Geminin deletion from hematopoietic cells causes anemia and thrombocytosis in mice

HSCs maintain the circulating blood cell population. Defects in the orderly pattern of hematopoietic cell division and differentiation can lead to leukemia, myeloproliferative disorders, or marrow failure; however, the factors that control this pattern are incompletely understood. Geminin is an unstable regulatory protein that regulates the extent of DNA replication and is thought to coordinate cell division with cell differentiation. Here, we set out to determine the function of Geminin in hematopoiesis by deleting the Geminin gene (Gmnn) from mouse bone marrow cells. This severely perturbed the pattern of blood cell production in all 3 hematopoietic lineages (erythrocyte, megakaryocyte, and leukocyte). Red cell production was virtually abolished, while megakaryocyte production was greatly enhanced. Leukocyte production transiently decreased and then recovered. Stem and progenitor cell numbers were preserved, and Gmnn^–/– HSCs successfully reconstituted hematopoiesis in irradiated mice. CD34⁺Gmnn^–/– leukocyte precursors displayed DNA overreplication and formed extremely small granulocyte and monocyte colonies in methylcellulose. While cultured Gmnn^–/– megakaryocyte-erythrocyte precursors did not form erythroid colonies, they did form greater than normal numbers of megakaryocyte colonies. Gmnn^–/– megakaryocytes and erythroblasts had normal DNA content. These data led us to postulate that Geminin regulates the relative production of erythrocytes and megakaryocytes from megakaryocyte-erythrocyte precursors by a replication-independent mechanism.

Stem cells maintain adult tissues by replacing cells that are lost through normal attrition, damage, or disease. Stem cell division patterns are unusual in that they produce 2 different types of daughter cells. Some daughters maintain their identity as stem cells, while others enter a pathway of terminal differentiation and ultimately become mature somatic cells. Stem cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The factors that control this balance are incompletely understood. In most cases, it is not even known whether stem cell division is symmetric, producing either 2 stem cells or 2 differentiating cells, or if it is asymmetric, generating 1 stem cell and 1 differentiating cell. One model proposes that the choice between self renewal and terminal differentiation is stochastic (i.e., random), while another proposes that the choice is driven by cytokines in response to environmental stimuli (1, 2).

The unstable regulatory protein Geminin (Gmnn) is thought to control patterns of cell division and differentiation (3, 4). Two different molecular functions have been described for Geminin. One function is to limit the extent of DNA replication to 1 round per cell cycle by binding and inhibiting the essential replication factor Cdt1 (5–7). Geminin is destroyed by ubiquitin-dependent proteolysis during mitosis, allowing for a new round of replication in the succeeding cell cycle. Overreplication is also suppressed by a redundant Geminin-independent mechanism: Cdt1 itself is destroyed by ubiquitin-dependent proteolysis when replication origins fire (8–11). Because of this redundancy, it is not known whether Geminin is absolutely required to prevent overreplication in all types of adult somatic cells.

In addition to regulating DNA replication, Geminin also affects cell differentiation in the central nervous system, the axial skeleton, and the eye. Using 2-hybrid assays, Geminin has been found to bind several different transcription factors in the Homeobox (Hox) and sine oculis families (12, 13). Geminin also binds 2 chromatin-remodeling proteins: Brg1, the ATPase subunit of a SWI/SNF remodeling complex; and Scmh1, a component of polycomb repressive complex 1 (13, 14). Overexpression and knockdown of Geminin in cultured cells and in various embryonic systems has suggested that Geminin influences cell fate by inhibiting the function of these proteins. Because it regulates both the cell cycle and tissue-specific transcription factors, it has been postulated that Geminin somehow coordinates cell division with cell differentiation.

This hypothesis has been tested by deleting Geminin from model organisms. Caenorhabditiselegans embryos that have been treated with Geminin RNAi grow to adulthood, but approximately 20% of them display cytological abnormalities in their germ cells and are sterile (15). A similar proportion of geminin (RNAi) worms show anaphase chromosome bridges in intestinal epithelial cells, suggesting a defect in DNA replication. Gmnn^–/–Drosophila embryos die at larval stages (16). They also show anaphase chromosome bridges, and an extended period of DNA replication has been detected in ovarian follicle cells. Geminin-deficient Xenopus embryos stop dividing after the 13th cleavage division and disintegrate during gastrulation (17). Their primary defect is overreplication of their DNA, which activates the DNA replication checkpoint and arrests the cells in G₂ phase. Gmnn^–/– mouse embryos also stop dividing at the early blastula stage, as soon as the maternal stockpile of Geminin is exhausted (18, 19). At the time of the arrest, their cells have a greater DNA content than normal. Intriguingly, all the blastomeres prematurely differentiate as trophoblast cells and none show markers of embryonic stem (ES) cells. Heterozygous Gmnn^+/– mice are phenotypically normal. Taken together, these studies provide good evidence that Geminin deficiency disrupts DNA replication and causes cell-cycle abnormalities. Geminin’s effects on cell differentiation have been difficult to assess in these systems because the population of differentiating cells is small and nonuniform.

To more rigorously examine the role of Geminin in regulating cell division and differentiation, we have developed a mouse model with a conditional floxed Geminin allele and deleted the protein from hematopoietic cells using an interferon-inducible Cre driver. The hematopoietic system is ideal for these studies because the stem cells have been well defined, their pattern of differentiation has been mapped out, and the numbers of different types of cells in the differentiation tree can be quantified by staining with cell-type–specific antibodies. We found that Geminin deletion caused only subtle abnormalities in DNA replication but had profound effects on the pattern of cell differentiation.

