The Rb tumor suppressor is required for stress erythropoiesis
2004; Springer Nature; Volume: 23; Issue: 21 Linguagem: Inglês
10.1038/sj.emboj.7600432
ISSN1460-2075
AutoresBenjamin T. Spike, Alexandra Dirlam, Benjamin Dibling, James Marvin, Bart O. Williams, Tyler Jacks, Kay F. Macleod,
Tópico(s)Cell death mechanisms and regulation
ResumoArticle30 September 2004free access The Rb tumor suppressor is required for stress erythropoiesis Benjamin T Spike Benjamin T Spike The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA The Committee on Cancer Biology, University of Chicago, Chicago, IL, USA Search for more papers by this author Alexandra Dirlam Alexandra Dirlam The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA The Committee on Immunology, University of Chicago, Chicago, IL, USA Search for more papers by this author Benjamin C Dibling Benjamin C Dibling The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author James Marvin James Marvin The Flow Cytometry Laboratory, University of Chicago, Chicago, IL, USA Search for more papers by this author Bart O Williams Bart O Williams Van Andel Research Institute, Grand Rapids, MI, USA Search for more papers by this author Tyler Jacks Tyler Jacks The Department of Biology & Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA The Howard Hughes Medical Institutes, Chevy Chase, MD, USA Search for more papers by this author Kay F Macleod Corresponding Author Kay F Macleod The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA The Committee on Cancer Biology, University of Chicago, Chicago, IL, USA The Committee on Immunology, University of Chicago, Chicago, IL, USA Search for more papers by this author Benjamin T Spike Benjamin T Spike The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA The Committee on Cancer Biology, University of Chicago, Chicago, IL, USA Search for more papers by this author Alexandra Dirlam Alexandra Dirlam The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA The Committee on Immunology, University of Chicago, Chicago, IL, USA Search for more papers by this author Benjamin C Dibling Benjamin C Dibling The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA Search for more papers by this author James Marvin James Marvin The Flow Cytometry Laboratory, University of Chicago, Chicago, IL, USA Search for more papers by this author Bart O Williams Bart O Williams Van Andel Research Institute, Grand Rapids, MI, USA Search for more papers by this author Tyler Jacks Tyler Jacks The Department of Biology & Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA The Howard Hughes Medical Institutes, Chevy Chase, MD, USA Search for more papers by this author Kay F Macleod Corresponding Author Kay F Macleod The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA The Committee on Cancer Biology, University of Chicago, Chicago, IL, USA The Committee on Immunology, University of Chicago, Chicago, IL, USA Search for more papers by this author Author Information Benjamin T Spike1,2, Alexandra Dirlam1,3, Benjamin C Dibling1, James Marvin4, Bart O Williams5, Tyler Jacks6,7 and Kay F Macleod 1,2,3 1The Ben May Institute for Cancer Research, The University of Chicago, Chicago, IL, USA 2The Committee on Cancer Biology, University of Chicago, Chicago, IL, USA 3The Committee on Immunology, University of Chicago, Chicago, IL, USA 4The Flow Cytometry Laboratory, University of Chicago, Chicago, IL, USA 5Van Andel Research Institute, Grand Rapids, MI, USA 6The Department of Biology & Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA 7The Howard Hughes Medical Institutes, Chevy Chase, MD, USA *Corresponding author. The Ben May Institute for Cancer Research, The University of Chicago, The Knapp Medical Research Building, R118, 924 East 57th Street, Chicago, IL 60637, USA. Tel.: +1 773 834 8309; Fax: +1 773 702 3701; E-mail: [email protected] The EMBO Journal (2004)23:4319-4329https://doi.org/10.1038/sj.emboj.7600432 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases. Introduction The retinoblastoma tumor suppressor gene (Rb) is essential