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The Journal of Immunology, 2008, 181, 5904 -5911
Copyright © 2008 by The American Association of Immunologists, Inc.
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A Quantitative Trait Locus on Chromosome 4 Affects Cycling of Hematopoietic Stem and Progenitor Cells through Regulation of TGF-β2 Responsiveness1

Serine Avagyan*, Ludmila Glouchkova{dagger}, Juhyun Choi{ddagger} and Hans-Willem Snoeck2,*

* Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029; {dagger} Institut für Zellbiologie, Universität Witten/Herdecke, Witten, Germany; and {ddagger} Department of Biological Sciences, Biomedical Research Center, Korea Advanced Institute of Science and Technology, Daejeon, South Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The hematopoietic stem and progenitor cell (HSPC) compartment is subject to extensive quantitative genetic variation. We have previously shown that TGF-β2 at low concentrations enhances flt3 ligand-induced growth of HSPCs, while it is potently antiproliferative at higher concentrations. This in vitro enhancing effect was subject to quantitative genetic variation, for which a quantitative trait locus (QTL) was tentatively mapped to chromosome 4 (chr.4). Tgfb2+/– mice have a smaller and more slowly cycling HSPC compartment, which has a decreased serial repopulation capacity, and are less susceptible to the lethal effect of high doses of 5-fluorouracil. To unequivocally demonstrate that these phenotypes can be attributed to the enhancing effect of TGF-β2 on HSPC proliferation observed in vitro and are therefore subject to mouse strain-dependent variation as well, we generated congenic mice where the telomeric region of chr.4 was introgressed from DBA/2 into C57BL/6 mice. In these mice, the enhancing effect of TGF-β2 on flt3 signaling, but not the generic antiproliferative effect of high concentrations of TGF-β2, was abrogated, confirming the location of this QTL, which we named tb2r1, on chr.4. These mice shared a smaller and more slowly cycling HSPC compartment and increased 5-fluorouracil resistance but not a decreased serial repopulation capacity with Tgfb2+/– mice. The concordance of phenotypes between Tgfb2+/– and congenic mice indicates that HSPC frequency and cycling are regulated by tb2r1, while an additional QTL in the telomeric region of chr.4 may regulate the serial repopulation capacity of hematopoietic stem cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The hematopoietic stem and progenitor cell (HSPC)3 compartment, which gives rise to all cells of the blood, is regulated by cell-intrinsic and -extrinsic mechanisms that endow the system with a capacity to maintain the steady-state production of multiple lineages of blood cells, each with widely differing lifespans, and to provide prompt replacement of these cells after hematopoietic stress (1, 2, 3, 4, 5).

A powerful approach to dissect the multiple pathways involved in the regulation of self-renewal, differentiation, and proliferation of HSPCs is the investigation of genetic variation in the HSPC compartment by quantitative trait analysis. Quantitative traits vary continuously across genetically different individuals and are inherited in a non-Mendelian fashion because of the contribution of multiple loci to the phenotype (6). These loci are called quantitative trait loci, or QTL. Several genes and pathways that show quantitative genetic variation in humans have been successfully modeled in mice (6). Importantly, many disease susceptibility QTL in mice could be translated to humans (7, 8, 9, 10, 11, 12). Therefore, the investigation of quantitative variation in the hematopoietic system of mice may not only lead to the identification of novel regulatory mechanisms, but it may also provide insight into susceptibility to hematological diseases and toxicity of drugs in the hematopoietic system.