Geminin is expressed in stem cells, erythroid precursors, and megakaryocytes. First, we measured the abundance of Geminin mRNA in different FACS-sorted hematopoietic cell populations by quantitative real-time PCR (RT-PCR). Geminin is expressed in the erythrocyte/megakaryocyte lineage, including megakaryocyte-erythrocyte progenitors (MEPs), CD71⁺Ter119⁺ erythroblasts, and mature CD41⁺ megakaryocytes (Figure 1A). Geminin is also broadly expressed in most progenitor cells, including common myeloid progenitors (CMPs), granulocyte monocyte progenitors (GMPs), and Lin^–Sca1⁺c-Kit⁺ (LSK) stem cells. LSK cells represent the most undifferentiated population and include the long-term HSCs that are able to reconstitute hematopoiesis in an irradiated animal (20). In contrast, mature marrow Gr1⁺Mac1⁺ wbc and peripheral blood leukocytes had very low levels of Geminin expression.

Figure 1

Targeted deletion of geminin from hematopoietic cells. (A) Geminin mRNA expression in sorted hematopoietic cell populations. CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, megakaryocyte-erythrocyte progenitor; E-blast, erythroblast; MK, megakaryocyte; Gr1⁺Mac1⁺, Gr1⁺Mac1⁺mononuclear cells; PB, peripheral blood. (B) Targeting strategy. The thick black line represents sequences encompassed by the targeting construct. (C) Southern blot of ES cell DNA showing homologous recombination of the targeting construct into the Geminin locus. The hatched box in B indicates the location of the hybridization probe. (D–G) Mice with the indicated genotypes were injected with pIpC, then analyzed. (D) PCR reaction showing deletion of exons 5–7 after pIpC injection. The first lane is a control with no DNA. (E) RT-PCR showing reduced Geminin expression pIpC-treated Mx1-Cre/Gem^fl/fl marrow and colonies. (F) Immunoblot showing undetectable Geminin expression in Mx1-Cre/Gmnn^fl/fl bone marrow cells after pIpC injection. TnT, in vitro transcription and translation. (G) Flow cytometry showing Mx1-Cre induction in the majority of marrow cells.

Construction of mice with a conditional targeted deletion of Geminin. The mouse genome contains a single Geminin gene that is composed of 7 exons (Figure 1B). We constructed a conditional Geminin floxed allele (Gmnn^fl) by flanking exons 5, 6, and 7 with loxP sites. These exons encode Geminin’s dimerization domain and its binding site for Cdt1, both of which are essential for the protein’s biological function (21). We inserted the Gmnn^fl allele into the mouse genome through homologous recombination in ES cells (Figure 1C), then generated a strain of Gmnn^fl/+ mice by injecting the targeted ES cells into mouse blastocysts. Gmnn^fl/+ and Gmnn^fl/fl mice are completely viable and fertile (not shown).

To specifically delete Geminin from hematopoietic cells, Gmnn^fl/fl mice were crossed to transgenic mice that express Cre recombinase from the interferon-responsive Mx1 promoter (22). To induce Cre expression, Mx1-Cre/Gmnn^fl/fl mice and littermate controls were injected with polyinosine-polycytosine (pIpC), which mimics a viral infection. With this protocol, Cre-mediated recombination through the loxP sites efficiently excises exons 5, 6, and 7 from the Geminin gene and generates a Geminin-null allele (Gmnn^Δ) (Figure 1D). RT-PCR of total marrow RNA showed that Cre induction brings about a 95%–99% reduction in the amount of Gmnn mRNA in marrow cells (Figure 1E). A time course demonstrated that Geminin RNA has largely disappeared by 48 hours after the first dose of pIpC (not shown). To document loss of the Geminin protein, we raised an antibody against mouse Geminin in rabbits. The antibody recognizes approximately 20 kDa protein on immunoblots that matches the predicted molecular weight of Geminin (23.3 kDa) and comigrates with Geminin that has been translated in vitro (Figure 1F). This protein was not detected in Mx1-Cre/Gmnn^fl/fl bone marrow cells after pIpC injection. This result attests to the specificity of the antibody and also documents disappearance of the Geminin protein after Cre induction. Finally, we crossed our Mx1-Cre/Gmnn^fl/fl mice to R26R-GFP mice, which carry a loxP-flanked transcription/translation STOP cassette inserted between the ROSA promoter and the GFP coding sequence (23). Cre-mediated recombination deletes the STOP cassette and brings about GFP expression. We found that after pIpC injection, Mx1-Cre(–)Gmnn^fl/fl marrow cells remained GFP negative, while 80–90% of Mx1-Cre/Gmnn^fl/fl cells became GFP positive (Figure 1G). Control experiments demonstrate that the GFP-negative cells consist of both nonhematopoietic (CD45^–) cells and hematopoietic (CD45⁺) cells that express low levels of Geminin mRNA by RT-PCR (Supplemental Figure 3).

Geminin deletion causes anemia and thrombocytosis. After pIpC injection, Mx1-Cre/Gmnn^fl/fl mice showed poor growth compared with littermate controls and appeared sickly. Most of them (~90%) died within 3 weeks, while few of the pIpC-injected control Mx1-Cre/Gmnn^fl/+ mice died (Figure 2A). We have not determined the cause of death in pIpC-injected Mx1-Cre/Gmnn^fl/fl mice (hereafter referred to as Gmnn^Δ/Δ mice). At the time of sacrifice, 2 of them were found to have multiple bacterial abscesses in their livers with little surrounding inflammatory reaction, suggesting that they may have died of overwhelming infection (not shown). All Gmnn^Δ/Δ mice exhibited pathological abnormalities in the peripheral blood, bone marrow, and spleen. Mx1-Cre/Gmnn^fl/fl mice had about one-third as many marrow cells as controls (Figure 2B). The loss of Geminin strikingly affected the production of mature blood cells in all 3 hematopoietic lineages.

