Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system
2001; Springer Nature; Volume: 20; Issue: 13 Linguagem: Inglês
10.1093/emboj/20.13.3402
ISSN1460-2075
Autores Tópico(s)Microtubule and mitosis dynamics
ResumoArticle2 July 2001free access Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system Marta M. Lipinski Marta M. Lipinski Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Search for more papers by this author Kay F. Macleod Kay F. Macleod Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY UK Search for more papers by this author Bart O. Williams Bart O. Williams Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI, 495030 UK Search for more papers by this author Tara L. Mullaney Tara L. Mullaney Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Search for more papers by this author Denise Crowley Denise Crowley Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Howard Hughes Medical Institute, 400 Jones Bridge Road, Chevy Chase, MD, 20815 USA Search for more papers by this author Tyler Jacks Corresponding Author Tyler Jacks Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Howard Hughes Medical Institute, 400 Jones Bridge Road, Chevy Chase, MD, 20815 USA Search for more papers by this author Marta M. Lipinski Marta M. Lipinski Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Search for more papers by this author Kay F. Macleod Kay F. Macleod Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY UK Search for more papers by this author Bart O. Williams Bart O. Williams Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI, 495030 UK Search for more papers by this author Tara L. Mullaney Tara L. Mullaney Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Search for more papers by this author Denise Crowley Denise Crowley Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Howard Hughes Medical Institute, 400 Jones Bridge Road, Chevy Chase, MD, 20815 USA Search for more papers by this author Tyler Jacks Corresponding Author Tyler Jacks Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK Howard Hughes Medical Institute, 400 Jones Bridge Road, Chevy Chase, MD, 20815 USA Search for more papers by this author Author Information Marta M. Lipinski1, Kay F. Macleod2, Bart O. Williams3, Tara L. Mullaney1, Denise Crowley1,4 and Tyler Jacks 1,4 1Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 UK 2Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY UK 3Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI, 495030 UK 4Howard Hughes Medical Institute, 400 Jones Bridge Road, Chevy Chase, MD, 20815 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3402-3413https://doi.org/10.1093/emboj/20.13.3402 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The retinoblastoma tumor suppressor (RB) plays an important role in the regulation of cell cycle progression and terminal differentiation of many cell types. Rb−/− mouse embryos die at midgestation with defects in cell cycle regulation, control of apoptosis and terminal differentiation. However, chimeric mice composed of wild-type and Rb-deficient cells are viable and show minor abnormalities. To determine the role of Rb in development more precisely, we analyzed chimeric embryos and adults made with marked Rb−/− cells. Like their germline Rb−/− counterparts, brains of midgestation chimeric embryos exhibited extensive ectopic S-phase entry. In Rb-mutants, this is accompanied by widespread apoptosis. However, in chimeras, the majority of Rb-deficient cells survived and differentiated into neuronal fates. Rescue of Rb−/− neurons in the presence of wild-type cells occurred after induction of the p53 pathway and led to accumulation of cells with 4n DNA content. Therefore, the role of Rb during development can be divided into a cell-autonomous function in exit from the cell cycle and a non-cell-autonomous role in the suppression of apoptosis and induction of differentiation. Introduction The retinoblastoma gene (RB) is best known for its role in tumor suppression. In humans, inheritance of a mutated allele of the RB gene predisposes to familial retinoblastoma. RB