Compound ablation of the Rb gene family in mice results in a severe hematopoietic phenotype with excessive expansion of myeloid 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
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−/−
condTKO) mice. Adult Mx1-Cre
condTKO mice were injected with pI-pC to activate the Cre recombinase and delete the Rb
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 (). Infiltrating leukocytes were present in the kidney, liver, lungs, and skin ( and data not shown). The BM structure was also altered, and showed a marked expansion of myeloid cells, particularly eosinophils ().
Mx1-Cre TKO mice develop a pathological myeloid eosinophilic expansion
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) (). 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) (). Cytospins from sorted BM subpopulations showed high heterogeneity in the pre-M population and well-differentiated granulocytes in the Mac-1+
population, as expected (Figure S2B
). In addition, decreased percentages of Mac-1+
granulocytes (), erythrocytes, and B lymphocytes ( 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 ( 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 (, 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.
Early hematopoietic progenitors and stem cells are actively cycling and more numerous in TKO 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 (). 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 (, 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 (). 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 (). In the lymphoid lineage, the number of Common Lymphoid Progenitor cells (CLP) was significantly decreased in Mx1-Cre TKO mice compared to controls (), and Annexin V staining experiments showed significantly higher numbers of apoptotic CLP in mutant mice (data not shown, see below).
Increased proliferative potential and altered fate of Rb family TKO early hematopoietic progenitor cells
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 (). 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 ().
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 (); 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.
Re-introduction of one wild-type allele of p107 rescues the TKO myeloproliferation
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 (). p107-Single mice had similar numbers of KLS, MP, and pre-M cells in the BM as control mice as determined by FACS analysis (), and no increase in pre-M cells in the spleen (). The cell cycle of KLS cells was also similar in control and p107-Single mice (). 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 (), suggesting that different cell types during hematopoiesis may be differentially sensitive to different levels of Rb family function.
Re-introduction of one wild-type allele of p107 rescues the TKO myeloproliferative phenotype
TKO HSCs display increased mobilization and are incapable of long-term reconstitution of the hematopoietic system
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 (). This was confirmed by the increase in the number of colonies formed by unfractioned splenocytes in methylcellulose (). Overall, there were approximately four times more Flk2−
HSCs in the spleen of mutant mice compared to their BM (compare and ). KLS and MP cells were also more frequent in the peripheral blood of mutant mice (), providing further evidence of their increased mobilization.
TKO hematopoietic progenitors cells are mobilized and less capable to home back to the BM
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) (). 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 , 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 (). 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 (). Despite this overall decreased cellularity, a majority of the mature hematopoietic cells were TKO and not wild-type competitors (). Importantly, hematopoietic progenitor cell numbers, including LT-HSCs, were significantly decreased in the BM of mice reconstituted with TKO cells ().
The long-term reconstitution potential of TKO HSCs is impaired
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.
The myeloproliferation present in TKO mice arises quickly and is transplantable
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
alleles and loss of the entire Rb
family in multiple cell types, including BM cells ( 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).
Loss of Rb family of genes induces rapid and cell-intrinsic defects in hematopoietic progenitors
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 ( 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 ( 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 (), 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 (). Both TKO CMP and GMP populations showed an increased proliferation (). 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 (). 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
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) ().
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 ().
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.
Rb family mutant early hematopoietic progenitors are aberrantly primed to produce myeloid cells
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
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 (). These data are consistent with the hyperproliferative status of the mutant cells.
The TKO myeloproliferation phenotype is primed by altered gene expression programs in Rb family mutant KLS 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 (). In addition, the expression of known regulators of lymphoid development, including the Lck, Rag1,
genes, was also decreased (). Conversely, genes coding for Gm-csfr
and the Gata-2
transcription factor, two critical promoters of myeloid development, were upregulated in TKO KLS cells (). 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 (). We also found that several genes that code for markers of the neutrophil lineage were downregulated in TKO KLS cells (). 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 ().