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Individual members of the retinoblastoma (Rb) tumor suppressor gene family serve critical roles in the control of cellular proliferation and differentiation but the extent of their contributions is masked by redundant and compensatory mechanisms. Here, we employed a conditional knockout strategy to simultaneously inactivate all three members, Rb, p107, and p130, in adult hematopoietic stem cells (HSCs). Rb family triple knockout (TKO) mice develop a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors and is accompanied by increased apoptosis in lymphoid progenitor populations. Loss of quiescence in the TKO HSC pool is associated with an expansion of these mutant stem cells, but also with an enhanced mobilization and an impaired reconstitution potential upon transplantation. The presence of a single p107 allele is sufficient to largely rescue these defects. Thus, Rb family members collectively maintain HSC quiescence and the balance between lymphoid and myeloid cell fates in the hematopoietic system.
Hematopoiesis initiates from multipotent hematopoietic stem cells (HSCs) that normally reside in the bone marrow. HSC populations are largely quiescent but have the capacity to proliferate and differentiate into progenitors whose amplification ensures the constant and rapid renewal of all blood compartments (Metcalf, 2007; Zon, 2008).
A number of recent studies have pointed to a role for cell cycle regulators and signaling pathways in the control of HSCs and hematopoiesis. The Bmi-1 polycomb-group protein plays a critical role in the proliferative capacity of normal HSCs and leukemic stem cells. Bmi-1 functions in HSCs at least partly through its repressive action of the p16INK4a, p19ARF, and p21CIP1 cell cycle inhibitors (Fasano et al., 2007; Oguro et al., 2006; Park et al., 2003). These cell cycle inhibitors and their family members generally restrain the ability of HSCs to proliferate and mediate the response of HSCs to stress signals (Cheng et al., 2000a; Cheng et al., 2000b; Janzen et al., 2006; Yuan et al., 2006). Similarly, HSCs with mutations in cyclin-dependent kinases and their cyclin partners display decreased proliferation (Kozar et al., 2004; Malumbres et al., 2004). Another critical pathway that controls the self-renewal potential and stress response of HSCs is the PTEN-FoxO signaling module (Miyamoto et al., 2007; Tothova et al., 2007; Yilmaz et al., 2006; Zhang et al., 2006). However, the mechanisms by which HSCs normally maintain their quiescence state and re-enter transiently the cell cycle to produce hematopoietic progenitors are still not fully understood. In addition, the functional links between the cell cycle machinery and the network of transcription factors regulating the fate of hematopoietic progenitors remain unclear (Orford and Scadden, 2008).
The three members of the retinoblastoma family of transcriptional regulators, pRB, p107, and p130, integrate multiple cellular signals to control cellular proliferation and differentiation (Wikenheiser-Brokamp, 2006). pRB family members act downstream of many of the critical cell cycle regulators previously studied in HSCs. Thus, pRB family proteins are strong candidates to regulate HSCs and hematopoiesis. But no blood phenotype is observed in p130 mutant mice (Cobrinik et al., 1996), and loss of p107 results in a mild myeloid hyperplasia that is only visible in a Balb/c genetic background (LeCouter et al., 1998). Rb-deficiency in mice does not provoke major blood defects except in erythropoiesis (Chen et al., 1993; Iavarone et al., 2004; Sankaran et al., 2008; Spike et al., 2004; Spike and Macleod, 2005; Walkley and Orkin, 2006; Walkley et al., 2007). Given its strong cell cycle inhibitory functions and broad tumor suppressor activity, loss of pRB function has surprisingly no direct effect on the cell cycle of HSCs (Walkley and Orkin, 2006; Walkley et al., 2007), except under stress conditions (Daria et al., 2008).
Activation of CyclinD/Cdk4 kinase complexes and loss of p16Ink4a function, two upstream regulators of pRB, p107, and p130, are common in leukemias and lymphomas (Leoncini et al., 2006; Spike and Macleod, 2005). Based on these observations, the absence of a significant hematopoietic phenotype in mice mutant with single Rb family genes, and the known extensive functional redundancy within this gene family in many cell types (Wikenheiser-Brokamp, 2006), we sought to inactivate Rb, p107, and p130 in adult mouse HSCs. To this end, we employed a conditional mutant strategy that bypasses the lethality of mutations in Rb family genes (Wikenheiser-Brokamp, 2006). We show that pRB family proteins collectively regulate programs of genes that normally inhibit the proliferation of HSCs and control the fate of hematopoietic progenitors. These experiments indicate that one key mechanism by which loss of pRB family function may initiate pre-neoplastic defects is by unrestricting the proliferation of adult stem cells.