is also mutated in many sporadic cancers, including retinoblastoma, sarcomas and various carcinomas (Goodrich and Lee, 1993). Mice heterozygous for germline mutation in the murine homolog of the retinoblastoma gene (Rb) are highly susceptible to pituitary and thyroid carcinomas, but do not develop retinoblastoma (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992; Williams et al., 1994a). Rb exerts its tumor suppressor function by controlling cell cycle progression from G1 into S-phase (Weinberg, 1995). Hypophosphorylated RB protein (pRB) prevents premature S-phase entry by binding to and inhibiting the E2F family of transcription factors. It can also recruit histone deacetylases to the E2F-bound promoters, thus actively inhibiting transcription of E2F target genes (Brehm and Kouzarides, 1999). When cells are stimulated to re-enter the cell cycle, pRB is phosphorylated by cyclin D–cdk4 and cyclin E–cdk2 complexes. Hyperphosphoryl ation of pRB reduces its affinity for E2Fs, leading to their release and the transcription of genes necessary for S-phase entry and progression (Nevins et al., 1997). In addition, pRB also functions in the regulation of terminal differentiation of many tissue types (Lipinski and Jacks, 1999). An early indication of the role of Rb in differentiation was the phenotype of Rb−/− mouse embryos (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). These embryos die between 13 and 15 days of gestation (E13–15) with pronounced defects in erythroid, neuronal and lens development. These lineages are able to initiate the differentiation process, but fail to achieve a fully differentiated state. For example, erythrocytes do not enucleate efficiently, and expression of some of the late neuronal and lens differentiation markers is decreased or absent (Lee et al., 1994; Morgenbesser et al., 1994). Additionally, ectopic cell cycle entry and elevated apoptosis levels are apparent in the ocular lens and in the peripheral (PNS) and central nervous system (CNS). These results indicate that Rb might assist in achieving and/or maintaining the post-mitotic state associated with terminal differentiation of many tissue types, as well as in the protection from apoptosis and induction of late differentiation markers. The phenotype in the nervous system of Rb−/− embryos has been most fully characterized. Extensive ectopic cell cycle entry and elevated apoptosis levels are apparent in both CNS and PNS by E12. The inappropriate cell cycle entry is accompanied by elevated activity of free E2F proteins and overexpression of E2F transcriptional targets, such as cyclin E (Macleod et al., 1996). Additionally, p53 protein levels and p53 DNA binding activity are enhanced in the brains of Rb−/− embryos, leading to increased expression of the p53 transcriptional target p21. In Rb−/− CNS, apoptosis has been shown to be p53 dependent, as cells in the CNS of Rb−/−p53−/− embryos continue to ectopically enter the S-phase, but do not die. In Rb−/− PNS, both p21 expression and apoptosis are p53 independent (Macleod et al., 1996). The Rb−/− CNS/PNS phenotype initiates at the time in mouse embryo development when neuronal precursor cells normally initiate exit from the cell cycle and begin neuronal differentiation, suggesting that Rb function might be specifically required in the process of neurogenesis. This has been confirmed by following expression of a β-galactosidase reporter gene driven by an early pan-neuronal promoter, Tα 1α-tubulin (Slack et al., 1998). Expression of this reporter is correctly initiated in Rb−/− embryos at E12.5, but declines by E14.5, indicating that differentiating neurons are dying in the absence of functional pRB. Cell-based studies have supported the importance of Rb in differentiation. For example, Rb−/− embryo lung bud fibroblasts can be induced to express early but not late adipocyte differentiation markers (Chen et al., 1996). Similarly, Rb−/− primary mouse fibroblasts induced to differentiate into muscle express early differentiation markers normally, but have attenuated expression of the late ones. Rb−/− myocytes