for normal mouse development (Clarke et al, 1992; Jacks et al, 1992; Lee et al, 1992) and Rb null mice die around E14.5 of gestation exhibiting defects in lens, placental, muscle, hematopoietic and nervous system development (Morgenbesser et al, 1994; Macleod et al, 1996; Zacksenhaus et al, 1996; Wu et al, 2003). The hematopoietic defect in Rb null mice is the least well characterized of these developmental phenotypes and is marked by a failure to produce enucleated red blood cells (Clarke et al, 1992; Jacks et al, 1992; Lee et al, 1992). Abnormalities in Rb null erythropoiesis have been attributed to non-cell autonomous effects and to defects in paracrine signaling (Whyatt and Grosveld, 2002). In particular, Rb-deficient cells were able to contribute normally to the peripheral blood of adult Rb null chimeric mice (composed of wild-type and Rb−/− cells) (Robanus-Maandag et al, 1994; Williams et al, 1994). Although these studies did not determine the relative contribution of Rb−/− cells to the bone marrow, or to specific lineages and stages of differentiation, they suggested that the red cell differentiation defects observed in fetal liver (FL) (low red cell count and a failure to enucleate) could be overcome by the presence of wild-type cells (Robanus-Maandag et al, 1994; Williams et al, 1994). Recent data have suggested that reduced nutrient exchange between maternal and fetal circulations, due to abnormal placental development, may explain non-cell autonomous defects in the Rb null mouse (Wu et al, 2003). Certainly, apoptosis in the developing nervous system of Rb null embryos appears to be the consequence of hypoxia, as shown recently when Rb loss was conditionally targeted to the nervous system (Ferguson et al, 2002; Macpherson et al, 2003). However, it is still not clear to what extent erythroid defects in Rb null mice contribute to non-cell autonomous defects in other tissues or whether aberrant placental function plays a causative role in red cell defects. To determine the exact nature of the red cell defect and the extent to which such defects were cell intrinsic, we examined the effects of Rb loss on erythropoiesis in vitro and in vivo. Our results showed that loss of Rb led to a block to erythroid differentiation, which is attributable to a unique cell intrinsic defect in erythroblast expansion and red cell enucleation under stress conditions. Results Erythroid defects in the Rb null fetal liver Erythropoiesis occurs in the FL between E11.5 and E16.5 of mouse embryonic development (Godin and Cumano, 2002). Erythropoiesis can be assessed in primary tissues by quantitating expression of c-Kit, CD71 and TER119 during the differentiation of erythroblasts to erythrocytes (Kina et al, 2000; Socolovsky et al, 2001). When we examined the profile of cKit, CD71 and TER119 expression in E12.5 FL, we observed increased relative numbers of cKit-expressing cells and fewer TER119hi-expressing cells in Rb null FL (Figure 1B, D and F) compared to wild-type littermate FL (Figure 1A, C and E). Specifically, there was a marked increase in the percentage of cKit+CD71+ cells (21.0%, Figure 1B), CD71+TER119− cells (43.5%, Figure 1D) and cKit+TER119− cells (34.5%, Figure 1F) compared to wild type (9.4%, Figure 1A; 14.5%, Figure 1C; 13.3%, Figure 1E), and a decreased percentage of cKit−CD71+ (68.2%, Figure 1B), CD71+TER119+ (34.7%, Figure 1D) and cKit−TER119hi (30.6%, Figure 1F) relative to wild type (83.8%, Figure 1A; 74.2%, Figure 1C; 69.4%, Figure 1E). These results demonstrated that loss of Rb resulted in a block to fetal erythropoiesis as cells became cKit− and TER119hi. Figure 1.Defective erythroid differentiation in Rb null FL and in vitro. FACS analysis (10 000 events) of E12.5 wild-type (A, C, E) and Rb−/− (B, D, F) FL labeled with fluorescent antibodies to CD71 and cKit (A, B), CD71 and TER119 (C, D) and cKit and TER119 (E, F). Less compact nuclei, disorganized chromatin structure and decreased enucleation in red cells of Rb null E13.5 embryos (H) compared to wild type (G). Day 2 CFU-Es are shown from wild-type (I) and