Similar to recent reports (Daria et al., 2008; Walkley and Orkin, 2006; Walkley et al., 2007), we found that Rb deletion in mouse HSCs does not significantly alter HSC functions but results in a mild myeloid neutrophilic expansion accompanied by extramedullary hematopoiesis (Figure S1 and data not shown). We surmised that the additional deletion of p107 and p130 may unmask novel roles for this gene family in the hematopoietic system. To inactivate the entire Rb gene family in adult mouse HSCs, we generated Mx1-Cre Rblox/lox;p130lox/lox;p107−/− (Mx1-Cre condTKO) mice. Adult Mx1-Cre condTKO mice were injected with pI-pC to activate the Cre recombinase and delete the Rb and p130 genes in HSCs. Real-time quantitative RT-PCR (QPCR) analysis showed that a significant number of mutant cells are present in the bone marrow (BM) of induced Mx1-Cre TKO mice (Figure S2A). TKO mice died within 1–3 months, whereas the viability of control mice was unaffected by pI-pC injections.
Histopathological analysis of moribund Mx1-Cre TKO mice revealed marked spleen enlargement, with a severe disruption of the splenic architecture. A majority of splenocytes from Mx1-Cre TKO mice exhibited an eosinophilic morphology (Figure 1A). Infiltrating leukocytes were present in the kidney, liver, lungs, and skin (Figures 1B and data not shown). The BM structure was also altered, and showed a marked expansion of myeloid cells, particularly eosinophils (Figure 1C).
Next, we examined the distribution of the different hematopoietic lineages in TKO mice. Cell counts from TKO BM cytospins showed a decrease in the percentage of lymphocytes and neutrophils, accompanied by an increase in the frequency of myeloid precursors and mature eosinophils (2% in controls versus 20% in mutants) (Figure 1D). FACS analysis for myeloid markers indicated that the BM from Mx1-Cre TKO contained a large population of cells positive for Mac-1 and expressing low levels of Gr-1 (which we refer to as pre-M population) (Figures 1E and 1F). Cytospins from sorted BM subpopulations showed high heterogeneity in the pre-M population and well-differentiated granulocytes in the Mac-1+, Gr-1high population, as expected (Figure S2B). In addition, decreased percentages of Mac-1+, Gr-1high granulocytes (Figures 1E and 1F), erythrocytes, and B lymphocytes (Figure 1F and Figure S2C) were counted in the BM of mutant mice. While the pre-M myeloid expansion was restricted to the BM of Mx1-Cre TKO mice at early time points (data not shown), sick mice displayed extramedullary hematopoiesis: FACS analysis of splenocytes showed an increase in the number of pre-M cells (Figure 1G and Figure S2D). In addition, we observed an increase in the number of splenic erythrocytes at various stages of differentiation (Figure S2D, middle panel, and Figure S3). The total number of B cells in the spleen of TKO mice was not altered compared to control mice (Figure 1G, right, and Figure S2D).
These observations indicate that deletion of Rb, p107, and p130 in Mx1-Cre TKO mice induces the development of a myeloproliferation characterized by the rapid expansion of a population of Mac-1+, Gr-1low (pre-M) myeloid cells with eosinophilic histological features. This disease originates from the BM, does not involve the peripheral blood (data not shown), but spreads to other organs, and is accompanied by general perturbations in the hematopoiesis of the mutant mice.
The expansion of pre-M cells in TKO mice could originate from an increase in the proliferative capacity of these cells or of Myeloid Progenitor (MP) cells. However, cell cycle analysis of pre-M and MP cells isolated from TKO and control mice did not reveal any significant differences in their cell cycle profiles (Figure 2A). This result led us to analyze the cell cycle of early hematopoietic progenitors of the KLS compartment (c-Kit+, negative for Lineage markers of differentiation, and Sca1+); KLS cells include HSCs and multipotent progenitors (MPP). Strikingly, we found that TKO KLS cells were significantly more proliferative than control KLS cells (Figure 2A, right). We also observed a significant increase in the number of KLS and MP cells in the bone marrow of sick Mx1-Cre TKO mice. Within the KLS population, both Flk2+ cells (MPP) and Flk2− cells (ST-HSC and LT-HSC) were increased (Figures 2B and 2C). Within the MP population, Common Myeloid Progenitor (CMP, FcγR+) and Megakaryocyte-Erythrocytes Progenitors (MEP, FcγR−) numbers were decreased in Mx1-Cre TKO mice, while Granulocyte-Monocytes Progenitors (GMP, FcγRhigh) numbers were increased (Figures 2B and 2D). In the lymphoid lineage, the number of Common Lymphoid Progenitor cells (CLP) was significantly decreased in Mx1-Cre TKO mice compared to controls (Figures 2E and 2F), and Annexin V staining experiments showed significantly higher numbers of apoptotic CLP in mutant mice (data not shown, see below).