Figure 2

Geminin deletion affects all 3 hematopoietic lineages. (A) Kaplan-Meier survival curve of pIpC-treated Mx1-Cre/Gem^fl/fl mice (red) and control littermates (black). (B–F) Total marrow cells and complete blood counts after pIpC induction. Red circles, pIpC-treated Mx1-Cre/Gem^fl/fl mice; black circles, pIpC-treated control littermates. Each circle represents a single mouse, and the line graph represents the average value at each time point. (G–J) Bone Marrow sections (G and H), blood smears (I), and spleen sections (J) from pIpC-treated Mx1-Cre/Gmnn^fl/fl mice (Gmnn^Δ/Δ) and pIpC-treated control littermates (control). Original magnification, ×400 (G, H, and J); ×1000 (I). (K–M) Typical flow cytometry profiles of marrow cells from pIpC-treated Mx1-Cre/Gem^fl/fl mice (Gem^Δ/Δ) and pIpC-treated control littermates (control). Quantification of data from several mice is shown in the right panels. The rectangles indicate the mean value and the error bars indicate the standard deviation of the mean. (N and O) EPO levels (N) and TPO levels (O) for control and pIpC-treated Mx1-Cre/Gem^fl/fl mice. The horizontal bar indicates the median, box indicates 25th to 75th percentiles, and whiskers indicate extremes. P values calculated from Student’s t test.

The erythrocyte and megakaryocyte lineages were the most severely affected. Gmnn^Δ/Δ mice developed a profound and progressive anemia; their average red cell count was 40%–60% less than littermate controls (Figure 2C). Their red cell indices (MCV, MCH, and MCHC) were normal (not shown). Immunocytochemistry revealed a virtual absence of Ter119⁺ red cell precursors in Gmnn^Δ/Δ bone marrow (Figure 2G). By flow cytometry, the numbers of all stages of erythroid precursors were greatly reduced compared with controls, including R1 CD71^hiTer119^lo proerythroblasts, R2 CD71^hiTer119^hi basophilic erythroblasts, R3 CD71^medTer119^hi polychromatophilic erythroblasts, and R4 CD71^loTer119^hi orthochromatophilic erythroblasts and reticulocytes (Figure 2K). The loss of Ter119⁺ progenitors was also apparent in red pulp of the spleen, a common site of erythropoiesis in younger mice. The red pulp was paucicellular compared with controls, while the white pulp appeared normal (Figure 2J). Gmnn^Δ/Δ mice had markedly elevated serum erythropoietin (EPO) levels compared with controls, consistent with their severe anemia (Figure 2N).

In striking contrast to the anemia, the number of platelets was vastly increased. Gmnn^Δ/Δ mice had platelet counts that were 5–10 times those of control mice, up to 6 × 10⁶/μl (Figure 2D). The platelets appeared morphologically normal (Figure 2I) and had a normal volume by flow cytometry (not shown). They also activated normally in response to thrombin (not shown). Reflecting the high peripheral platelet count, both the marrow and spleen were densely infiltrated with large numbers of megakaryocytes (Figure 2H). Flow cytometry showed that the number of CD41⁺ megakaryocytes in the marrow was increased by about 10-fold (Figure 2L). The number of Lin^–Sca1^–c-Kit⁺CD9⁺CD41⁺ megakaryocyte precursors was also increased (Supplemental Figure 2; supplemental material available online with this article; doi: 10.1172/JCI43556DS1). Serum thrombopoietin (TPO) levels were not significantly different in Gmnn^Δ/Δ mice and littermate controls, indicating that the thrombocytosis was not being driven by TPO (Figure 2O).

The wbc were also affected by Geminin deletion, but not to the same extent as the erythroid cells and megakaryocytes. Control mice showed a sharp rise in peripheral wbc after pIpC injection, but Gmnn^Δ/Δ mice showed a decrease (Figure 2, E and F). The number of neutrophils was more severely affected than the number of lymphocytes or monocytes. Near the nadir, flow cytometry showed that the number of mature Gr1⁺Mac1⁺ leukocytes in Gmnn^Δ/Δ marrow was greatly decreased (Figure 2M). These differences were transient and normalized within 2–3 weeks (Figure 2, E and F). Furthermore, when Gmnn^Δ/Δ marrow cells were transplanted to an irradiated host to bypass the mortality, the number of peripheral Gr1⁺Mac1⁺ leukocytes normalized with time (see below).

Gmnn^Δ/Δ stem and progenitor cells show reduced proliferation in vitro. Next we tested how Geminin deletion affects HSCs, multipotent progenitor cells, and committed precursor cells. By flow cytometry, the absolute number of LSK (Lin^–Sca1⁺c-Kit⁺LSK) stem cells was increased in Gmnn^Δ/Δ mice (Figures 3, A and B). The number of SLAM⁺ LSK cells, which represent a more definitive HSC population, was also preserved (Supplemental Figure 1). There was also a nonsignificant trend toward greater numbers of CMPs and MEPs but not GMPs (Figure 3B). Stem and progenitor cells constituted a greater fraction of the marrow in Gmnn^Δ/Δ mice because of the loss of more mature types of cells (not shown). Because stem and progenitor cell populations are preserved, Geminin must affect hematopoiesis at later stages.