Among inbred mouse strains there is extensive genetically determined variation in the function and kinetics of HSPCs (5, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Although multiple suggestive QTL have been mapped, for only one has the underlying gene been identified thus far, latexin, which is involved in the regulation of stem cell pool size (24). However, its mechanism of action is not yet understood. We have shown that signaling by the TGF-β isoform TGF-β2 plays a role in mouse strain-dependent variation in the HSPC compartment (25, 26). There are three isoforms of TGF-β (TGF-β1, -β2, and -β3), which are encoded on different chromosomes, but bind to the same receptors although with different binding mechanisms and relative affinities (27, 28, 29, 30). TGF-βs are potent inhibitors of HSPC proliferation in culture (31, 32, 33, 34), although studies in mice with conditional deletion of the type I TGF-β receptor questioned the importance of these observations, as these mice had normal steady-state hematopoiesis (35, 36). Our data suggest, however, that one of these isoforms, TGF-β2, is in fact a positive regulator of hemopoietic stem cells (HSCs) both in vitro and in vivo. In vitro, TGF-β2 has a biphasic dose response on the proliferation of purified HSPCs, defined as lineage–Sca1+c-kit+ or LSK cells (37). At low concentrations, the effect of this factor is stimulatory and requires factor(s) present in mouse and fetal calf sera (26), while at higher concentrations inhibition of proliferation occurs. This stimulatory effect of TGF-β2 is caused by a specific interaction with flt3 signaling, and not with any other early acting hematopoietic cytokine (S. Avagyan and H.-W. Snoeck, submitted for publication). Studies in mice with a heterozygous deletion of Tgfb2 (Tgfb2–/– mice die at birth) (38) revealed that the frequency of LSK cells as well as their cycling activity and the serial repopulating capacity of HSCs were lower than in wild-type (wt) littermates (25). Interestingly, while the stimulatory effect of exogenously added TGF-β2 on the proliferation of HSPCs was relatively subtle and only detected over a limited concentration range, Tgfb2+/– LSK cells showed a profound proliferation defect in response to flt3 ligand (flt3L; S. Avagyan and H.-W. Snoeck, submitted for publication) or to growth factor combinations containing flt3L (25). These data strongly suggest that the stimulatory effect of TGF-β2 is predominantly, although not exclusively, cell autonomous and is based on a specific enhancement of flt3 signaling. This effect of TGF-β2 on HSPCs is subject to genetically determined variation in inbred mouse strains. Using BXD recombinant inbred stains of mice a suggestive QTL for this trait was mapped to the telomeric region of chromosome 4 (chr.4) (25). This region also contains a QTL that contributes to genetic variation in the frequency of LSK cells (20). While it is probable that the phenotypes of Tgfb2+/– mice can be attributed to the enhancing effect of TGF-β2 on HSPC proliferation observed in vitro, and are therefore also likely subject to mouse strain-dependent variation, definitive proof of this contention requires the generation of mouse strains where specifically the proliferative effect of TGF-β2 was absent.

A classical strategy to demonstrate the veracity of a mapped QTL is the construction of congenic mice, where a chromosomal region from one inbred mouse strain is introgressed into the genetic background of a different mouse strain, leading to a change in the phenotype of the acceptor strain for this trait. In this context, we generated appropriate congenic mice, which allowed us to confirm the location of a QTL on chr.4 that regulates TGF-β2 responsiveness of HSPCs. Additionally, we show herein that these congenic mice shared several phenotypes with Tgfb2+/– mice, including a decreased responsiveness to flt3L, a more slowly cycling and smaller HSPC compartment, and decreased lethality from cell cycle-specific cytotoxic drug 5-fluorouracil (5-FU). Collectively, our findings strongly suggest that genetic variation in flt3 signaling caused by variation in isoform-specific TGF-β2 signaling mechanistically underlies these potentially clinically relevant quantitative traits. Another trait, decreased serial repopulation capacity of HSC, however, was not shared between congenic and Tgfb2+/– mice, suggesting the presence of distinct QTL in this region of chr.4 that are involved in the regulation of the repopulation capacity of HSC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Four- to 6-wk-old C57BL/6J mice were purchased from The Jackson Laboratory. B6.D2-chr.4 mice were generated through the Speed Congenic Service of The Jackson Laboratory, using 150 single nucleotide polymorphism (SNP) markers (39, 40). After the initial C57BL/6 and DBA/2 mating, 80–160 animals of each generation after the F1 crossbreeding were scanned using 108 Mit markers, five of which, d4mit155, d4mit37, d4mit251, d4mit234 and d4mit190, were on chr.4. Of these, 20–25 samples with the telomeric chr.4 genotype of DBA/2 were then scanned using 150 SNP markers to select the animals with the highest percentage of C57BL/6 in the remaining part of the genome and mated those to C57BL/6 mice to produce the next generation. After six generations (N6) animals were found to be 100% C57BL/6 for all SNP markers except in the telomeric 20 cM of chr.4, which was DBA/2. N6 animals were mated to produce homozygous DBA/2 chr.4 telomeric region at generation N6F1. The sex chromosomes were fixed via mating. Animals were housed in a specific pathogen-free facility. Experiments and animal care were performed in accordance with the Mount Sinai Institutional Animal Care and Use Committee.