Based on these observations, we further tested the cell cycle profile of subpopulations of stem/progenitors cells. Within the KLS population, both CD34+ (MPP and ST-HSC) and CD34− (LT-HSC) displayed enhanced proliferation (Figures 2G). This increased proliferation was not associated with changes in the apoptotic rates of these cells (data not shown). CMP cells, while decreased in numbers, cycled more rapidly, while GMP cells, which are more numerous in TKO mice, did not cycle more (Figure 2G).
We next performed methylcellulose cultures using unfractioned BM cells and sorted KLS, MP, and pre-M cells. Unfractioned BM cells from Mx1-Cre TKO mice displayed a reduced number of colonies compared to controls (Figure 2H); this decrease is likely due to the dilution of the cells that have the capacity to form colonies by expanding pre-M cells. Nevertheless, the relative number of TKO granulocytic colonies (G-CFU) was increased compared to control cells, while the number of mixed colonies (GEMM-CFU) was decreased, which suggests a differentiation bias towards the granulocytic myeloid lineage. Purified Mx1-Cre TKO KLS cells produced a higher number of colonies compared to control cells, with again an increase in G-CFU and a decrease in GEMM-CFU. We observed a similar trend towards G-CFU with sorted myeloid progenitor MP populations. Only colonies obtained from KLS cells, but not MP cells, could be serially replated (data not shown). In addition, we did not observe colony formation from the more mature TKO pre-M cells, confirming that these mutant cells had not acquired a significant proliferation potential (data not shown). Together, these data suggest that the disease observed in TKO mice may result from the abnormal proliferation of early hematopoietic progenitors in the KLS and CMP compartments as well as from a change in the fate of these progenitors towards the myeloid lineage, and not by the increased proliferation potential of more mature myeloid cell populations.
To assess the importance of the simultaneous inactivation of the three Rb family members for these phenotypes, we genetically re-introduced one wild-type allele of p107 and analyzed the BM of these p107-Single mice (Mx1-Cre Rblox/lox;p130lox/lox;p107+/−). As expected, p107-Single mice developed splenomegaly indicative of extramedullary hematopoiesis (data not shown). Strikingly, however, the BM of p107-Single mice did not exhibit a myeloproliferation (Figure 3A). p107-Single mice had similar numbers of KLS, MP, and pre-M cells in the BM as control mice as determined by FACS analysis (Figure 3B and 3C), and no increase in pre-M cells in the spleen (Figure 3D). The cell cycle of KLS cells was also similar in control and p107-Single mice (Figure 3E). The absence of a myeloproliferative phenotype illustrates the extent of the functional overlap within the Rb gene family and the necessity to inactivate all three genes to severely disrupt early hematopoietic development. Interestingly, however, the decrease in the number of lymphoid cells (both CLP and B cell populations) observed in TKO mice is still present in p107-Single mice (Figures 3B–D), suggesting that different cell types during hematopoiesis may be differentially sensitive to different levels of Rb family function.
The hyperproliferative phenotype of TKO KLS populations led us to further investigate the properties of these mutant stem cells. Since increased proliferation of hematopoietic progenitor cells is associated with their increased mobilization (Passegue et al., 2005), we examined the mobilization of TKO hematopoietic progenitors. The spleen of Mx1-Cre TKO mice contained increased numbers of HSC and progenitor populations, indicative of extramedullary hematopoiesis (Figures 4A, 4B, and 4C). This was confirmed by the increase in the number of colonies formed by unfractioned splenocytes in methylcellulose (Figure 4D). Overall, there were approximately four times more Flk2− HSCs in the spleen of mutant mice compared to their BM (compare Figure 4C and Figure 2C). KLS and MP cells were also more frequent in the peripheral blood of mutant mice (Figure 4E), providing further evidence of their increased mobilization.