Figure 3

Defective stem and progenitor cell proliferation in Gmnn^Δ/Δ mice. (A) Typical stem cell flow cytometry profiles from pIpC-treated Mx1-Cre/Gem^fl/fl mice (GmnnΔ/Δ) and pIpC-treated control littermates (control). (B) Quantification of stem cell populations. (C) Typical appearance of myeloid colonies grown from control and Gmnn^Δ/Δ marrow cells. Original magnification, ×200. (D and E) Number of erythroid and myeloid colonies (D) or megakaryocyte colonies (E) grown from control or Gmnn^Δ/Δ marrow cells. (F) Control or Gmnn^Δ/Δ MEPs were plated for erythroid colonies. Left, photographs of colony plates; right, number of colonies produced. Scale bar is 5 mm. (G) Control or Gem^Δ/Δ MEPs were plated for megakaryocyte colonies. Left, photographs of colony plates (megakaryocyte colonies are circled); right, number of colonies produced. Scale bar is 0.5 mm. For box-and-whisker plots, horizontal bar indicates the median, box indicates 25th to 75th percentiles, and whiskers indicate extremes. P values calculated from Student’s t test.

Although Gmnn^Δ/Δ stem and progenitor cells were maintained, they had a dramatically reduced ability to form colonies in methylcellulose (Figure 3, C and D). Gmnn^Δ/Δ cells did not form erythroid colonies at all, reflecting the loss of marrow Ter119⁺ erythroid precursor cells seen in vivo. They formed granulocyte and monocyte colonies, but both the total number of colonies and the size of individual colonies were severely reduced (Figure 3, C and D). The distribution of different types of myeloid colonies was normal. When the Gmnn^Δ/Δ myeloid colony cells were recovered from the plate, we found that the Geminin message was still undetectable by RT-PCR, indicating that the Geminin gene had been deleted from the committed precursors (Figure 1F).

In contrast, Gmnn^Δ/Δ marrow cells formed about 3 times as many megakaryocyte colonies as control cells (Figure 3E). To see if this was caused by an increased number of MEPs or by a change in their differentiation pattern, we plated a fixed number of purified MEPs from Gmnn^Δ/Δ and littermate control mice in methylcellulose. When plated in erythrocyte growth medium, we found that control MEPs produced 25 times as many erythroid colonies as Gmnn^Δ/Δ MEPs (Figure 3F). When plated in megakaryocyte growth medium, the ratio was reversed: Gmnn^Δ/Δ MEPs produced 3–4 times as many megakaryocyte colonies as control MEPs (Figure 3G). These data indicate that the thrombocytosis in Gmnn^Δ/Δ mice is due to an increased propensity of the MEPs to differentiate as megakaryocytes.

In summary, the ability of marrow cells to form colonies in culture paralleled the hematological abnormalities seen in intact mice. These results indicate that the abnormalities are intrinsic to the marrow cells themselves and not driven by changes in the hematopoietic microenvironment. They also indicate that, except for the megakaryocyte lineage, there is a defect in the ability of individual Gmnn^Δ/Δ stem and precursor cells to proliferate in vitro.

Geminin is not required for maintenance of HSCs. We next performed transplantation experiments to determine whether Geminin is required for self renewal and long-term maintenance of HSCs. Marrow cells from Mx1-Cre/Gmnn^fl/fl/R26R-GFP/CD45.2 mice were injected into lethally irradiated WT CD45.1 mice along with a small rescue dose of WT CD45.1 marrow cells (Figure 4A). We also transplanted cells from Mx1-Cre/Gmnn^fl/+/R26R-GFP/CD45.2 littermates as a control. We monitored engraftment by flow cytometry of peripheral wbc, using the CD41 polymorphism to distinguish the transplanted CD45.2⁺ cells from the recipient’s endogenous CD45.1⁺ cells (Figure 4B). Five weeks after transplantation, approximately 65% of the peripheral white cells were CD45.2⁺, indicating successful engraftment in all cases (Table 1). We then deleted the Gmnn gene by injecting the mice with pIpC following the same regimen as before. Some mice in each group were left uninjected as a control. After pIpC treatment, 70%–90% of the peripheral CD45.2⁺ cells became GFP positive, indicating successful Cre induction (Figure 4C). Immediately after induction, the mice transplanted with Mx1-Cre/Gmnn^fl/fl marrow showed a precipitous drop in the peripheral wbc count and the fraction of CD45.2⁺ cells decreased to 30%–40% of the total (Figure 4, D and E). After approximately 5 weeks, however, the white cell count normalized and the fraction of CD45.2⁺ cells returned to approximately 65%. Mice transplanted with the 3 different types of control marrow showed only a slight drop in the total white count and no significant change in the fraction of CD45.2 cells after pIpC injection. These results mirror what we observed before: that the white count drops transiently in response to Geminin deletion, then recovers.