are also able to re-enter the cell cycle following serum stimulation (Novitch et al., 1996). In this system, pRB has been shown to induce myogenesis through inhibition of E2F function to prevent cell cycle re-entry and through augmentation of muscle-specific transcription factor activity (Novitch et al., 1999). Neural precursor cells derived from Rb−/− embryos exhibit delayed exit from mitosis and deregulated E2F activity (Callaghan et al., 1999). Inactivation of pRB and the closely related p107 and p130 proteins, by expression of a mutant adenovirus E1A oncoprotein can prevent cell cycle withdraw and lead to death of in vitro differentiating cortical progenitor cells (Slack et al., 1998). Because germline Rb−/− embryos die too early to achieve a terminally differentiated state in many tissues, we and others studied the role of Rb in development and differentiation using chimeric mice composed of wild-type and Rb−/− cells (Rb−/− chimeras) (Maandag et al., 1994; Williams et al., 1994b). Surprisingly, Rb−/− chimeras are viable and fertile even with extensive contribution of Rb−/− cells to all tissues, including brain, liver and blood. They develop pituitary tumors with reduced latency as compared with Rb+/− mice, but otherwise show only mild histopathological abnormalities, including hyperplasia of the adrenal medulla, abnormal lens architecture, cataracts, pleomorphic Purkinje cells and enlarged liver cells. Rb−/− chimeric embryos have delayed enucleation of erythrocytes, ectopic mitosis in the lens, and slightly increased pycnosis in the retina and the spinal ganglia. No abnormalities were observed in the developing CNS (Maandag et al., 1994; Williams et al., 1994b). The lack of prominent developmental defects in Rb−/− chimeras contrasts with the pronounced phenotype of germline Rb−/− embryos and with the cell-based data suggesting an important role for pRB in differentiation. The data from chimeras have been interpreted to indicate that the defects in cell cycle control and apoptosis observed in the nervous system of Rb−/− embryos are non-cell autonomous in nature, as they are suppressed by the presence of wild-type cells. This contrasts with the known function of pRB in cell cycle regulation, where it is thought to restrict entry into S-phase in a cell-autonomous manner. In order to reconcile these discrepancies, as well as to characterize further the role of pRB in neuronal differentiation in vivo, we have performed a detailed analysis of the CNS phenotype in chimeric embryos and adults produced from marked Rb−/− cells. Results Contribution of Rb−/− cells to chimera CNS A major limitation of earlier analyses of Rb−/− chimeras (Maandag et al., 1994; Williams et al., 1994b) was the inability to identify and follow the fate of individual mutant cells. To overcome this limitation, we have created male Rb−/− 129sv ES cells expressing β-galactosidase from a ubiquitous Rosa26 promoter (Zambrowicz et al., 1997). We injected these cells into wild-type C57BL/6 blastocysts, obtaining chimeric progeny with up to 90% Rb−/− cell contribution, as judged by coat color. Given the extensive analysis of the CNS of germline Rb−/− embryos, we concentrated our analysis on this tissue. To assess Rb−/− cell contribution, dissociated cells from E13.5 embryo brains were incubated with a fluorescent β-galactosidase substrate, 5-chloromethylfluorescein di-β-galactopyranoside (CMFDG), and analyzed by fluorescence-activated cell sorting (FACS). CMFDG-positive cells constituted up to 45% of all cells, with an average contribution of 20.5% (n = 10; Table I). Table 1. Summary of FACS data E13.5 # % Rb−/− wt G1 wt S wt G2/M Rb−/− G1 Rb−/− S Rb−/− G2/M 1 17.8 86.4 5.1 8.3 67.4 9.7 22.5 2 45.6 98 1.2 0.4 85.1 6.6 8.4 3 22.5 95.7 3 1.6 79.1 6.4 14.9 4 27.7 96.9 2.3 1.2 82.5 7.2 10.3 5 11.3 86.3 5.8 8.1 59.6 14.9 25.3 6 6.8 92.1 3.8 4.2 66.2 16.7 16.9 7 14.3 93 5.5 1.5 53.9 23.9 22 8 27.8 96.6 3.4 0.2 71.2 19 9.8 9 21.4 77.3 14.7 7.6 65.1 15.4 19.6 10 9.6 91.8 4.5 3.9 72.4 9.5 17.8 Average 20.48 91.41 4.93 3.7 70.25 12.93 16.75 SD 11.43 6.43 3.73 3.24 9.92 