To test the homing capacity of TKO progenitors, lineage-negative cells, enriched in hematopoietic progenitors, were labeled with the fluorescent vital dye CFSE, and transplanted into sublethally irradiated SCID mice. Under these conditions, fewer CFSE-labeled cells from Mx1-Cre TKO mice homed to the BM of the recipient mice (n=10) (Figure 4F). Together, these data indicate that hyperproliferative TKO hematopoietic progenitors are constitutively mobilized and display a decreased homing capacity.
The long-term engraftment potential of hematopoietic cells resides predominantly in the quiescent pool of HSCs (Fleming et al., 1993; Passegue et al., 2005). Because TKO HSCs are less quiescent than wild-type HSCs, we sought to test their reconstitution properties. To this end, 106 control or Mx1-Cre TKO BM cells expressing the Ly5.1 surface marker were transplanted together with 106 Ly5.1/Ly5.2 wild-type competitor cells into lethally irradiated Ly5.2 Rag2/commonγchain double mutant immunodeficient mice. Four weeks after transplantation, leukocyte chimerism was assessed in the peripheral blood (PB). As shown in Figure 5A, Mx1-Cre TKO cells outcompeted control competitor cells in this short-term reconstitution setting. As expected given the expansion of these cells in TKO mice, this advantage was more pronounced in myeloid populations (Figure 5B). Strikingly, however, all the recipients of Mx1-Cre TKO cells died by week 5, while recipients of control cells survived (n=5). Because irradiated mice that were not transplanted with any cells died ~2 weeks after irradiation (data not shown), the death of mice reconstituted with TKO cells after 5 weeks suggested that TKO hematopoietic progenitors were only transiently able to reconstitute the hematopoietic system of recipient mice. Consistent with this idea, the BM cellularity was reduced in recipients of TKO cells compared to controls (Figure 5C). Despite this overall decreased cellularity, a majority of the mature hematopoietic cells were TKO and not wild-type competitors (Figure 5D). Importantly, hematopoietic progenitor cell numbers, including LT-HSCs, were significantly decreased in the BM of mice reconstituted with TKO cells (Figure 5E).
Taken together, these results suggest that TKO early hematopoietic progenitors are very efficient at short-term repopulation, outcompeting wild-type cells and preventing their engraftment, probably by producing an outburst of myeloid cells. This observation may in part reflect the increased numbers of TKO KLS and mature myeloid cells transplanted, as well as the hyperproliferative status of early progenitors. Nevertheless, hyperproliferative TKO HSCs are severely impaired in their long-term reconstitution potential in vivo, as illustrated by the decrease of LT- and ST-HSC populations and the rapid death of the transplanted recipients. This inability of TKO HSCs to reconstitute the blood system may have several non-exclusive causes, including, but not limited to, a reduced homing potential, an increased mobilization and an incapacity to stably reside in the BM that may be due to their hyperproliferative status, and a loss of self-renewal upon the stress of the transplantation.
To further determine the kinetics and the cell-autonomy of the TKO phenotype, we sought to investigate if these defects arose both rapidly and in transplanted mice. Low leaky expression of the Mx1 promoter in the absence of pI-pC injections and expression of this promoter in many cell types upon induction (data not shown) (Kuhn et al., 1995) precluded the use of the Mx1Cre TKO model for this purpose. To bypass this problem, we crossed condTKO mice to Rosa26Cre-ERT2 mice (Ventura et al., 2007). In these mice, Cre expression is very broad but its activity is strictly dependent on tamoxifen injection (data not shown). Rosa26Cre-ERT2 TKO mice died on average two to three weeks after tamoxifen injection due to efficient deletion of the Rb and p130 alleles and loss of the entire Rb family in multiple cell types, including BM cells (Figure 6A and data not shown). Rosa26Cre-ERT2 TKO mice may die from digestive defects as they show a rapid weight loss, but at these early time points, the spleen, thymus and liver of mutant mice displayed normal appearance (data not shown).
Two weeks after Cre induction, KLS, MP, and GMP cell numbers remained similar to control numbers in the BM of Rosa26Cre-ERT2 TKO mice. However, both CMP and CLP populations were already significantly decreased in Rosa26Cre-ERT2 TKO mice (Figure 6B and Figure S4A). In addition, analysis of mature BM cells already showed a significant increase in the pre-M population and a decrease in the B cell lineage (Figure 6C and Figure S4B). Thus, the shift toward the myeloid lineage is very rapid in TKO mice.