Abs and cytokines

Unconjugated anti-CD2, -CD3{epsilon}, -CD8{alpha}, -CD4, -B220, -Gr-1, -Mac1, PE-conjugated anti-CD45.1, biotinylated anti-Thy1, and FITC-conjugated goat anti-rat Ab were purchased from SouthernBiotech. FITC-conjugated anti-CD2, -CD3{epsilon}, -CD8{alpha}, -CD4, -CD19, -B220, -Gr-1, and -Mac1, PE-conjugated anti-flt3, PE-Cy7-conjugated streptavidin, and allophycocyanin-Alexa Fluor 750-conjugated anti-c-kit were purchased from eBiosciences. Unconjugated anti-Ter119, biotinylated anti-Sca1 and -CD34, PE-conjugated anti-Sca1 and -CD34, PerCP-Cy5.5-conjugated streptavidin and anti-CD45.2, allophycocyanin-conjugated anti-c-kit and goat anti-rat Ab, PerCP-conjugated streptavidin, PE-Cy7-conjugated anti-CD19, allophycocyanin-Cy7-conjugated streptavidin, and anti-CD19 were purchased from BD Pharmingen. Pacific Blue-conjugated anti-Sca1 was purchased from BioLegend. Recombinant cytokines were purchased from R&D Systems. Recombinant human FLT3 ligand was received from Amgen.

Cell sorting and flow cytometry

Bone marrow (BM) cells were prepared by flushing the femora and tibia of mice with cold DMEM (Cellgro/Mediatech) containing 2% FBS (HyClone) and penicillin/streptomycin (Cellgro). Mononuclear cells were obtained after gradient centrifugation using lymphocyte separation medium (Cellgro). Low-density BM cells were stained with Abs for lineage Ags (CD2, CD3{epsilon}, CD8{alpha}, CD4, CD19, B220, Ter119, Gr-1, Mac1), Sca1, and c-kit and isolated by cell sorting, as shown in supplemental Fig. S1,4 using FACSVantage SE (BD Biosciences), Cytopeia (Advanced Cytometry Systems) or MoFlo (Dako) sorters, to obtain LSK cells. Flow cytometric analysis was performed on a three-laser LSRII or a special order five-laser LSRII with DiVa software (BD Biosciences). Data were analyzed using FlowJo software. Doublets were excluded by plotting with forward scatter pulse area vs side scatter both for sorting and FACS analysis.

Culture of LSK cells

Sorted LSK cells were cultured in triplicate at 20–60 cells per well in flat-bottom 96-well plates in StemPro34 medium (Invitrogen), 10% FCS (HyClone), and penicillin/streptomycin in the presence of various cytokines, as mentioned for each experiment. Within an hour after plating, the exact number of cells per well was determined by visually counting the cells at x200 magnification. After 5 days of liquid culture in a humidified incubator with 5% CO2 at 37°C the cells were again counted.

Cell cycle analysis of LSK cells

BM mononuclear cells were stained with lineage markers (CD2, CD3{epsilon}, CD8{alpha}, CD4, B220, Gr-1, Mac1, Ter119, unlabeled Abs), followed by goat anti-rat polyclonal IgG-FITC, PE-conjugated anti-Sca1, and allophycocyanin-conjugated anti-c-kit. The cells were fixed in 1% paraformaldehyde (Sigma-Aldrich) and 0.2% Nonidet P-40 (Sigma-Aldrich) for 1 h at 4°C. Fixed cells were washed in PBS and labeled with Hoechst 33342 (Sigma-Aldrich), final concentration of 5 µg/ml, for 1 h at 37°C. Analysis was performed on a triple laser LSRII flow cytometer with DiVa software (BD Biosciences). Double exclusion using pulse shape was done by plotting area vs height of the UV-excited Hoechst fluorescence. Cell cycle analysis was done using up to 3000 singlet LSK cells.

5-FU administration

5-FU (Sigma-Aldrich) was dissolved in PBS at concentration of 20 mg/ml for survival and of 11.25 mg/ml for recovery studies, and filtered through 0.2-µm SFCA (surfactant-free cellulose acetate) filters (Corning). The effect of sublethal doses of 5-FU (150 mg/kg, i.p.) was followed in six mice per experiment, of which three were bled alternately every other day. Blood counts were performed using a Beckman Coulter Ac · Tdiff automated hematology machine.