5.95 3.83 Rb/wt t-test 2.3 × 10−5 0.0021 7.7 × 10−6 %Rb−/−, Rb−/− cell contribution; wt, wild-type cell population; Rb−/−, Rb−/− cell population; Rb/wt t-test, p value for two-tailed T-test for the difference between wild-type and Rb−/− populations. In order to confirm the FACS data, we visualized Rb−/− cells in situ. Because staining with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) did not give satisfactory results, we determined the Rb−/− cell contribution to chimeric brains by visualizing these cells using fluorescent in situ hybridization (FISH). For approximately half of the embryos examined, we were able to distinguish injected Rb−/− male cells from the wild-type female host embryo cells by examining their sex chromosomes. Cy3-labeled XIST RNA probe was used to visualize the inactive X-chromosome in female (wild-type) cells, and biotinylated Y-paint DNA probe followed by avidin–fluorescein isothiocyanate (FITC) detection to mark the Y-chromosome in male (Rb−/−) cells (Figure 1). At E13.5, Rb−/− and wild-type cells in the chimeric embryo brains were intermixed or present in small patches without any obvious large cell clusters of homogeneous genotype. XIST- and Y-paint-positive cells were counted in 3–4 non-overlapping fields in the hindbrain (area around the fourth ventricle, including medulla, pons and cerebellar primordium) of chimeric embryos to estimate contribution. By this method, up to 72% of all cells were Rb−/−, with an average contribution of 55.9% (n = 8; Table II). The discrepancy between the levels of Rb−/− contribution obtained from FACS as compared with FISH analysis can be accounted for by differences between the two methods. By FACS analysis, the Rb−/− contribution was underestimated because of a high FDG fluorescence gate set to ensure exclusion of all wild-type cells from further analysis. In FISH experiments, only ∼80% of cells in female and male embryo controls stained with XIST probe or Y-paint, respectively (Figure 1). Therefore, this method can be used only to estimate the Rb−/− contribution. Additionally, for FACS analysis, we used cells from the entire embryo brain, while in the FISH experiments only the hindbrain was counted. The contribution of Rb−/− cells could vary in different regions of the chimeric brain. Cell-type-dependent variance in Rosa26-driven expression of β-galactosidase might also contribute to underestimation of Rb−/− contribution by FACS. Despite these differences, both methods confirmed that, unlike in germline Rb−/− embryos, Rb−/− cells were able to persist in chimeric CNS at E13.5. The Rb−/− cell contribution to chimeric brains continued at later stages of development. From FISH analysis, we have estimated the contribution at E15.5 to be on average 58.68% (n = 3; Figure 1). Since neither FACS nor FISH analysis gave satisfactory results in postnatal animals, Rb−/− cell contribution to adult brains was demonstrated by staining with X-gal (Figure 1). Owing to poor expression of β-galactosidase in adult brain, we were unable to quantitate the extent of Rb−/− cell contribution to the chimeric adult CNS. However, previous studies have used GPI analysis to demonstrate that Rb−/− cells contribute on average 20% to adult chimeric brains (Maandag et al., 1994; Williams et al., 1994b). This contribution is comparable to that obtained using wild-type instead of Rb-deficient cells for chimera generation. Figure 1.Rb−/− cells contribute to chimeric embryo and adult brain. (A) XIST and Y-chromosome paint FISH analysis on E13.5 and E15.5 embryo sections. In chimeras, male Rb−/− cells are marked with green Y-chromosome paint and female host cells with red XIST probe. Wild-type (wt) female and male control embryos are shown along with chimeras. Magnification 60×. (B) X-gal staining on 5-week-old (5wk) chimeric brain. Blue-staining, Rb−/− Rosa26 cells are evident. Magnification 40×. Download figure Download PowerPoint Table 2. Summary of immunohistochemistry data E13.5 # Genotype Sex % Rb−/− TUNEL Ect. BrdU PH3 1 chimera m nd 53.5 nd 30 2 chimera f 36.3 58.5 nd nd 3 chimera f 67.5 182 