Figure 4

Geminin is not required for HSC self renewal. (A) Experimental protocol. Recipient CD45.1 mice were irradiated and transplanted with CD45.2 marrow from R26R-GFP/Mx1-Cre/Gem^fl/fl or R26R-GFP/Mx1-Cre/Gem^fl/+ donors. After engraftment, the mice were either left untreated or treated with pIpC. (B) Typical flow profile quantifying CD45.1⁺ and CD45.2⁺ populations. (C–G) Time-dependent changes in peripheral blood GFP⁺ cells (C), CD45.2⁺cells (D), wbc count (E), rbc count (F), and platelet count (G) in transplanted mice after pIpC injection. Black, uninjected control mice; green, plpC-injected control mice; blue, uninjected Mx1-Cre/Gemfl/f mice; red, plpC-injected Mx1-Cre/Gem^fl/f mice. Average of 6 measurements (pIpC-treated mice) or 4 measurements (untreated mice). *P < 0.05; **P < 0.01. (H) Geminin mRNA levels in CD45.1 and CD45.2 cells recovered from transplanted mice. Number of measurements is shown above each column. Black bar, uninjected control mice; blue bar, uninjected Mx1-Cre/Gem^fl/f mice; red bar, plpC-injected Mx1-Cre/Gem^fl/f mice. (I) Absolute numbers of recovered CD45.2⁺ marrow cells by flow cytometry. Color code is the same as in (H). (J) Colony types grown from CD45.1 or CD45.2 cells recovered from transplanted mice. Number of mice is shown above each column. Red, erythroid colonies (E); yellow, granulcoyte colonies (G); green, monocyte colonies (M); blue, granulocyte-monocyte colonies (GM); lavender, granulocyte-erythrocyte-megakaryocyte colonies (MK). (K) Recovered marrow cells (CD45.1⁺CD45.2) were retransplanted into irradiated CD45.1 recipients. Time-dependent changes in CD45.2 peripheral blood cells in individual mice transplanted with cells from pIpC-treated control mice (green curves) or with cells from pIpC-treated Mx1-Cre/Gem^fl/fl mice (red curves). Cross symbol indicates death. Green lines, donor cells from plpC-injected control mice; red lines, donor cells from plpC-injected Mx1-Cre/Gem^fl/fl mice. (L) Right, experimental protocol. Mice were treated with pIpC and 4 weeks later marrow cells were transplanted into irradiated recipients. Left, number of CD45.1⁺ and CD45.2⁺ cells in the peripheral blood 2 weeks after transplantation. Black, percentage of CD45.1⁺ cells; red, percentage of CD45.2⁺ cells. n = 6 for both groups. P values calculated by ANOVA.

Table 1

Transplantation and recovery of control and Gmnn^Δ/Δ CD45.2⁺ cells

The mice transplanted with Mx1-Cre/Gmnn^fl/fl/R26R-GFP/CD45.2 marrow developed anemia and thrombocytosis after pIpC injection, while the control mice did not (Figure 4, F and G). This confirms that the hematological abnormalities associated with Geminin deletion are caused by a primary defect within the marrow cells themselves and not from a change in the hematopoietic microenvironment. The anemia and thrombocytosis were not as severe as in Mx1-Cre/Gmnn^fl/fl mice; they were probably ameliorated by the presence of normal CD45.1⁺ marrow cells. None of the mice transplanted with Mx1-Cre/Gmnn^fl/fl marrow and then injected with pIpC died during the course of the experiment.

The Gmnn^Δ/Δ CD45.2⁺ cells were maintained in the peripheral blood for 22 weeks after transplantation, indicating long-term survival of the Geminin-deficient stem cells and precursor cells (Figure 4D). After 22–24 weeks, the mice were sacrificed and the marrow was examined. In the mice that were transplanted with Mx1-Cre/Gmnn^fl/fl cells and injected with pIpC, CD45.2⁺ cells constituted 90%–100% of the marrow, confirming that the Gmnn^Δ/Δ cells were able to persist (Table 1). The CD45.2⁺ cells were purified by flow cytometry, and RT-PCR confirmed that these cells lacked Geminin RNA (Figure 4H). In contrast, the small number of remaining CD45.1⁺ cells and both the CD45.1⁺ and the CD45.2⁺ cells from controls had normal amounts of Geminin message. This confirms that the Geminin gene had been deleted from the stem and precursor cells and that the marrow had not become repopulated by rare CD45.2⁺ cells that did not delete Geminin. The absolute number of CD45.2⁺ LSK stem cells and CD45.2⁺ Lin^–Kit⁺Sca1^– progenitors was the same in all 4 groups of mice (Figure 4I). These results indicate that Gmnn^Δ/Δ CD45.2⁺ stem and progenitor cells can persist in the marrow for at least 22 weeks. Geminin does not seem to be required for long-term marrow reconstitution or for self renewal of HSCs.

In other respects, the marrow of the mice transplanted with Mx1-Cre/Gmnn^fl/fl cells resembled the marrow from pIpC-injected Mx1-Cre/Gmnn^fl/fl mice. It was densely infiltrated with megakaryocytes (not shown), and the number of CD45.2⁺Ter119⁺CD71⁺ erythrocyte precursors was reduced compared with controls (Figure 4I). One difference was that the number of mature Gr1⁺Mac1⁺ leukocytes had partially recovered, in accordance with the transient nature of the reduction in wbc numbers (Figure 4I). When the recovered CD45.2⁺Gmnn^Δ/Δ cells were plated in methylcellulose, they readily produced megakaryocyte colonies but not erythroid colonies (Figure 4J). The number of leukocyte colonies was severely reduced, and the few that formed were small and contained few cells. The reduction in colony number was more pronounced than in pIpC-injected Mx1-Cre/Gmnn^fl/fl mice (compare Figure 3D and Figure 4J). In contrast, CD45.1⁺ cells from the same mice formed near normal numbers of colonies, as did both CD45.2⁺ and CD45.1⁺ cells from all 3 groups of control mice. These results confirm that the hematopoietic defects are intrinsic to the Gmnn^Δ/Δ cells.

Gmnn^Δ/Δ cells engraft poorly. To see if the Gmnn^Δ/Δ cells could persist indefinitely, marrow from each of the transplanted mice was infused into a second lethally irradiated CD45.1 recipient. In this experiment, we noted that Gmnn^Δ/Δ cells displayed a severe engraftment defect. Three of the 6 irradiated mice infused with Gmnn^Δ/Δ marrow died within 3 weeks of the procedure (Figure 4K). One mouse that survived had a very low percentage (2%–5%) of CD45.2⁺ cells in his peripheral blood, indicating that the marrow had been reconstituted largely by Gmnn^+/+ CD45.1 cells. Engraftment was successful in only 2 of the 6 cases. In contrast, all 14 recipient mice that were infused with Mx1-Cre/Gmnn^fl/+ marrow or Mx1-Cre/Gmnn^fl/fl marrow without pIpC treatment had normal engraftment and survival. In these mice, the peripheral blood showed about the same proportion of CD45.2⁺ cells as was present in the donor (Figure 4K). These results indicate that Gmnn^Δ/Δ stem and progenitor cells engraft poorly when transferred to a new host.