To further probe the mechanisms underlying this phenotype, we tested the apoptotic activity in control and TKO hematopoietic stem/progenitor cells using Annexin V staining. We found that the number of apoptotic KLS and MP TKO cells was low but that TKO CLP cells displayed a significant increase in cell death compared to controls (Figure 6D), suggesting that enhanced death is one mechanism leading to decreased numbers of lymphoid cells in TKO mice.
Increased proliferation was also observed in the BM of Rosa26Cre-ERT2 TKO mice two weeks after Cre induction, most strikingly in the KLS population, which exhibited a high replicative rate (Figures 6E). Both TKO CMP and GMP populations showed an increased proliferation (Figure 6F). Control experiments showed that loss of p130 only in induced Rosa26Cre-ERT2 p130lox/lox mice did not affect the proliferation of KLS cells while the acute loss of Rb had only a mild effect (Figure 6G). The fact that no cell cycle defect was observed in Rb deficient KLS populations in longer-term experiments (Daria et al., 2008; Walkley and Orkin, 2006; Walkley et al., 2007) is again suggestive of compensatory mechanisms similar to what was observed in mouse fibroblasts (Sage et al., 2003). This result further underscores the extent of the functional overlap within the Rb gene family. Together, these results show that loss of Rb family genes rapidly induces the proliferation of hematopoietic progenitors and initiates a myeloproliferation similar to that observed in aged Mx1-Cre TKO mice.
To explore a possible role for the microenvironment in the development of the TKO phenotype, we transplanted wild-type Ly5.1/Ly5.2 BM cells into lethally irradiated Ly5.1 control and Rosa26Cre-ERT2 condTKO mice. Following establishment of hematopoiesis, mice were treated with tamoxifen, thereby deleting Rb family genes in multiple cell types, except donor HSCs and their progeny. As expected, TKO recipient mice died approximately 2–3 weeks after induction while control mice remained healthy. Analysis of the progenitor compartment did not reveal significant changes between control and TKO populations (Figure S4C). In addition, wild-type donor cells transplanted into TKO recipients did not show a significant increase in the pre-M population (Figure S4D).
To further investigate the cell-intrinsic nature of the TKO phenotypes, we then tested the ability of Rosa26Cre-ERT2 TKO BM cells to recapitulate the disease when Cre-mediated recombination occurred after transplantation in wild-type recipient mice. To this end, we transplanted 4×106 unfractioned control or Rosa26Cre-ERT2 condTKO BM cells into sublethally irradiated (1.5G) SCID mice, and injected these recipient mice with tamoxifen five days after transplantation. Within 4 weeks, all recipients of Rosa26Cre-ERT2 TKO cells displayed an expansion of pre-M myeloid cells (n=4) (Figure 6H).
Similarly, longer-term experiments showed that 4 months after induction of Cre, lethally irradiated immunodeficient Rag2−/− mice transplanted with Rosa26Cre-ERT2 TKO BM cells displayed a myeloproliferation phenotype with infiltration of myeloid cells in the skin and the liver, and enlargement of the spleen and disruption of its normal architecture (Figure S5A–H). Accordingly, FACS analysis indicated that the mice with mutant blood cells had increased number of pre-M cells and decreased numbers of B cells (Figure S5I–J). Cell cycle analysis of TKO KLS and MP populations showed a reproducible increase in the proliferative status of these cells compared to control cells (Figure 6I).
Taken together, these results demonstrate that the hyperproliferative phenotype of TKO hematopoietic progenitors and the myeloproliferation associated with Rb family deficiency are primarily cell autonomous.
Given the broad role of pRB family proteins as transcriptional modulators, we assessed the molecular basis of the TKO myeloid disease by gene expression profiling. A first analysis revealed that the gene programs affected by loss of Rb family genes in KLS cells were significantly similar to those affected by loss of Rb alone in early erythroid progenitors (Sankaran et al., 2008) (p<2×10−5 and p<2×10−4 for upregulated and downregulated genes, respectively), although the fold changes were generally greater in TKO cells (data not shown). These observations validate the presence of an “Rb mutant” signature in TKO KLS cells.
Assignment of differentially expressed genes to functional annotation groups further revealed that a significant number of genes associated with cell cycle progression were upregulated in TKO cells when compared to control cells, including members of the E2f gene family and genes coding for structural components of mitotic chromosomes (Figure 7A). These data are consistent with the hyperproliferative status of the mutant cells.