Rb null (J) E12.5 FL. Cytospin of cells from day 2 CFU-Es, wild-type (K) or Rb null (L) E12.5 FL. Download figure Download PowerPoint FL erythropoiesis in Rb null mice is marked by reduced red blood cell count, an increased proportion of which are nucleated (Clarke et al, 1992; Jacks et al, 1992). Closer inspection revealed a failure of Rb null red cell nuclei to compact and significant numbers of erythroblasts with condensed chromosomes in their benzidine-positive cytoplasm (Figure 1H) compared to wild type (Figure 1G). Similar defects in nuclear condensation and enucleation were observed in Rb null erythroblasts derived from in vitro colony assays (Figure 1L). When cKit+CD71+ erythroid progenitors from either wild-type or Rb−/− E12.5 FL were plated in semisolid media, they gave rise to similar numbers of CFU-E erythroid colonies by 2 days in culture. However, Rb null CFU-Es (Figure 1J) were qualitatively different from wild-type CFU-Es (Figure 1I). Rb−/− CFU-Es were more varied in size, were physically disorganized and displayed irregular shape (Figure 1J), in contrast to wild-type CFU-Es (Figure 1I). When erythroblasts from day 2 CFU-Es were examined cytologically, we observed high levels of red cell enucleation in wild-type CFU-E cultures (Figure 1K). In contrast, Rb null CFU-E-derived erythroblasts were deficient for enucleation (Figure 1L). Furthermore, Rb null erythroblasts were increased in size, showed increased nuclear to cytoplasmic ratio, and condensed chromosomes were visible in many cells (Figure 1L) reminiscent of observations in vivo (Figure 1H). These observations suggest that in vitro erythroid differentiation recapitulates the defects observed in vivo. Rb null erythroblasts exhibit defects in cell cycle exit during terminal differentiation To determine whether Rb null erythroblasts undergo normal cell cycle exit during terminal differentiation, disaggregated wild-type and Rb null E12.5 FL cells were labeled with antibodies to cKit, CD71 and TER119 and sorted by FACS. Purified populations were fixed, stained with propidium iodide (PI) and analyzed for DNA content as a measure of cell cycle phase distribution. As shown in Figure 2A, we observed three distinct populations when we examined the TER119 expression profile of cKit+CD71+ E12.5 wild-type FL erythroblasts (red profile): TER119neg (39.0%), TER119lo (36.0%) and TER119hi (24.7%). In Rb null FL (blue profile), TER119hi cells made up only 10.7% of cKit+CD71+ erythroblasts and Rb null cKit+CD71+ erythroblasts accumulated as TER119lo cells (66%, Figure 2A, blue profile). These data suggest that Rb null cells are defective for the end stages of erythroid differentiation including upregulation of TER119 from TER119lo to TER119hi that normally accompanies cell cycle exit (Kina et al, 2000). Figure 2.Rb null erythroblasts show deregulated cell cycle. Wild-type (red) and Rb−/− (blue) E12.5 cKit+CD71+ erythroblasts can be subdivided into TER119neg, TER119lo and TER119hi populations (A, 10 000 events). PI staining of TER119lo- (B) and TER119hi- (C) expressing cells from E12.5 wild-type (red profile) or Rb null FL (blue profile) revealing % cells in each population with G0/G1 (2N), S (2N–4N) or G2/M (4N) DNA content. Immunohistochemical staining of wild-type (D) and Rb−/− (E) FL for PH3 expression. GPI analysis of contribution by Rb null cells to the TER119neg, TER119lo and TER119hi populations in an E12.5 chimeric mouse embryo (F). FACS profile of TER119lo E12.5 chimeric (bold profile) and E12.5 wild-type control FL (gray profile) stained with CMFDG to distinguish β-galactosidase-expressing Rb−/− TER119lo erythroblasts (CMFDG+) from nonexpressing wild-type TER119lo erythroblasts (CMFDG−) (G). FACS analysis of CMFDG− (red) and CMFDG+ (blue) cells labeled with Draq5 to assess DNA content as a measure of cell cycle profile (H). Download figure Download PowerPoint When we examined the cell cycle of E12.5 cKit+CD71+ erythroblasts, we observed that wild-type cells accumulated in G0/G1 with a 2N DNA content as they passed from the TER119lo state of