Competitive repopulation assays

LSK cells from donor mouse strain (C57BL/6 or B6.D2-chr.4, CD45.2+) were injected into lethally (700 cG followed by 500 cG 3 h later) irradiated recipient strains (C57BL/6, CD45.1+). Donor cells were mixed with 2 x 105 BM cells from CD45.1+CD45.2+ C57BL/6 mice in case of primary competitive repopulation transplantations. Peripheral blood cells were analyzed for the expression of CD45.1, CD45.2, and lineage Ags (Thy-1, Gr-1/Mac1, and CD19) 12 wk after transplantation. Competitive serial transplantations were performed at least 16 wk after reconstitution by injecting 2 x 106 BM cells from primary recipients into lethally irradiated secondary recipients (CD45.1+). Three secondary recipients were reconstituted with BM from one primary donor (n = 9 primary donors per donor strain). After 3 mo, the ratio between the competing cell populations was measured in the peripheral blood of the secondary recipients (n = 27 total recipients per donor strain). The data were presented as ratios between the two donor populations determined by the expression CD45 alleles. Changes in reconstitution ratios between primary and secondary were analyzed by evaluating the difference in the logarithm of the reconstitution ratios in the respective recipients. The reason for this strategy was described previously (25). Briefly, the change in the reconstitution ratio between primary and secondary recipients is a better measure of a shift in reconstitution capacity upon serial transplantation than a change in the percentage contribution of CD45.2+ cells between primary and secondary recipients, as the latter method does not weigh the same percentage change at high or low contribution levels equally (i.e., a 5% change from 20 to 25% is a change in reconstitution ratio from 0.25 to 0.33, whereas a 5% change from 90 to 95% represents a much larger change in reconstitution ratio from 9 to 19). A more accurate measure is the difference of CD45.2+(donor)/CD45.1+CD45.2+(competitor) ratios in primary recipients and secondary recipients. With ratiometric data, the difference between log(CD45.2+/CD45.1+CD45.2+) can be used, which added the advantage of log transformation where a ratio smaller than 1 will give a negative value, and negative ratios will extend over the same numerical ranges as positive ones (e.g., a ratio of 0.01 gives a log ratio of –2, a ratio of 100 gives a log ratio of 2). Thus, serial repopulation data are presented as the difference between log(CD45.2+/CD45.1+CD45.2+) of primary transplantation recipients and the log(CD45.2+/CD45.1+CD45.2+) of secondary transplantation recipients, and will be referred to as {Delta} log ratio.

Statistical analysis

Student’s two-tailed t test for paired samples was used in the calculation of all p-values unless indicated otherwise. Data represent means ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A locus on chr.4 regulates the stimulatory effect of TGF-β2 on HSPC growth, number and cycling, and 5-FU lethality

The isoform-specific proliferative effect of TGF-β2 on HSPCs is subject to genetically determined variation in inbred mouse strains, and a suggestive QTL for this trait was mapped to the telomeric region of chr.4 (25). To confirm the location of this QTL, we constructed congenic mice by introgressing 20 cM of the telomeric region of chr.4 from DBA/2 into C57BL/6 mice. Although there was wide variation in TGF-β2 responsiveness of HSPCs among various inbred mouse strains, the TGF-β2 dose response was similar in DBA/2 and C57BL/6 mice (26). Constructing congenic mice from these strains may therefore seem ill justified at first sight. However, this QTL was mapped using BXD recombinant inbred mice, which are derived from the allele pool of C57BL/6 and DBA/2 mice. In BXD recombinant inbred strains, where the genome is made up of a patchwork of segments that are homozygously inherited from either progenitor strain, significant variation in TGF-β2 responsiveness was observed (25, 26). This phenomenon is explained by the fact that if multiple loci are involved in determining a phenotype, some BXD strains can accumulate predominantly "high" or "low" alleles for this trait and acquire more extreme phenotypes than either of the progenitor strains. In other BXD strains and in the progenitor C57BL/6 and DBA/2 strains, the phenotypic effects of high and low alleles balance each other. These observations therefore proved that TGF-β2 responsiveness is a quantitative trait, and they justified generating congenic mice. As only one or a limited number of QTL are transferred from the DBA/2 to the C57BL/6 background in the congenic mice, we anticipated that if the QTL mapping was correct, the phenotype of the congenic mice and the background C57BL/6 strain would differ. Using C57BL/6 as the acceptor strain also facilitates analysis of in vivo transplantation assays by taking advantage of allelic variation at the CD45 locus to track donor and host contribution to hematopoiesis.