586.5 55 4 chimera f 72.5 174.5 468.5 67 5 chimera m nd 178.5 652.5 46.3 6 chimera m nd 100 548 43 7 chimera f 63.2 63.5 nd 51.3 8 chimera f 39.8 28 320 48.5 Average 55.9 111.1 535.4 48.7 SD 16.6 64 127.8 11.3 9 Rb−/− nd 100 nd nd 154 10 Rb−/− nd 100 nd nd 148.5 11 Rb−/− nd 100 526 585 141.5 12 Rb−/− nd 100 492 484 183 13 Rb−/− nd 100 444.5 nd nd 14 Rb−/− nd 100 nd 404 nd 15 Rb−/− nd 100 247 277 nd Average 427.4 437.5 156.7 SD 124.8 130.1 18.2 16 wt nd 0 3.5 21 62 17 wt nd 0 1.5 30 52.3 18 wt nd 0 6 54 28.7 19 wt nd 0 nd nd 30.6 20 wt nd 0 3 nd nd 21 wt nd 0 4.5 nd nd 22 wt nd 0 2.5 nd nd Average 3.5 31.7 43.4 SD 1.6 17.1 16.4 wt/ch t-test 0.0023 0.0007 0.5364 Rb/ch t-test 0.0001 0.3989 6.2 × 10−7 E15.5 # Genotype Sex % Rb−/− TUNEL 23 chimera f nd 20 24 chimera f 31.3 19 25 chimera f 70.9 28.5 26 chimera m nd 73 27 chimera m nd 108 28 chimera m nd 34 29 chimera m nd 57.5 30 chimera m nd 29 31 chimera f nd 36 32 chimera f 58.7 46.5 Average 53.6 45.1 SD 20.3 27.8 33 wt nd 0 10 34 wt nd 0 18.5 35 wt nd 0 17 36 wt nd 0 12 Average 14.4 SD 4 wt/ch t-test 0.0521 Neonate # Genotype Sex % Rb−/− TUNEL 37 chimera nd nd 225 38 chimera nd nd 183 39 chimera m nd 377.5 40 chimera f nd 275.3 41 chimera m nd 485 42 chimera m nd 275 Average 303.5 SD 110.2 43 wt nd nd 133.5 44 wt nd nd 131.5 Average 132.5 SD 1.4 wt/ch t-test 0.0825 Adult # Genotype Sex % Rb−/− TUNEL 45 chimera m 60 12.5 46 chimera f 90 12.5 47 chimera m 80 27.5 48 chimera m 15 16.5 Average 61.2 17.2 SD 33.3 7.1 49 wt m 0 15 50 wt f 0 7.5 Average 11.2 SD 5.3 wt/ch t-test 0.2849 Ect. BrdU, ectopic BrdU incorporation; PH3, phospho-histone 3; f, female; m, male; nd, not determined; Rb/ch t-test, two-tailed T-test for the difference between Rb−/− and chimeric animals; wt/ch t-test, p-value for two-tailed T-test for the difference between wild-type and chimeric animals. For other abbreviations see Table I. Low levels of apoptosis in chimeric CNS The presence of Rb−/− cells in the CNS of Rb−/− chimeras contrasted with the extensive apoptosis in germline Rb−/− embryo brains (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992, 1994). In order to examine specific levels of apoptosis in Rb−/− chimera brain, TdT biotin-16-dUTP nick end labeling (TUNEL) staining was performed on paraffin sections of E13.5 embryos. The total number of TUNEL-positive cells was counted in the hindbrain on 3–4 sagittal sections of every embryo (Figures 2 and 3; Table II). As previously described, the number of TUNEL-positive cells was greatly increased in germline Rb−/− embryos (average of 427.4 cells/section, n = 4) as compared with wild type (3.4 cells/section, n = 6). In chimeric embryos, TUNEL levels (111.1 cells/section, n = 8) were higher than in wild type, but despite the high contribution of Rb−/− cells to chimeric brains, were significantly lower than in Rb−/− embryos (p = 0.00012). For chimeras with known Rb−/− contribution, TUNEL levels were found to be significantly lower (p = 0.0045) than these calculated to be expected in an equivalent mixture of Rb−/− and wild-type cells (239 cells/section, n = 5) based on the average TUNEL levels observed in Rb−/− and wild-type embryos. Thus, the contribution of Rb−/− cells to the CNS of chimeras at midgestation can be accounted for by a reduction in the levels of apoptosis in the presence of wild-type cells. The levels of cell death in chimeric embryos decreased at later stages of embryonic development. At E15.5, there was only a small increase in the number of TUNEL-positive cells in the hindbrain of chimeric embryos (45.2 cells/section, n = 10) as compared with wild type (14.4 cells/section, n = 4). In neonates, because of low levels of apoptosis in the CNS, the number of TUNEL-positive cells per sagittal section was determined for all areas of the brain [303.5 (n = 6) and 132.5 cells/section (n = 2) in chimeras and wild type, respectively]. As the mice matured, total TUNEL levels gradually declined even further and the difference became insignificant [17.3 (n = 4) and 11.3 cells/section (n = 2), respectively]. Figure 2.Apoptosis, but not ectopic cell cycle