In the 2 cases where the Gmnn^Δ/Δ cells successfully engrafted, they came to constitute 30%–70% of the recipient’s marrow. The proportion of CD45.2⁺ cells generally increased with time in these mice, confirming that Gmnn^Δ/Δ stem cells can persist after transplantation. Both mice showed severe thrombocytosis, anemia, and transient leukopenia (Table 2), again confirming that these abnormalities are intrinsic to the Gmnn^Δ/Δ cells. These 2 mice died at 45 and 120 days after the procedure. These results suggest that pIpC-injected Mx1-Cre/Gmnn^fl/fl mice die primarily from their hematological abnormalities and not because of deletion of Geminin from some other cell type.

Table 2

Complete blood counts after retransplantation

To confirm that Gmnn^Δ/Δ cells engraft poorly, we performed a direct transplantation experiment. We injected CD45.2/Mx1-Cre/Gmnn^fl/fl mice and CD45.2/Mx1-Cre/Gmnn^fl/+ controls with pIpC to delete the Geminin gene, and 4 weeks later, we transplanted the cells into irradiated CD45.1 recipients along with a rescue dose of WT CD45.1 cells (Figure 4L). After 2 weeks, we found that the mice transplanted with pIpC-treated CD45.2/Mx1-Cre/Gmnn^fl/fl cells had only CD45.1⁺ cells in their bloodstream, while the mice transplanted with control CD45.2/Mx1-Cre/Gmnn^fl/+ cells contained an equal mixture of CD45.1⁺ and CD45.2⁺ cells. This confirms that Gmnn^Δ/Δ cells engraft poorly.

Gmnn^Δ/Δ wbc precursor cells overreplicate their DNA. One well-established function of Geminin is to limit the extent of DNA replication to 1 round per cell cycle (24). We next sought to determine whether abnormalities in Gmnn^Δ/Δ marrow could be caused by overreplication of the DNA. Two weeks after pIpC injection, Mx1-Cre/Gmnn^fl/fl mice and littermate controls were given a single injection of ethinyl-deoxy uridine (EdU) to label cells in S-phase and sacrificed 90 minutes later. We then determined the fraction of the marrow cells in each phase of the cell cycle by measuring the amount of EdU incorporation and the total DNA content by flow cytometry (Figure 5, A and B). Both WT and Gmnn^Δ/Δ cells vigorously incorporated EdU. Slightly fewer Gmnn^Δ/Δ cells had entered S phase, but otherwise the cell-cycle distribution was normal. We could not detect any evidence of overreplication in Gmnn^Δ/Δ cells, which would be manifest as an increased number of cells with DNA contents greater than 4n. We also analyzed the DNA content of Gmnn^Δ/Δ cells in the first few days after pIpC injection, since this is the time when the cell populations are changing the most. Under these conditions, we again could not detect an increased number of cells with DNA content greater than 4n (Supplemental Figure 4A).

Figure 5

Replication defects in Geminin-deficient cells. (A) pIpC-treated Mx1-Cre/Gem^fl/+ (Control) or Mx1-Cre/Gem^fl/fl (Gem^Δ/Δ) mice were injected with EdU, then sacrificed. Flow cytometry profile of EdU incorporation versus DNA content. (B) The percentage of cells in each phase of the cell cycle is graphed. n = 3 for each genotype. *P < 0.05 (Student’s t test). (C) Bone marrow cells from pIpC-treated Mx1-Cre/Gem^fl/+ (control) or Mx1-Cre/Gem^fl/fl (Gem^Δ/Δ) mice were stained with antibodies to CD41 and for DNA content. The histogram of DNA contents for CD41⁺ megakaryocytes is shown at the right. (D) Ploidy analysis of megakaryocytes from control or Gmnn^Δ/Δ mice. P > 0.30 for all DNA contents (Student’s t test). (E) DNA content of CD34⁺ cells from pIpC-treated Mx1-Cre/Gem^fl/+ (control) or Mx1-Cre/Gem^fl/fl (Gmnn^Δ/Δ) mice after culture in vitro for 2 days. (F) Cell-cycle distribution of control or Gem^Δ/Δ CD34⁺ cells after culture. n = 3 for each genotype. (G and H) Control and Mx1-Cre/Gmnn^fl/fl mice were treated with pIpC, and at various times afterwards the percentage of apoptotic cells was measured by annexin V or FLICA staining. The right shows a graph of the percentage of apoptotic cells versus time. Black dots represent individual control mice, and red dots represent individual Mx1-Cre/Gem^fl/fl mice. P values calculated by Student’s t test.

We also measured the extent of replication in subpopulations of hematopoietic cells that were identified by staining with specific antibodies. CD41⁺ megakaryocytes can become polyploid with DNA contents up to 32n (Figure 5C). We found that there was no significant difference in the distribution of DNA content between control and Gmnn^Δ/Δ megakaryocytes (Figure 5C). The small number of remaining Gmnn^Δ/Δ CD71⁺Ter119⁺ red cell precursors vigorously incorporated EdU but did not detectably overreplicate their DNA (not shown), but so few cells could be analyzed that it was difficult to form a firm conclusion. To circumvent this problem, we measured the DNA content of Gmnn^Δ/Δ CD71⁺Ter119⁺ cells in the first few days after pIpC injection and again found no evidence of overreplication (Supplemental Figure 4, B and C).