differentiation (Figure 2B, red profile, 37.8%) to TER119hi (Figure 2C, red profile, 41.4%), whereas Rb null erythroblasts retained an abnormally high 4N DNA content (33.9% as TER119lo; 33.5% as TER119hi), consistent with accumulation in the G2/M phases of cell cycle (Figure 2C, blue profile). As shown in Figure 2A, very few Rb null erythroblasts had made the transition from TER119lo to TER119hi, consistent with an arrest of Rb null erythroblasts in G2/M at the TER119lo stage of erythroid maturation. Consistent with arrest in M phase, we detected substantially increased numbers of cells expressing high levels of phosphohistone H3 (PH3) in the Rb null FL (Figure 2E) compared to wild-type FL (Figure 2D). These results suggested that pRb was required to ensure proper cell cycle arrest of FL erythroblasts prior to, or coincident with, upregulation of TER119 expression. Loss of Rb resulted in mitotic arrest, a failure to upregulate TER119 and to enucleate. Differentiation defects in Rb null red cells are cell intrinsic To determine whether defects in cell cycle control during end-stage Rb null erythroid differentiation were cell intrinsic or not, we examined Rb null erythroblasts from E12.5 FL derived from chimeric mouse embryos generated by injection of wild-type blastocysts with Rb null-LacZ+(ROSA 26) transgenic ES cells (Lipinski et al, 2001). TER119neg-, TER119lo- and TER119hi-expressing erythroblasts were sorted from chimeric FLs at E12.5 and glucose phosphate isomerase (GPI) assays were conducted to determine the relative contribution of Rb null cells to each distinct erythroid population. As shown in Figure 2G in a representative GPI assay, the contribution of Rb null cells to the TER119lo population (lane 2, 51.6%) was greater than the contribution to the TER119neg population (lane 1, 18.9%), while the contribution to the TER119hi end-stage erythroid population (lane 3, 1.1%) was negligible. These percentages reflect the data shown in Figure 2A indicating that even in chimeric FL with comparatively low overall chimerism, Rb null erythroblasts show defects in end-stage erythroid maturation to TER119hi status and accumulate as TER119lo-expressing cells. We compared the cell cycle status of Rb null TER119lo erythroblasts (5-chloromethylfluoroscein di-β-galactosipyranoside (CMFDG)-positive, Figure 2H) with wild-type (CMFDG-negative, Figure 2H) TER119lo erythroblasts from the same E12.5 chimeric FL. As shown in Figure 2I, increased numbers of Rb null (75.3%, CMFDG+, blue profile) erythroblasts possessed a greater than 2N DNA content compared to wild type (66.1%, CMFDG−, red profile), suggesting that Rb null TER119lo erythroblasts in the context of a low chimeric FL demonstrated similar cell cycle defects to that seen in the homozygous Rb mutant FL at E12.5 (Figure 2B and C), namely a G2/M arrest. These observations suggested that the erythroid cell cycle defect observed in the Rb null FL was cell intrinsic and independent of the placental defect since extraembryonic tissues in chimeric mice are blastocyst derived. Furthermore, the presence of wild-type cells was unable to confer the ability on Rb null erythroblasts to exit cell cycle or to upregulate TER119, suggesting that the erythroid defect in Rb null chimeric embryos was cell intrinsic. To further explore the cell intrinsic nature of red cell defects in Rb null erythropoiesis, we examined the effects of acute deletion of Rb from erythroblasts in vitro. E12.5 FL erythroblasts were recovered from Rbflox/flox embryos (Marino et al, 2000), infected with either control retrovirus (pMSCV-IRES-GFP) or with Cre recombinase-expressing retrovirus (pMSCV-Cre-IRES-GFP) and then cultured for 2 days in the presence of dexamethasone, Kit ligand and erythropoietin to promote proliferation of erythroid progenitors (Bauer et al, 1999). GFP-positive TER119lo erythroblasts were FACS sorted for control cells (Figure 1A) and Cre-expressing cells (Figure 1B). Sorted cells were analyzed by PCR for the floxed allele to confirm that Cre-mediated deletion had taken place successfully in