entry, is suppressed in Rb−/− chimeric brains. (A) S-phase activity and apoptosis levels in E13.5 chimeras and controls. BrdU incorporation demonstrates elevated ectopic S-phase entry in germline Rb−/− and chimeric embryos [brown-stained cells present in the intermediate zone (iz), white arrows]. Hindbrain area around the fourth ventricle (v) is shown, with the ventricular zone (vz) and iz indicated. TUNEL assay (brown-stained cells, black arrows) demonstrates reduced level of apoptosis in chimeric CNS as compared with germline Rb-mutant. (B) Analysis of older chimeric embryos, neonates and adults. At E15.5, ectopic cell cycle entry was present in the chimeric CNS, while levels of apoptosis remained low. In neonatal and adult chimeras, levels of S-phase entry declined in both wild-type and chimeric brains. Levels of TUNEL-positive cells were also low. Magnification 40×. Download figure Download PowerPoint Figure 3.Quantification of cell cycle and cell death analysis in the CNS of E13.5 germline Rb−/− and chimeric embryos. (A) S-phase activity (assessed by BrdU incorporation) and apoptosis (by TUNEL analysis) in hindbrain region on sagittal sections of germline Rb−/−, chimeric and wild-type (wt) E13.5 embryos. S-phase entry was increased in the brains of both germline Rb-deficient and chimeric embryos as compared with wild type. However, apoptosis was significantly suppressed in chimeras as compared with germline Rb−/− embryos. (B) FACS cell cycle profile analysis of Rb−/− and wild-type cells in chimeric CNS at E13.5. Rb−/− cells show reduced G1, and increased S-phase and G2/M fraction. (C) PH3 (a mitotic marker) expression in wild-type, germline Rb-deficient and chimeric embryo CNS at E13.5. M-phase entry was increased in germline Rb−/− embryos but not in chimeras as compared with wild type. Download figure Download PowerPoint Ectopic cell cycle entry Suppression of apoptosis of Rb−/− cells in chimeric brains could be due to a complete rescue of the Rb mutant phenotype in the presence of wild-type cells. Alternatively, wild-type cells could specifically suppress Rb−/− cell death without affecting other aspects of the mutant phenotype. Therefore, we analyzed chimeric brains at E13.5 for inappropriate S-phase entry. Pregnant females carrying chimeric or control embryos were injected with bromodeoxyuridine (BrdU) and killed after 1 h; embryo sections were examined for BrdU incorporation by immunohistochemistry. The number of BrdU-positive cells was counted in the hindbrain of every embryo in the same manner as for TUNEL analysis (Figures 2 and 3). We observed an increase in overall BrdU incorporation and especially in ectopic BrdU incorporation away from the ventricular area in both germline Rb−/− [437.5 intermediate zone (iz) cells/section, n = 4; Table II] and Rb−/− chimeric embryo brains (535.4 iz cells/section, n = 5) as compared with wild type (31.7 iz cells/section, n = 3). Levels of ectopic cell cycle entry in chimeric brains were comparable to those observed in germline Rb−/− embryos, and higher than those expected in wild type and Rb−/− cell mixture of the same composition (277 iz cells/section, n = 3). Although non-significant (p = 0.027), this increased BrdU incorporation could potentially suggest a non-cell-autonomous increase in cell cycle entry of Rb−/− cells in the presence of wild type. However, it is more likely to be due to the fact that many of the inappropriately cycling cells died in germline Rb−/− embryos, while in chimeras the majority of them survived. The increased and ectopic S-phase entry in chimeric CNS continued at E15.5, but eventually decreased to levels indistinguishable from wild type in neonates and adults (Figure 2). In order to assess the cell cycle distribution of Rb−/− cells in chimeric brains, we performed a two-color FACS analysis. Dissociated brain cells from E13.5 Rb−/− Rosa26 chimeras were labeled with CMFDG, followed by paraformaldehyde fixation. Cells were than stained with propidium iodide (PI) to assess the DNA content. This allowed us to analyze cell cycle distribution separately among