Construction of congenic mice was accomplished by repeated backcrossing of DBA/2 onto C57BL/6 mice and selecting offspring where the telomeric region of chr.4 was heterozygous (see Material and Methods). These congenic mice will be referred to as B6.D2-chr.4 mice hereafter. The transition between the C57BL/6 and DBA/2-derived genome occurred between D4Mit37 (57 cM) and D4Mit251 (66 cM) or between chromosome base pairs 04-114064127 and 04-135804867 (Fig. 1A). Any difference in the effect of TGF-β2 on the proliferation of HSPCs between B6.D2-chr.4 and C57BL/6 mice must be caused by one or more alleles in the introgressed region of chr.4. If this is the case and if the antiproliferative effect of higher concentration of TGF-β2 is similar in B6.D2-chr.4 and C57BL/6 mice, then any phenotype shared by B6.D2-chr.4 mice and Tgfb2+/– mice can with a very large likelihood be assigned to the stimulatory effect of low concentrations of TGF-β2 on HSPC proliferation. To test this, we cultured LSK cells (Fig. S1) from C57BL/6 and B6.D2-chr.4 mice in serum-containing media for 5 days in the presence of flt3L, kit ligand, thrombopoietin, and increasing concentrations of TGF-β2. While the dose response of exogenously added TGF-β2 on the proliferation of LSK cells from parental C57BL/6 mice was biphasic with a stimulatory effect at low concentrations, this stimulatory effect was absent in B6.D2-chr.4 LSK cells (Fig. 1B). Importantly, the inhibitory effect of higher concentrations of TGF-β2 (Fig. 1B) and the dose response of TGF-β1 (data not shown) on the proliferation of LSK cells were similar in B6.D2-chr.4 and C57BL/6 mice. We have shown that TGF-β2 specifically enhances flt3 signaling in HSPCs (submitted for publication). Similar to Tgfb2+/– mice, in the absence of any exogenously added TGF-β2, the response of LSK cells to increasing concentrations of flt3L was ~50% lower in B6.D2-chr.4 than in C57BL/6 mice (Fig. 1C). In contrast, the response to IL-3 was similar in both mice (Fig. 1D). Collectively, these data indicate that only the predominantly, athough not exclusively, cell-autonomous enhancing effect of TGF-β2 on flt3L responsiveness of HSPCs, and not the generic antiproliferative effect of TGF-β2, is regulated by a QTL on chr.4. These results in fact suggest that most genetic variation in the stimulatory effect of TGF-β2 is regulated by this QTL. As our observation provides unequivocal confirmation for the location of this QTL, we call this QTL tb2r1 (for TGF-β2 responsiveness 1).


Figure 1
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  		FIGURE 1. Tb2r1 is located in the telomeric region of chr.4. A, Schematic diagram of generation of B6.D2-chr.4 mice. White portion of chr.4 represents the 20-cM telomeric region of DBA/2 introgressed into C57BL/6. B, TGF-β2 responsiveness of C57BL/6 and B6.D2-chr.4 LSK cells in vitro, in the presence of flt3L, kit ligand, and thrombopoietin, each at 50 ng/ml (n = 7, *, p < 0.00005). C, B6.D2-chr.4 and C57BL/6 LSK cell proliferation in response to flt3L only in the presence of 10% serum (n = 5, *, p < 0.02). D, B6.D2-chr.4 and C57BL/6 LSK cell proliferation in response to IL-3 only in the presence of 10% serum (n = 2, p > 0.3); 100% response is defined as that of C57BL/6 LSK cells at 50 ng/ml of flt3L or IL-3.

 
As Tgfb2+/– mice display a hematopoietic defect, we further analyzed the hematopoietic phenotype of the B6.D2-chr.4 mice. We had previously mapped a QTL regulating the frequency of LSK cells to the telomeric region of chr.4 (20). Consistent with these mapping data, the frequency of LSK cells was significantly lower in B6.D2-chr.4 than in C57BL/6 mice (Fig. 2A, Table I), confirming that the same region on chr.4 also contains a QTL regulating LSK frequency. This finding cannot be explained by intrinsic variation in c-kit expression, as the overall c-kit expression level in the BM was similar in C57BL/6 mice and congenics. The frequency of a subpopulation of LSK cells containing the more primitive stem cells, the CD34–flt3–LSK population, was also decreased in B6.D2-chr.4 mice BM (Fig. 2B). BM cellularity, peripheral blood counts, and spleen weight were similar in B6.D2-chr.4 and C57BL/6 mice (Table I). Tgfb2+/– mice have a more slowly cycling HSPC compartment than do wt littermates (25). Therefore, we examined cell cycle activity of LSK cells in B6.D2-chr.4 mice. Similar to Tgfb2+/– mice, fewer LSK cells in B6.D2-chr.4 mice than in control C57BL/6 mice were in cell cycle, as measured by staining fixed BM cells with DNA-binding dye Hoechst 33342 (Fig. 2C), while the fraction of S/G2/M phase cells in the total BM cell population did not differ between B6.D2-chr.4 and C57BL/6 mice (data not shown). We hypothesized that a slower cycling HSPC compartment may render Tgfb2+/– and B6.D2-chr.4 mice more resistant to the effects of cell cycle-specific cytotoxic agents, such as 5-FU. The lethal effects of this drug are due to hematopoietic failure as mice are rescued from high doses of 5-FU by BM transplantation (41). Consistent with this notion, the survival of both Tgfb2+/– and B6.D2-chr.4 mice after an i.v. injection of a highly lethal dose of 5-FU (500 mg/kg, i.v.) was better than that of control mice (p < 0.0001 and p = 0.0001, respectively; Fig. 3A). However, recovery from a sublethal dose of 5-FU was similar in B6.D2-chr.4 and C57BL/6 mice (Fig. 3B) and in wt and Tgfb2+/– mice (Fig. 3C). We conclude that B6.D2-chr.4 mice and Tgfb2+/– mice share several hematopoietic phenotypes, including the frequency and cycling activity of HSPCs in vivo, and their response to flt3L in vitro. Furthermore, both mouse strains show enhanced resistance to the lethal effects of 5-FU. These observations suggest that these phenotypes of B6.D2-chr.4 mice are likely due to regulation of TGF-β2 responsiveness by tb2r1.