the pMSCV-Cre-IRES-GFP-infected cells (Figure 3C). Efficient (100%) deletion of the floxed allele was measured by reduction of the 700 bp PCR product (lanes 2 and 3) to a 260 bp PCR product (lanes 4 and 5) in the Cre-expressing cells (lanes 4 and 5) but not in the control virus-infected cells (lanes 2 and 3). Figure 3.Acute inactivation of pRb in cultured erythroblasts. FACS sort profile of E12.5 FL erythroblasts harvested from Rbflox/flox mice, infected in vitro with control pMSCV-IRES-GFP retrovirus (A) or with Cre recombinase-expressing pMSCV-Cre-IRES-GFP (B) and sorted as GFP-positive/TER119neg- or GFP-positive/TER119lo-expressing cells. PCR analysis (C) of DNA harvested from sorted GFP-positive/TER119neg- (lanes 3 and 5) or GFP-positive/TER119lo- (lanes 2 and 4) expressing cells from either control virus-infected (lanes 2 and 3) or Cre-expressing virus-infected cells (lanes 4 and 5). Excision of floxed exon 19 reduces the size of the Rb PCR product in Rbflox/flox erythroblasts from 700 to 260 bp in erythroblasts infected with Cre-expressing retrovirus (lanes 4 and 5) but not with control retrovirus (lanes 2 and 3). PCR analysis of DNA from E13.5 Rb−/flox FL carrying an Rb null allele (650 bp) and a floxed exon 19 allele (700 bp) (lane 7) and following in vivo excision of the floxed exon 19 on the Meox2-Cre transgenic background generates a 260 bp fragment (lane 6). Peripheral red blood cells from Meox2-Cre/Rb−/exon 19flox E13.5 embryos show abnormal nuclear structure and reduced enucleation (D, arrows). FACS profile of cultured TER119lo/GFP+ erythroblasts following Cre-mediated excision of exon 19 (F) or control cultures (E) labeled with Draq5. Cytospin analysis of TER119lo/GFP+ erythroblasts following 2 days in culture showing extensive red cell enucleation (G, arrows) in the control cultures but not in the Cre-expressing cultures (H). Download figure Download PowerPoint After 2 days in differentiation medium, Rbflox/flox erythroblasts in which exon 19 had been acutely deleted (RbΔexon19) showed a marked decrease in enucleation rate (28.2%, Figure 3F) compared to control Rbflox/flox erythroblasts (44.5%, Figure 3E). This is supported by cytological analysis showing extensive enucleation in control cultures (Figure 3G, arrows) but not in RbΔexon19 erythroblasts (Figure 3H). RbΔexon19 erythroblasts also showed increased cell size and nuclear:cytoplasmic ratio (Figure 3H) compared to control erythroblasts (Figure 3G), which is reminiscent of the defects observed in vivo (Figure 1H) and in vitro (Figure 1L) for constitutively Rb null erythroblasts. In addition to the enucleation defect, RbΔexon19 erythroblasts showed increased numbers of cells with a 4N DNA content (12.1%, Figure 3F) compared to control Rbflox/flox erythroblasts (6.4%, Figure 3E). These observations are consistent with those obtained from both homozygous Rb mutant FLs (Figure 2B and C) and Rb null chimeric FLs (Figure 2I) and suggest that the accumulation of Rb null erythroblasts with G2/M DNA content is determined by cell intrinsic defects. Cre-mediated deletion of exon 19 in vitro was markedly more efficient than the deletion effected in vivo in the FL by crossing Rb−/flox mice to Meox2-Cre mice (lane 6), suggesting that these mice are chimeric for Rb loss. Nevertheless, we consistently observed red cell enucleation defects (Figure 3D) at E13.5 in Meox2-Cre/Rb−/exon 19flox embryos that mirrored those seen in the E13.5 homozygous null embryos (Figure 1H). Mice rescued with Rb null FL die exhibiting anemia Further evidence to support a cell intrinsic aspect to the erythroid defect came from hematopoietic reconstitution experiments carried out using wild-type, Rb heterozygous, Rb homozygous mutant or Rb null chimeric FL as donor tissue (Figure 4). All host mice rescued with wild-type FL survived beyond 12 months postirradiation (Figure 4A, red profile). In contrast, none of the mice rescued with Rb null FL survived more than 5 months after irradiation (Figure 4A, blue profile). Interestingly, heterozygosity for pRb reduced the reconstituting