium. Evi1 heterozygosity diminished the colony-forming efficiency of clone-sorted Flk-2− CD34− LSK cells, and single Evi1+/− Flk-2− CD34− LSK cells generated smaller colonies compared with control cells (), which indicates that the disruption of Evi1 gene not only decreases the number of clonogenic HSCs but also impairs the functional output per cell.
To assess the repopulating capacity of Evi1+/− LT-HSCs in vivo, we performed a CRA using purified Flk-2− CD34− LSK cells from Evi1+/+ and Evi1+/− mice. Notably, Evi1+/− Flk-2− CD34− LSK cells were almost unable to efficiently repopulate all mature lineages as well as stem and progenitor cells 16 wk after transplantation (), suggesting that LT-HSC function is critically dependent on Evi1 gene dosage. In a noncompetitive setting, although recipients of Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells had similar survival after transplantation (not depicted), Evi1+/− Flk-2− CD34− LSK cells showed impaired engraftment (), suggesting that Evi1+/− HSCs were outcompeted by residual host HSCs. However, some of those recipients exhibited long-term multilineage reconstitution ( and not depicted), confirming that the multipotent differentiation capacity is not abrogated in Evi1+/− mice. To further explore the competitive disadvantage of Evi1+/− HSCs, we transplanted WT BM cells into unirradiated Evi1+/+ or Evi1+/− mice. We found no engraftment in both mice (), which indicates that the resistance to the donor HSC engraftment during steady-state hematopoiesis is maintained in Evi1+/− mice. Collectively, these data suggest that Evi1 is dispensable for the regulation of proliferative and differentiation capacity of ST-HSCs/MPPs, but is strictly required for the maintenance of LT-HSC activity.
To investigate the mechanism behind the impaired HSC activity, we performed cell-cycle and apoptosis assays, but found no differences in the cell-cycle profile or apoptotic rates of Flk-2− CD34− LSK cells between Evi1+/+ and Evi1+/− mice (unpublished data). Collectively, in consideration of the accelerated loss of LT-HSC activity in Evi1+/− mice, it is supposed that Evi1 heterozygosity directs LT-HSCs from self-renewal toward differentiation to generate more committed progenitors, which is uncoupled from cell-cycle progression or apoptosis.
Forced expression of Evi1 prevents HSPC differentiation and enhances their expansion
The findings noted above led us to hypothesize Evi1 has the potential to inhibit differentiation and enhance HSC self-renewal independent of cell-cycle progression. To clarify this, we adopted a gain-of-function approach, in which WT LSK cells were transduced with Evi1, and then incubated in serum-free medium. Although forced expression of Evi1 gave no apparent growth advantage for the first 10 d of culture, Evi1-transduced LSK cells subsequently manifested a mild but significant increase in proliferation rate (). Moreover, we found a substantial increase in the frequency of the remaining LSK fraction in cultured Evi1-transduced cells compared with control cells (). In parallel, the number of colonies derived from cultured Evi1-transduced LSK cell was drastically increased (). These results suggest that Evi1 activation restricts lineage differentiation and enhances self-renewal activity of HSPCs. Collectively, our data provide compelling evidence that Evi1 regulates the developmental transition from HSPCs to more committed progenitors, suggesting a crucial role of Evi1 in controlling the balance between self-renewal and differentiation.
Figure 8. Forced expression of Evi1 prevents HSPC differentiation and promotes their expansion. (A) Proliferation of 3,000 control- or Evi1-transduced LSK cells cultured in serum-free medium with 20 ng/ml SCF and 20 ng/ml TPO for 14 d (*, P < 0.05; n = (more ...)
A recent work suggests that the longer, PR domain-containing isoform Mds1-Evi1 (ME) deficiency alone causes a reduction in the number of HSCs with a loss of long-term repopulation capacity (Zhang et al., 2011
). 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. For this purpose, we transduced Evi1 or ME into Evi1+/−
LSK cells and examined whether they could maintain stem cell phenotype after in vitro culture. Reintroduction of Evi1 led to a significant increase in the proportion that remained in the LSK fraction, similar to observations made in Evi1+/+
cells (). However, retroviral transfer of ME was unable to normalize the frequency of the remaining LSK fraction (), indicating that Evi1 preferentially rescues Evi1+/−
LT-HSC defects. Given that ME has broader effects on the hematopoietic system than Evi1 and acts in part by maintaining HSC quiescence through up-regulation of Cdkn1c
transcription (Zhang et al., 2011
), Evi1 and ME may exert their functions in regulating hematopoiesis at different stages and by different mechanisms.