Because Gmnn^Δ/Δ cells formed smaller and fewer granulocyte and monocyte colonies in vitro, we also tested whether cultured Gmnn^Δ/Δ wbc precursors overreplicated their DNA. We purified CD34⁺ leukocyte precursors from pIpC-injected Mx1-Cre/Gmnn^fl/fl and control mice, cultured them in liquid medium for 48 hours, and then measured their DNA content by flow cytometry (Figure 5, E and F). In this case, we found a significant population of Gmnn^Δ/Δ cells with DNA content greater than 4n, indicating overreplication, while control cells showed no such population (P = 0.003). The distribution of cells in different phases of the cell cycle was otherwise normal. We also detected overreplication in CD34⁺ cells isolated directly from the marrow without culture and in granulocytes and monocytes recovered from colonies in methylcellulose (Supplemental Figure 5). These results indicate that the poor growth of Gmnn^Δ/Δ wbc in culture is caused by a replication defect.

To see whether Gmnn^Δ/Δ cells undergo apoptosis, we measured the number of annexin V⁺ cells at different times after Mx1-Cre induction. We could detect no consistent difference in the total number of apoptotic cells between Mx1-Cre/Gmnn^fl/fl and control mice at any time (Figure 5, G and H). These results indicate that the changes in marrow cell populations after Geminin deletion are not caused by widespread cell death.

Geminin is an unstable regulatory protein that is thought to coordinate cell division and cell differentiation (3, 4). Geminin limits the extent of DNA replication to 1 round per cell cycle by binding and inhibiting the replication factor Cdt1 (5–7). In several systems, Geminin has also been found to inhibit cell differentiation by binding and inhibiting various transcription factors and chromatin remodeling proteins. In this study, we examined the effect of Geminin deletion on the proliferation and differentiation of HSCs. Surprisingly, Geminin is not required for accurate DNA replication in most marrow cells; deletion of the protein does not cause overreplication, apoptosis, or a cell-cycle arrest. Geminin is probably dispensable because of redundant mechanisms that inhibit a second round of DNA synthesis during S and G₂ phase, such as the ubiquitin-dependent proteolysis of Cdt1 (8–11). We did find, however, that Geminin profoundly affects the production of blood cells in all 3 hematopoietic lineages.

Geminin’s effects on the wbc lineage are the least extreme. The neutrophil count drops precipitously when Mx1-Cre/Gmnn^fl/fl mice are given pIpC, then recovers within a few weeks. The drop in leukocyte production seems to be caused by a replication defect. Gmnn^Δ/Δ CD34⁺ myeloid precursor cells overreplicate their DNA in vivo and in vitro, and Gmnn^Δ/Δ marrow cells show a markedly reduced ability to form granulocyte and monocyte colonies when plated in methylcellulose. These results indicate that Geminin is required to suppress overreplication in leukocytes. Although Gmnn^Δ/Δ white cells grow poorly in culture, overreplication of the DNA appears to be well tolerated in vivo. Gmnn^Δ/Δ stem and progenitor cells are able to produce mature wbc for at least 6 months after transplantation into an irradiated host. The reason for the difference in phenotype between in vivo and in vitro conditions is unclear. We hypothesize that when Gmnn^Δ/Δ cells are stressed to divide rapidly, either by growth factors in the culture medium or during a pIpC-inducted interferon response, they may develop more severe replication abnormalities or tolerate them less well. Geminin does not appear to regulate the pattern of cell differentiation in the wbc lineage. Normal proportions of G, M, and GM colonies are produced when Gmnn^Δ/Δ cells are plated in methylcellulose, and we have not been able to detect an abnormal population of developmentally arrested white cells in Gmnn^Δ/Δ marrow.

Our most striking finding is that Geminin deletion strongly influences the relative production of erythrocytes and megakaryocytes, corresponding with the high level of Geminin expression seen in these cells. The 2 lineages are affected in opposite directions — the erythroid cells are virtually eliminated from the marrow while the megakaryocytes are greatly expanded. Gmnn^Δ/Δ mice develop both a severe anemia and a massive thrombocytosis. Geminin’s effects on erythropoiesis and megakaryopoiesis are intrinsic to the marrow cells themselves since they are reproduced in colony-plating assays and persist through 2 serial transplantations. Red cell production is blocked at a very early stage, since Gmnn^Δ/Δ marrow has decreased numbers of both immature CD71⁺Ter119^– and more mature CD71⁺Ter119⁺ precursors. In contrast, all stages in the megakaryocyte pathway are increased, including megakaryocyte precursors, mature megakaryocytes, and platelets. These results suggest that Geminin regulates cell fate in the common progenitor cell of both these lineages, the megakaryocyte-erythrocyte progenitor (MEP). In colony-plating assays, Gmnn^Δ/Δ MEPs produce far fewer erythroid colonies and far more megakaryocyte colonies than control MEPs, indicating that Gmnn^Δ/Δ MEPs are intrinsically more likely to differentiate as megakaryocytes. Our functional studies complement previous reports that Geminin is downregulated when megakaryocytic cell lines are induced to differentiate with TPA (25).