potential of FL (Figure 4A, green profile), suggesting that Rb may be haploinsufficient for aspects of hematopoietic homoeostasis. Figure 4.Decreased survival and aplastic anemia in mice reconstituted with Rb null FL. Survival curve of mice reconstituted with wild-type (32, red), Rb null (34, blue), Rb heterozygous (34, green) or Rb null chimeric (24, yellow) FL (A). Rb null rescued hosts at experimental end point (loss of >20% of their body weight) and matching wild-type rescued hosts were assessed for blood cell parameters including red blood cell number (RBC, × 106/ml), hemoglobin levels (HgB, g/l), hematocrit (Hct, %) and platelet numbers (Plt, × 103/ml). Spleen weights in mg are also indicated. Normal value ranges for each parameter are also shown (B). Peripheral blood of mice reconstituted with Rb null FL showing the appearance of abnormal erythrocytes (C). Cytospin of bone marrow from mice transplanted with Rb null FL shows the increased presence of benzidine-positive (yellow stain) nucleated erythroblasts with condensed nucleic acid in the cytoplasm (D, arrows). FACS analysis of bone marrow from representative wild-type and Rb null rescued mice at experimental end point (5 months post-IR) (E). Donor FL, peripheral blood (1, 4, 8 and 10 months post-IR rescue) and bone marrow (end point) of recipient mice were assessed by GPI analysis, for contribution by wild-type and Rb null cells (F). Download figure Download PowerPoint Toward the end of their lives, mice rescued with Rb null FL showed a hunched appearance, overall weight loss and a general failure to thrive. Blood analysis of these animals revealed a dramatically reduced hematocrit, reduced hemoglobin (Figure 4B) and abnormal erythrocyte morphology evident in all blood smears (Figure 4C). These defects in adult erythroid differentiation were very similar to those observed in Rb null fetal erythropoiesis. Most mice rescued with chimeric FL (Figure 4A, yellow profile) lived longer than mice reconstituted with Rb null FL (blue profile), but not as long as wild-type FL reconstituted mice (red profile). We observed that there was an initial decrease in contribution by Rb null cells to peripheral blood in all chimeric rescued mice, as measured by GPI contribution (Figure 4F). However, by 4–8 months postirradiation, Rb null cells reappeared in the peripheral blood and by experiment end point, showed high contribution to blood and bone marrow, irrespective of initial FL chimerism (Figure 4F). The dynamic contribution to peripheral blood by Rb null cells over time may reflect a growth advantage conferred on erythroid progenitors by Rb loss under conditions of stress. However, these mice ultimately suffered the same erythroid defects as Rb null rescued mice (low hematocrit, low hemoglobin levels, abnormally shaped RBCs) and also died of anemia. Progressive hematopoietic depletion in mice reconstituted with Rb null FL Bone marrow from mice reconstituted with Rb null FL or with Rb null chimeric FL contained increased numbers of benzidine-positive, nucleated red cells indicative of a maturation defect similar to that observed in the Rb null FL (Figure 4D). In addition to an enucleation defect, FACS analysis revealed that erythroblasts from bone marrow reconstituted with Rb null FL failed to downregulate the CD71 transferrin receptor (Figure 4E) in contrast to wild-type erythroblasts (Socolovsky et al, 2001). Loss of Rb resulted in markedly reduced numbers of bone marrow CD71−TER119hi erythroid cells (4.3%, Figure 4E) compared to wild type (16.9%, Figure 5A) and increased numbers of CD71+TER119hi erythroblasts (32.8%, Figure 4E) compared to wild type (24.4%, Figure 4E). The failure to downregulate CD71 in bone marrow erythroblasts suggested a critical role for Rb in the maturation of bone marrow red cells. Figure 5.Bone marrow and spleen aplasia in mice rescued with Rb null FL. H&E-stained sections of bone marrow from mice rescued with Rb null FL showed various stages following adoptive transplant. Stage I: We initially observe reduced erythroid contribution and