Figure 2
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  		FIGURE 2. B6.D2-chr.4 mice have a smaller and slower cycling HSPC compartment. A, Representative flow cytometric analysis of LSK frequency in C57BL/6 and B6.D2-chr.4 mice. B, Frequencies of CD34–flt3–LSK, CD34+flt3–LSK, and CD34+flt3+LSK cells in BM of C57BL/6 and B6.D2-chr.4 mice (n = 8, *, p < 0.001). C, Fraction of LSK cells in S/G2/M phase of cell cycle in C57BL/6 and B6.D2-chr.4 mice as determined by Hoechst 33342 staining of fixed BM cells (n = 4, p = 0.039). Lines connect data obtained from each individual experiment, where two or three mice of each genotype were pooled. p-value calculated using two-tailed Student’s t test for paired experiments.

 

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  		Table I. Hematologic parameters in C57BL/6 and B6.D2-chr.4 micea

 

Figure 3
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  		FIGURE 3. Decreased 5-FU lethality in B6.D2-chr.4 and Tgfb2+/– mice. A, Analyses of the survival of C57BL/6 and B6.D2-chr.4 mice (n = 26 per group, p = 0.0001) and of Tgfb2+/– and wt littermate mice (n = 20 and n = 23, respectively, p < 0.0001) after administration of 5-FU (500 mg/kg, i.v.). p-values calculated using Kaplan-Meier statistics. B, Peripheral leukocyte (left panel) and platelet (right panel) counts after injection of a sublethal dose of 5-FU (150 mg/kg, i.p.) in C57BL/6 and B6.D2-chr.4 mice (n = 3 for each data point, p > 0.1). C, Peripheral leukocyte (left panel) and platelet (right panel) counts after injection of a sublethal dose of 5-FU (150 mg/kg, i.p.) in Tgfb2+/– mice and wt littermates (n = 3 for each data point, p > 0.1).

 
Decreased serial reconstitution capacity is not shared between B6.D2-chr.4 and Tgfb2+/– mice

HSC from Tgfb2+/– mice show a defect in serial repopulation capacity compared with HSC from wt mice (25). Therefore, we tested the serial competitive repopulation capacity of LSK cells from B6.D2-chr.4 mice. Five hundred purified CD45.2+ LSK cells from C57BL/6 or B6.D2-chr.4 mice were competed with 2 x 105 CD45.1+CD45.2+ C57BL/6 BM cells in lethally irradiated CD45.1+ C57BL/6 recipients. In the primary transplantation, LSK cells from B6.D2-chr.4 mice competed equally well compared with LSK cells from C57BL/6 mice as determined by the donor CD45.2+ contribution to PBMC after 12 wk (p > 0.1; Fig. 4A). This was not surprising, as the content of most primitive CD34–flt3– stem cell fraction was the same in the LSK population of C57BL/6 and B6.D2-chr.4 mice (Fig. S2). For serial transplantation, we injected 2 x 106 BM cells from competitively repopulated primary recipients into lethally irradiated secondary CD45.1+ recipients, 4 mo after the primary transplantation. Twelve weeks later, the contribution of CD45.2+ and CD45.1+CD45.2+ cells to hematopoiesis in peripheral blood was assessed in secondary recipients. The data are presented as the logarithm of the ratio of the two donor populations determined by the expression CD45 alleles. The difference in the log reconstitution ratios ({Delta} log ratio; see Materials and Methods) between primary and secondary recipients was significantly higher for LSK cells from B6.D2-chr.4 mice than for LSK cells from C57BL/6 mice (Fig. 4B). These data indicate that HSC from B6.D2-chr.4 mice performed significantly better than HSC from C57BL/6 mice in serial transplantation assays. This phenotype of B6.D2-chr.4 mice is not shared with Tgfb2+/– mice, which show a subtle defect in competitive repopulation capacity that increases with serial transplantation.