Rb−/− (CMFDG+) and wild-type (CMFDG–) cells within each chimeric embryo brain (n = 10). As shown in Table I, the average Rb−/− contribution was 20.5%. The S-phase cells comprised on average 4.9% of the wild type and 12.9% of the Rb−/− population (Figure 3). These data confirm that there is an increase in S-phase population in E13.5 Rb−/− chimeric embryo brains and that Rb−/− cells are, indeed, the cells that are inappropriately continuing to progress through the cell cycle. The cell cycle profile of wild-type cells from Rb−/− chimeric brains was comparable to that of cells from wild-type embryos, indicating that these cells were unaffected by the presence of their Rb−/− neighbors (see Supplementary data available at The EMBO Journal Online). E2F and p53 activity Inappropriate cell cycle entry in germline Rb−/− E13.5 embryos is accompanied by increased activity of the E2F transcription factors and induction of E2F target genes, such as cyclin E (Macleod et al., 1996). We investigated E2F activity in the brains of E13.5 chimeric embryos by in situ hybridization using 35S-labeled antisense RNA probe to cyclin E. cyclin E mRNA levels were increased in Rb−/− chimera embryos as compared with wild type (Figure 4), consistent with elevated E2F transcriptional activity. Figure 4.Analysis of cell cycle and p53 pathway in E13.5 chimeric embryos. (A) In situ hybridization with antisense cyclin E mRNA probe (green). Cyclin E expression was increased in chimeric embryo brain as compared with wild-type (wt) controls, suggesting elevated E2F activity. Hindbrain area around the fourth ventricle (v) is shown (false color, 40× magnification). (B) In situ hybridization with antisense p21 mRNA probe. Elevated levels of p21 expression in chimeric embryo CNS compared with wild-type controls suggest increased p53 transcriptional activity. (C) p53 gel shift analysis on chimeric embryo brain extracts demonstrates that p53 DNA binding activity correlates with the degree of Rb−/− chimerism. (D) Expression of the mitotic marker PH3 (brown-stained cells, black arrows) demonstrates increased and ectopic [in the intermediate zone (iz)] M-phase entry in CNS of germline Rb−/− but not chimeric embryos as compared with wild type. Download figure Download PowerPoint In germline Rb−/− embryos, neuronal cell death in CNS is p53 dependent, and accompanied by elevated p53 protein levels and transcriptional activity (Macleod et al., 1996). We performed a p53 gel shift assay using E13.5 chimeric embryo brain extracts, and determined that p53 DNA binding activity was increased and correlated with the extent of Rb−/− contribution (determined by GPI analysis) in each embryo. Elevated p53 activity was confirmed in chimeras by in situ hybridization against p21, a transcriptional target of p53 (Figure 4). These data are consistent with the suppression of apoptosis by wild-type cells occurring at a point downstream of activation of the p53 pathway in the Rb−/− cells. G2 arrest of Rb−/− cells In germline Rb−/− embryos, aberrant cell cycle entry of neuronal precursor cells is believed to lead to their death. The fact that apoptosis was significantly suppressed in Rb−/− chimeras raised a question about the fate of the ectopically cycling Rb−/− cells. In addition to elevated S-phase, our two-color FACS analysis demonstrated an increase in the G2/M-phase population among Rb−/− cells (16.8% on average) as compared with wild type (3.7%). Importantly, there was no increase in the G2/M population in germline Rb−/− embryos as compared with wild type (see Supplementary data). These data suggest that Rb−/− cells in E13.5 chimeric embryos might not progress through M-phase, but instead arrest in G2 with 4n DNA content. Although the total number of Rb−/− cells with 4n DNA content in chimeric CNS remained relatively low, it has to be taken into account that only cells that have ectopically entered S-phase would be expected to arrest in G2. In order to confirm this result, we stained sections from E13.5 embryos with antibodies against the mitotic
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