increased relative numbers of myeloid elements in the bone marrow (A), which is accompanied by extramedullary hematopoiesis to the spleen (D). Stage II: Loss of myelopoiesis from the bone marrow as defective erythropoiesis takes over (B). If this is also accompanied by displacement from the spleen (E), the mouse succumbs to anemia, prior to Stage III: the complete exhaustion of all elements from the bone marrow (C, G) and spleen (F, H). Download figure Download PowerPoint More detailed histological examination of bone marrows and spleens from mice transplanted with Rb null FL (Figure 5) or Rb null chimeric FL revealed discrete changes in lineage contribution to these organs over time. Bone marrow pathologies could be subdivided into three categories: (I) reduced erythroblast numbers (and progenitors) but abundant production of granulocytes and megakaryocytes (Figure 5A); (II) erythroblast hyperplasia (Figure 5B) and (III) bone marrow depletion marked by extensive spaces and bone deposits (Figure 5C and G). The spleen was enlarged in most of the Rb null rescued mice, from early to late stages following transplant, weighing approximately three to four times more than spleens from control mice rescued with wild-type FL (Figure 4B). Extramedullary hematopoiesis (EMH) in the spleen is indicative of defects in bone marrow hematopoiesis. We noted that multilineage EMH (Figure 5D) was most frequently observed in mice during the early stages of reconstitution with Rb null FL and not in moribund mice. As mice became moribund, the spleens of most Rb null rescued mice (26 out of 34) showed loss of follicular structure as erythroid cells (judged by their size and dark staining nuclei) outpopulated other elements, including B cells. However, erythropoiesis in the spleens of these mice was ineffective (Figure 5E shows extensive hemosiderin deposition). Given the timing of the changes in bone marrow and spleen pathology, we propose that initial ineffective erythropoiesis in the bone marrow promotes multilineage EMH in the spleen, which is also ineffective due to defects in end-stage erythroid differentiation. This is followed by a period in which erythroblasts expand in the bone marrow and spleen as homeostatic mechanisms respond to anemia. A significant proportion of mice (eight mice out of 34) rescued with Rb null FL showed very small spleens when they became moribund. These spleens were found to be aplastic and showed loss of hematopoietic progenitors (Figure 5F and H). Mice that showed fibrotic spleens also had aplastic bone marrow (Figure 5C and G). These observations suggested that loss of Rb conferred a cell intrinsic growth advantage on CD71+ erythroblasts, which coupled with defective red cell maturation depleted the bone marrow and spleen of hematopoietic progenitors and cell types of other lineages. Age-dependent erythroid defects in adult Rb null chimeric mice All of our data presented so far suggested that loss of pRb precipitated cell intrinsic defects in red cell maturation. However, these observations appear to contradict previous results showing high contribution of Rb null cells to the peripheral blood of adult Rb null chimeras with no apparent defects in red cell maturation (Robanus-Maandag et al, 1994; Williams et al, 1994). We re-examined erythropoiesis in adult Rb null chimeras and observed effective contribution to the peripheral blood of young mice by Rb null cells, consistent with published data (Robanus-Maandag et al, 1994; Williams et al, 1994). However, as adult chimeric mice approached 4 months of age and became sick with pituitary tumors (marked by increased coat pigmentation), we noticed substantial increases in peripheral blood contribution by Rb null cells, reduced hematocrits and increasing numbers of morphologically abnormal red cells, similar to that observed in transplanted mice (Figure 4C). When we examined the contribution of Rb null cells to specific lineages within the bone marrow of 4-month-old adult chimeras by FACS, we observed th
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