We also found that many genes belonging to mature lymphoid cells annotation groups, such as genes coding for immunoglobulins and major histocompatibility class (MHC) II molecules, were downregulated in TKO KLS cells (Figure 7B). In addition, the expression of known regulators of lymphoid development, including the Lck, Rag1, and IL7R genes, was also decreased (Figure 7B). Conversely, genes coding for Gm-csfr and the Gata-2 transcription factor, two critical promoters of myeloid development, were upregulated in TKO KLS cells (Figure 7C). Gata-2 overexpression in KLS cells is sufficient to direct their differentiation towards eosinophils (Hirasawa et al., 2002). QPCR analysis confirmed that expression of Gata-2 is significantly increased in purified KLS cells (Figure 7C). We also found that several genes that code for markers of the neutrophil lineage were downregulated in TKO KLS cells (Figure 7C). Together, these gene expression data strongly correlate with the hematopoietic phenotype of TKO mice and indicate that gene programs that are altered upon loss of Rb family genes in KLS cells may prime these early hematopoietic progenitors to generate a myeloproliferation characterized by the expansion of an eosinophilic population (Figure 7D).
Here we report the in vivo phenotype of adult mouse hematopoietic cells with compound genetic inactivation of Rb, p107, and p130. Loss of the entire Rb gene family induces loss of HSC quiescence, an increase in progenitor and stem cell populations accompanied by extramedullary hematopoiesis, and a large expansion of eosinophilic granulocytes. These results underscore the role of pRB, p107, and p130 in stem and progenitor cells, and provide a cellular mechanism by which loss of RB family functions may initiate cancer in humans.
We found that loss of all Rb family genes activates the proliferation and increases the total numbers of mouse HSCs. The RNA levels for genes involved in the self-renewal of stem cells (e.g. Bmi1, Hoxb4, and Hes1) did not reveal any differences between TKO and control KLS cells (Figure S6). When serially replated in methylcellulose, KLS cells from control and Mx1-Cre TKO mice both replated each three times (data not shown). Together, these observations suggest that TKO HSCs do not have a major self-renewal defect. However, TKO mutant mice become rapidly sick and cannot be aged to study the fate of TKO HSCs in the long-term. In addition, TKO HSCs fail to stably reconstitute a stable hematopoietic system in transplantation experiments. Thus, it is possible that, under certain stress conditions, loss of the entire Rb gene family may affect the self-renewal potential of HSCs.
Increased proliferation is often associated with loss of self-renewal in HSCs, and alterations in some upstream regulators of the RB pathway and other key cell cycle inhibitors such as PTEN and FoxOs have been associated with self-renewal defects (Tothova et al., 2007; Zhang et al., 2006). However, increased proliferation in HSCs without loss of self-renewal has been observed in p18Ink4c deficient mice (Yu et al., 2006) and it has been proposed that mutations in genes controlling the early G1 phase of the cell cycle – such as p18Ink4c and Rb family genes – in quiescent stem cells may result in increased proliferation with no effects on the self-renewal (Orford and Scadden, 2008). More studies are clearly required to better comprehend the molecular links between cell cycle and self-renewal potential in HSCs and other stem cells, including cell autonomous and non-cell autonomous aspects (Zon, 2008).
Increased proliferation of HSCs has also been linked to an inability to transplant, although the molecular mechanisms underlying this observation are still largely unknown (Passegue et al., 2005). It is not clear if the mobilization, decreased homing potential, and overall incapacity to transplant of TKO HSCs are only direct consequences of their hyperproliferative status or if other cellular functions are altered in these mutant cells. Future experiments with Rb family mutant mice and other mutants will help to explore the mechanisms by which HSCs can reconstitute the blood system upon transplantation.
The rapidity of development of the TKO myeloproliferation and its recapitulation in transplanted animals show that it is largely an intrinsic consequence of loss of Rb family genes in hematopoietic cells. Rb-only mutant mice display a mild neutrophilic myelodysplasia that is largely induced by the BM microenvironment (Spike et al., 2004; Walkley et al., 2007). p107 mutant mice in a Balb/c genetic background also develop a mild myeloid hyperplasia (LeCouter et al., 1998). Together, these observations directly implicate the Rb gene family in restricting myeloid cell expansion in vivo.
