Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system

Abstract

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.

Fig. 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×.

Fig. 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×.

Fig. 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.

Fig. 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.

Fig. 5. Neuronal differentiation of Rb-deficient cells in chimeric brains. (A) Double staining of E13.5 chimeric brain with Y-chromosome paint and antibodies against the neuronal differentiation marker MAP2. Male Rb^–/– cells (marked with green Y-paint FISH probe) are present in CNS regions expressing MAP2 (red). (B) Dissociated Rb^–/– cell from E13.5 chimeric CNS marked with CMFDG (green) and expressing neuronal differentiation marker neurofilament 165 kDa subunit (red). (C) Sections of 5-week-old (5wk) chimeric brain stained with X-gal and nuclear fast red (left panel) or cresyl echt violet (right panel). Rb^–/–; Rosa26 cells (marked blue, arrows) in chimeric brain display normal neuronal morphology. (D) Section of cerebellum from the same chimera stained with X-gal and cresyl echt violet. Rb^–/– cerebellar Purkinje neurons (arrow) are enlarged and display nuclear pleomorphism as compared with normal wild-type Purkinje cells (arrowhead). The internal granular (g) and molecular (m) cell layers of the cerebellum are marked. Magnification: (A, C and D) 60×; (B) 100×.

Fig. 6. Model of the fate of Rb^–/– cells in CNS of germline Rb^–/– and chimeric embryos. In both settings, Rb^–/– neuronal precursors ectopically enter the cell cycle in a cell-autonomous fashion, with elevated E2F activity and high levels of expression of E2F transcriptional targets such as cyclin E. In both settings, inappropriate S-phase entry is accompanied by increased p53 activity and elevated expression of the p53 target p21. In germline Rb-mutant embryos, these events lead to ectopic M-phase entry and apoptosis. However, in chimeric CNS, ectopic M-phase progression and apoptosis of Rb^–/– cells are suppressed in a non-cell-autonomous fashion. In the presence of wild-type neighbors, Rb^–/– cells survive and differentiate into neuronal fates.