Geminin might influence cell fate in MEPs by a passive replication–based mechanism. Cells in the megakaryocyte lineage, being naturally polyploid, might tolerate overreplication of the genome better than erythroid cells. In contrast to the white cell lineage, however, we could not detect any evidence of overreplication in Gmnn^Δ/Δ megakaryocytes or erythroid precursors. Furthermore, a passive mechanism cannot easily explain why the megakaryocyte number is so vastly increased in Gmnn^Δ/Δ mice. The thrombocytosis does not seem to be driven by hematopoietic growth factors because the serum TPO levels are only slightly increased. The EPO levels are significantly higher (P < 0.01), but EPO only mildly stimulates megakaryopoiesis (26, 27) and the effects we observe seem too extreme to be explained by this mechanism. Furthermore, in our cell culture experiments, both Gmnn^Δ/Δ and control MEPs were exposed to the same levels of exogenous growth factors, yet Gmnn^Δ/Δ MEPs formed more megakaryocyte colonies and fewer erythroid colonies. These results strongly suggest that Geminin influences erythrocyte and megakaryocyte production by a replication-independent mechanism.

We postulate that Geminin actively regulates a transcription factor or a chromatin remodeling protein that determines MEP cell fate. According to this model, the normal function of Geminin is to promote erythrocyte differentiation at the expense of megakaryocyte differentiation. A phenotype of anemia and thrombocytosis has previously been observed in mice that carry hypomorphic mutations in the transcription factor c-Myb or a mutation in the KIX domain of the transcriptional coactivator p300 (28–32). We have not been able to demonstrate a physical interaction between Geminin and either c-Myb or p300 by coprecipitation in several different lines of hematopoietic cells, nor have we been able to demonstrate an effect of Geminin on either c-Myb or p300 transcriptional activity in reporter assays (data not shown). This suggests that Geminin controls MEP cell fate by a Myb-independent mechanism.

Geminin is not strictly required for the self renewal of long-term HSCs. Flow cytometry demonstrates that stem and progenitor cell numbers are preserved after Geminin deletion and Gmnn^Δ/Δ HSCs that are generated in a host animal continue to supply the blood with mature white cells and platelets for at least 6 months. Nevertheless, Gmnn^Δ/Δ HSCs may have a subtle stem cell defect. After 6 months, the number of wbc derived from the transplant was significantly less in animals that harbored Gmnn^Δ/Δ marrow cells compared with controls (Table 1). Furthermore, the Gmnn^Δ/Δ cells recovered from the marrow after 6 months are less efficient at forming colonies than the Gmnn^Δ/Δ cells isolated shortly after Mx1-Cre induction. In their unperturbed state, HSCs divide on average once every 57 days, so that it might take many months to exhaust the pool if there were a slight cell-cycle defect. We are now observing transplanted mice for longer periods of time to see whether Gmnn^Δ/Δ HSCs undergo senescence. A subtle deficiency in the HSCs may account for the observed engraftment defect in Gmnn^Δ/Δ cells, although we cannot rule out a homing defect.

Our model of how Geminin regulates hematopoiesis is summarized in Figure 6. One function of Geminin is to prevent overreplication of the DNA, but different types of hematopoietic cells might have differing requirements for Geminin depending on how fast they cycle, the efficiency of DNA repair pathways, and the effectiveness of redundant Geminin-independent mechanisms that prevent rereplication. The wbc precursors rely on Geminin to suppress overreplication, but under baseline conditions, they seem to tolerate the excess DNA. Stem and progenitor cells may also depend on Geminin to suppress overreplication. When stressed to divide rapidly by growth factors or after transplantation, cells with overreplicated DNA may fail to divide further or undergo apoptosis. Although we could not detect an overall increase in apoptotic cells in Gmnn^Δ/Δ mice, if most apoptosis occurs within the small population of stem and progenitor cells, it would have little effect on the overall number.

Figure 6

Model. Geminin is required for stress production of granulocytes and monocytes and amplification of HSCs after engraftment (thick red arrows). Geminin may also be required for proliferation of erythroblasts (small red arrows). Geminin is not required for self renewal of HSCs or baseline production of granulocytes or monocytes (black arrows). Geminin also influences the relative production of megakaryocytes and erythroid cells from MEPs (green arrows). MPP, multipotent progenitor; CLP, common lymphocyte progenitor.

A replication-based mechanism, however, cannot readily explain why the megakaryocyte population is so vastly expanded when Geminin is deleted. We hypothesize that Geminin has a second replication-independent function in controlling the relative production of erythroid cells and megakaryocytes from MEPs. Geminin has previously been shown to physically bind and regulate several different Homeobox (Hox) transcription factors, the SWI/SNF chromatin remodeling ATPase Brg1, and the Polycomb protein Scmh1 (13, 14). More recently, Geminin has been shown to inhibit the histone acetylase HBO1 (33). Many of these proteins are known to regulate hematopoiesis (34–37), and abnormal regulation of one or more of these factors may be responsible for the anemia and thrombocytosis seen in Gmnn^Δ/Δ mice. We are now conducting experiments to evaluate these possibilities.

View Supplemental data

ES cell electroporation and blastocyst injection were performed at the Transgenesis and Targeted Mutagenesis Facility at the Feinberg School of Medicine. Flow cytometry was performed at the Flow Cytometry Core Facility at the Robert H. Lurie Cancer Center. We thank Kelly Barry, Iwona Konieczna, Lisa Hurley, Gina Kirsammer, Marissa Suchyta, Ruben Lastra, Sol Misener, Jeremy Wen, and John Crispino for advice and technical assistance. T.J. McGarry was supported by grants from the National Heart, Lung, and Blood Institute, the Illinois Division of the American Cancer Society, and the American Heart Association.

Address correspondence to: Thomas J. McGarry, 303 E. Chicago Avenue, Tarry 14-721, Chicago, Illinois 60611, USA. Phone: 312.503.4386; Fax: 312.503.0137; E-mail: t-mcgarry@northwestern.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2010;120(12):4303–4315. doi:10.1172/JCI43556.

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