Interestingly, the TKO myeloproliferation does not come from an increased proliferation of mature myeloid cells but rather originate from altered gene programs and cell fate decisions in early hematopoietic progenitors. These data also suggest that loss of Rb family genes is not sufficient to prevent cell cycle arrest in some differentiating hematopoietic populations, a phenomenon that will require additional studies in other cell lineages in vivo.
TKO mice display a decrease in lymphoid progenitors and B cell populations, a phenotype likely due to increased cell death in CLP populations and decreased expression of a program of genes that normally prime KLS cells to generate lymphoid cells, similar to what has been described for aging hematopoietic progenitors (Rossi et al., 2008).
Several recent reports have characterized the consequences of loss of Rb only in hematopoiesis (Daria et al., 2008; Spike et al., 2004; Walkley and Orkin, 2006; Walkley et al., 2007). These studies and our own characterization of Rb mutant mice all show an increased mobilization of Rb mutant hematopoietic progenitors accompanied by extramedullary hematopoiesis. These data indicate that p107 and p130 do not always functionally compensate for loss of pRB. However, TKO mice have many different phenotypes compared to Rb mutant mice. While Rb mutant mice develop a neutrophilic myeloproliferation, TKO mice develop an eosinophilic disease. Walkley et al. clearly showed that the neutrophilic expansion in Rb mutant mice has a strong non-cell autonomous component. In contrast, the TKO eosinophilic expansion is largely cell-intrinsic. TKO mice might eventually also develop a neutrophilic defect in the absence of the rapid eosinophilic disease. Alternatively, loss of Rb, p107, and p130 may reprogram myeloid progenitors to produce eosinophils specifically. In both cases, the additional loss of p107 and p130 generates a novel phenotype.
Our data also indicate that the TKO myeloproliferation is driven by the KLS compartment, while the origin of the defect is still unclear in Rb-deficient mice, as the Lyz-M Cre model used by Walkley et al. is mostly expressed in mature myeloid cells but also in a subset of early progenitors (Ye et al., 2003).
But the most striking difference between the TKO and Rb mutant models lies in the HSC phenotype. Rb mutant HSCs are not continually hyperproliferative, highlighting the compensatory role of p130 and p107 in controlling the cell cycle of these stem cells. TKO, but not Rb−/− HSCs have impaired homing activity and fail to transplant (Daria et al., 2008; Walkley et al., 2007). These observations suggest that mobilization of hematopoietic progenitors out of the BM and homing back to the BM may be controlled by different mechanisms, but that both processes are regulated by the three members of the Rb gene family.
Together, our results indicate that p107 and p130, in addition to pRB, have essential functions during hematopoiesis, including in HSCs. These compensatory roles between pRB, p107, and p130 may also explain why human blood malignancies often have alterations in upstream regulators of the entire pRB family ((Barista et al., 2001; Leoncini et al., 2006; Maru, 2001; Mihara et al., 2006; Spike and Macleod, 2005) and references included).
This analysis of the phenotype of Rb family TKO HSCs and hematopoietic progenitors complements recent in vivo studies with mice mutant for families of mammalian genes, especially cell cycle regulators (Geng et al., 2003; Kozar et al., 2004; Krimpenfort et al., 2007; Santamaria et al., 2007). Future experiments will continue to investigate the role of Rb family genes in the control of cell cycle re-entry in quiescent and differentiated cells to probe the cellular mechanisms of cancer initiation.
Rb family conditional TKO mice were obtained by crossing Rb conditional mutant mice (Sage et al, 2003) with p107−/− mice (Lee et al., 1996) and p130 conditional mutant mice (to be described elsewhere) in a mixed 129Sv/J and C57/BL6 background.
Induction of Cre in Mx1-Cre mice (Kuhn et al., 1995) was performed by five consecutive daily injections of 200 µg of pI-pC (Sigma P-0913). In Rosa26-CreERT2 TKO mice, Cre was induced by five consecutive injections of 1 mg of tamoxifen (Sigma T-5648) in corn oil. Inductions were performed on 6-weeks old mice.
Ly5.2-expressing Rag2/Commonγchain double KO mice were obtained from the Weissman laboratory at Stanford. SCID and Rag2 mutant mice were purchased from Stanford’s Research Animal Facility.
All the experiments with mice were approved by Stanford IACUC (protocol 13565).