Figure 4
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  		FIGURE 4. Serial competitive reconstitution capacity of HSCs from B6.D2-chr.4 and C57BL/6 mice. A, Log ratio of donor/competitor contribution in primary and secondary transplantations of C57BL/6 and B6.D2-chr.4 LSK cells (donor, CD45.2+) and C57BL/6 competitor BM cells (CD45.1+CD45.2+) into lethally irradiated recipients (CD45.1+ C57BL/6) (n = 5, p > 0.1, primary transplantation; n = 9, p = 0.019, secondary transplantation). p-values calculated using Mann-Whitney U test. B, The difference in the log ratio, {Delta} log ratio, of reconstitution ratios between secondary and primary transplantations for recipients of BM from primary recipients repopulated with LSK cells from C57BL/6 or B6.D2-chr.4 mice (n = 9, p = 0.042). p-value calculated using two-tailed Student’s t test for unpaired samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show herein that a strong QTL in the telomeric region of chr.4, tb2r1, regulates the enhancing effect of TGF-β2 on HSPC proliferation in vitro. We have shown that this effect of TGF-β2 is caused by a specific enhancement of flt3 signaling (submitted for publication). As in B6.D2-chr.4 mice, the positive regulatory effect of TGF-β2 is absent while the antiproliferative effect of high concentrations of TGF-β2 was similar to C57BL/6 mice, and any traits shared between B6.D2-chr.4 and Tgfb2+/– mice are most likely caused by a defect in TGF-β2-mediated enhancement of flt3 signaling. These traits are the frequency and cycling activity of HSPCs in vivo and 5-FU lethality. Alternatively, it is possible, although unlikely, that these hematopoietic phenotypes of B6.D2-chr.4 mice are not caused by a defect in TGF-β2 signaling, although they must be caused by reduced TGF-β2 signaling in Tgfb2+/– mice. In that case, this narrow region of chr.4 contains a remarkable number of QTL that independently affect hematopoiesis.

The lower cycling activity of HSPCs in both Tgfb2+/– and B6.D2-chr.4 mice compared with control mice is likely linked to the decreased lethality from 5-FU in these mouse strains, as it has been shown that mice die of hematopoietic failure after high doses of 5-FU (41). As the cycling activity of total BM was similar in Tgfb2+/– and B6-D2-chr.4 mice compared with their appropriate control groups, our data suggest that the lethality from high doses of 5-FU is due to loss of the more primitive cells in the HSPC compartment. On the other hand, recovery from a sublethal dose, which did not appear to be subject to genetically determined variation in the mouse strains tested here, is likely dependent on more mature progenitors that are less affected by TGF-β2 signaling. The similar rate of recovery from sublethal 5-FU dose in C57BL/6, Tgfb2+/–, and B6.D2-chr.4 mice also indicates that 5-FU metabolism is similar in these mouse strains. It cannot be fully excluded that additional QTL in the introgressed region of chr.4 regulated lethality from 5-FU, for example by affecting intestinal toxicity. A QTL regulating lethality from 5-FU is clinically important, however. As genes and pathways that show quantitative genetic variation in mice often also do so in humans (6, 7, 8, 9, 10, 11, 12), it is possible that similar variation exists in humans, and that the underlying mechanism is the same.

The only phenotype not shared between Tgfb2+/– and congenic mice was the serial repopulating capacity of HSC, which was enhanced in the latter but decreased in the former. Traits not shared between B6.D2-chr.4 mice, where at least 174 alleles of genes with known protein product might be distinct (Table SI), and Tgfb2+/– mice, where only one allele of one gene is deleted, may be caused by additional QTL in the introgressed region. One explanation for this observation may therefore be that a separate QTL on chr.4 is responsible for the enhanced serial repopulation capacity of HSC from B6.D2-chr.4 mice. In particular, a stronger QTL in this region may override the effect of tb2r1 in regulating serial repopulation capacity. However, as in B6.D2-chr.4 mice, only the stimulatory TGF-β2 signaling is compromised, while in Tgfb2+/– mice both stimulatory and inhibitory TGF-β2 signaling are equally affected. Another explanation for this discrepancy may be that the defect in serial repopulation capacity of Tgfb2+/– HSC is due to the lower level of antiproliferative TGF-β2 signaling in these mice, which might lead to accelerated exhaustion of HSC. Only gene identification of tb2r1, for which the generation and analysis of the congenic mice described herein is a critical step, will shed light on this issue.