Cells from the bone marrow (BM), spleen and peripheral blood (PB) were filtered, and red blood cells were lysed with the ACK buffer (NH4Cl/KHCO3). Mature white blood cells (WBC) were subsequently stained with antibodies against B220, Mac-1, Gr-1, and Ter119 for 30 minutes. Progenitors cells were stained with a cocktail of lineage-cy5PE antibodies and subsequently stained with antibodies against c-Kit, Sca1, Flk2, CD34, FcγR or Il7Rα. All the antibodies were purchased from eBioscience. All Analysis was performed at the Stanford FACS Facility on FacsScan or LSR, using the CellQuest software for data acquisition. Data were analyzed using the FlowJo software (Tree Star). Experiments displayed are representative of 3 or more independent experiments. To sort progenitor cells, WBC were first incubated with c-Kit beads (Miltenyi Biotech) and purified on columns (Automacs, Miltenyi Biotech). The c-Kit-enriched fraction was subsequently stained with lineage, c-Kit, CD34, Flk2, and Sca1 antibodies. Cells were sorted on a Vantage machine.
Mice were injected with BrdU 1 hour before sacrifice. Unfractioned BM cells or sorted subpopulations were fixed in 70% ethanol overnight and stained with an anti-BrdU antibody (BD) and Propidium Iodide (PI) as described before (Passegue et al., 2005). Cell survival was analyzed by Annexin V staining (Roche).
Unfractioned splenocytes (50,000) or BM cells (20,000), KLS (100), Myeloid Progenitors (100) or mature Mac-1+/Gr-1Low (10,000) cells were plated in Methocult M3434 from Stem Cell Technologies. Colonies were evaluated 8 to 10 days after plating. Replating was performed with 20,000 cells. Every experiment was performed independently three times in duplicate. For cytospin analysis, 20,000 cells were centrifuged onto a glass slide. Slides were then stained with Giemsa (Fisher Scientific).
As a result of their 129Sv/J:C57BL/6 mixed genetic background, Ly5.1 WT and Mx1Cre TKO cells could not be transplanted in F1 mice generated by breeding Ly5.1 WT and Ly5.2 C57BL/6 mice. To circumvent this caveat, we transplanted unfractioned BM cells (2 or 4 millions) into SCID or Rag2/commonγchain KO immunodeficient mice. Prior to transplantation, SCID mice were sublethally irradiated with 1.5 G, while Rag2/commonγchain mutant were lethally irradiated with 9 G. We successfully transplanted Ly5.1/Ly5.2 F1 BM cells into Ly5.1 control and Rosa26-CreERT2 TKO mice. Prior to transplantation, recipient mice were lethally irradiated with 9 G and allowed to recover overnight. Mice were fed with antibiotics for 3 weeks and the transplantation efficiency was monitored after 4 weeks by analyzing Ly5.1/Ly5.2 expression in the PB. Transplantations were performed retro-orbitally in anesthetized mice.
For homing experiment, we isolated lineage negative cells by staining WBC from BM with unconjugated lineage antibodies (raised in rat). Next, cells were incubated with anti-rat beads and subsequently run on an Automacs (Miltenyi). Positively selected cells were stained with a fluorescent dye (CFSE, Invitrogen) for 30 min in the dark. An aliquot of the cells was analyzed by FACS to confirm appropriate staining. 2×106 cells were injected into SCID mice and recipient mice were sacrificed 16 hours later. The bone marrow was flushed separately from each lower limb and CFSE+ WBC from each limb were analyzed.
RNA were processed and PCR reactions were prepared as described before (Passegue et al., 2005). Primer sequences are available upon request. A total of 10,000 KLS cells from either control or Mx1-Cre TKO were used for microarray analysis. Samples processing and data analysis are described in Supplementary Methods.
Statistical significance was assayed by t-test. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.005; ns: not significant.
The authors thank Tom Serwold, Anne Brunet, and Steven Artandi for critical reading of the manuscript, Jinkuk Choi for his help with the initial microarray analysis, Michael Cleary for his support and helpful discussions, and Tyler Jacks for the generous provision of mutant mice generated in his laboratory. This work was supported by the Lucile Packard Foundation for Children’s Health (J.S. and A.B.), the Damon Runyon Cancer Research Foundation and NIH-NCI RO1 CA114102 (J.S.), fellowships from the Human Frontier Science Program, the European Molecular Biology Organization, the Fonds de la Recherche Scientifique, and the Leon Fredericq Foundation (P. V.), the California Institute for Regenerative Medicine and NIH-NCI PO1 CA049605 (A.B.). S. K. is a Leukemia and Lymphoma Society scholar.
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