TGF-β2 responsiveness of LSK cells was similar in DBA/2 and C57BL/6 mice. It may therefore seem unexpected that in congenic mice, where a region of chr.4 was introgressed from DBA/2 to C57BL/6, responsiveness to the flt3-specific proliferative effect of TGF-β2 was abrogated. However, TGF-β2 responsiveness is a quantitative trait, and, as discussed previously (see Results), there is likely an unbalanced contribution of high and low responsiveness alleles in B6.D2-chr.4 mice. Additionally, the effect of certain alleles can depend on the genetic background, leading to unexpected epistatic interactions (42). Hence, the isolation of a DBA/2 allele on a C57BL/6 background may alter the balance between high and low alleles and/or change allele-specific epistatic interactions, leading to the observed near complete abrogation of the phenotype in B6.D2-chr.4 mice (42). The phenotype of the B6.D2-chr.4 mice was still somewhat unexpected, however, as according to our quantitative trait analysis DBA/2 mice carried the high allele for LSK frequency and TGF-β2 responsiveness on chr.4 (26). Consequently, a higher frequency of LSK cells and a higher responsiveness to TGF-β2 were anticipated in B6.D2-chr.4 mice. Surprisingly, exactly the opposite was observed. Two explanations are possible. One possibility is that the telomeric region of chr.4 contains more than one QTL with opposing effects, and that the introgressed 20 cM contains only one of those, with the other one(s) lying just outside of the terminal 20 cM of chr.4. Another possibility is that the region only contains one QTL, but, because of epistatic interactions with other genes, has a different effect on a C57BL/6 than on a DBA/2 background. The first scenario, implying multiple QTL within the same region, is surprisingly common (43), while evidence in plants supporting the plausibility of the second explanation has been published recently (44).

Identifying the gene underlying tb2r1 is a critical step toward undestanding the mechanism of the effect of TGF-β2 on HSPCs. There are more than 170 known gene products in the introgressed portion of chr.4 in B6.D2-chr.4 mice (Table SI). Some of these, such as Ski (45) and Prdm16 (46), have been described to play a role in TGF-β signaling via their interaction with Smad4 and Smad3, respectively. Others, like Tnfrsf9 (47) and Tnfrsf14 (48), have been implicated in hematopoiesis by knockout mouse models. However, speculating on potential candidates could be misleading as the gene underlying QTL can be very surprising. For example, latexin, regulating HSC pool size in mice (24), is an inhibitor of metalocarboxypeptidases in neurons. The mechanism of its role in the biology of HSC is still unknown. HPE, the hereditary hemochromatosis protein in humans, is highly homologous to MHC class I proteins but has no immunological function (49) and is important in iron intake regulation (50). Hence, we have adopted an unbiased approach to achieve gene identification through gene expression profiling in C57BL/6 and B6.D2-chr.4 mice, and through positional cloning using fine-congenic lines with smaller introgressed regions that have been generated from B6.D2-chr.4 mice.

In conclusion, we have unequivocally shown that responsiveness of HSPCs to the proliferative effect of TGF-β2 is regulated by the QTL tb2r1 on chr.4. Given the concordance of phenotypes between Tgfb2+/– and B6.D2-chr.4 mice, our observations indicate that frequency and cycling of HSPCs, in addition to lethality from high doses of 5-FU, are also quantitative traits regulated by tb2r1. Finally, an additional QTL in the telomeric region of chr.4 may regulate the serial repopulation capacity of HSCs.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01 AG016327 and HL073760. Back

S.A. performed most of the experiments and wrote the manuscript. L.G. performed the initial analysis of B6.D2-chr.4 mice. J.C. performed the 5-FU survival studies on Tgfb2/– mice. H.W.S. designed and supervised the experiments and co-wrote the manuscript with S.A.

2 Address correspondence and reprint requests to Dr. Hans-Willem Snoeck, Department of Gene and Cell Medicine, Mount Sinai of School of Medicine, Gustave L. Levy Place, Box 1496, New York, NY 10029. E-mail address: hans.snoeck{at}mssm.edu Back

3 Abbreviations used in this paper: HSPC, hematopoietic stem and progenitor cell; BM, bone marrow; chr.4, chromosome 4; 5-FU, 5-fluorouracil; flt3L, flt3 ligand; HSC, hematopoietic stem cell; LSK, lineage–Sca1+c-kit+; QTL, quantitative trait loci; SNP, single nucleotide polymorphism; wt, wild type. Back

4 The online version of this article contains supplemental material. Back

Received for publication May 30, 2008. Accepted for publication August 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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