Volume 2, Issue 3, Pages 293-304 (September 1998) Mutation of E2f-1 Suppresses Apoptosis and Inappropriate S Phase Entry and Extends Survival of Rb-Deficient Mouse Embryos  Kenneth Y Tsai, Yanwen Hu, Kay F Macleod, Denise Crowley, Lili Yamasaki, Tyler Jacks  Molecular Cell  Volume 2, Issue 3, Pages 293-304 (September 1998) DOI: 10.1016/S1097-2765(00)80274-9

Figure 1 Effects of E2f-1 Mutation on Apoptosis and S Phase Entry in the Rb-Deficient Lens Transverse sections through the eye of wild-type, Rb mutant, and double mutant E13.5 embryos are shown, stained for apoptotic cells (TUNEL method, panels A–F) or cells in S phase (BrdU incorporation, panels G–I); sections include both retina (R) and lens (L) as indicated. Note the marked reduction in the number of TUNEL-positive (darkly staining) cells in the double mutant lens (panels C and F) compared to the Rb mutant (panels B and E). Low, background levels of apoptosis are seen in the wild-type lens (panels A and D). The number of BrdU-positive (darkly staining) cells in the lens is also reduced in the double mutant (panel I) compared to the Rb mutant (panel H), but the double mutant S phase activity is still higher than that present in wild-type (panel G). Magnification, panels A–C and G–I = 10×; panels D–F = 40×. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 2 Quantitative Analysis of Apoptosis and S Phase Activity in Lens, CNS, and PNS (A) Mutation of E2f-1 efficiently suppresses apoptosis in Rb-deficient ocular lens and central nervous system but not in peripheral nervous system. The frequency of apoptotic cells (measured as number of TUNEL-positive cells per unit area of tissue) in the three tissue types was determined from multiple E13.5 embryos of different genotypes. For each tissue type, the frequency of apoptotic cells in the Rb mutant samples was set at 1.0 and relative amount of apoptosis in those tissues in the other genotypes plotted against this value. Error bars indicate the standard error of the mean. (B) Differential effects of E2f-1 mutation on S phase entry in Rb-deficient ocular lens and nervous system. The frequency of cells in S phase (measured as the number of BrdU-positive cells per unit area of tissue) was determined in the lens fiber cell compartment, intermediate zone of the cortex in the CNS (ectopic S phase entry), and sensory ganglia of the PNS for multiple E13.5 embryos of different genotypes. For each tissue type, the frequency of BrdU-positive cells in the Rb mutant sample was set at 1.0 and the relative number of BrdU-positive cells in those tissues in the other genotypes were plotted against that value. Error bars indicate the standard error of the mean. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 3 Effects of E2f-1 Mutation on Apoptosis and S Phase Entry in the Rb-Deficient CNS Sections of the fourth ventricle of the brain of E13.5 wild-type, Rb mutant, and double mutant embryos are shown, stained for apoptotic cells (TUNEL method, panels A–C) or cells in S phase (BrdU incorporation, panels D–I). Compared with the abundant TUNEL-positive (darkly staining) cells in the Rb mutant sections (panel B), the double mutant section (panel C) has only background levels, similar to that seen in wild-type (panel A). High levels of ectopic S phase activity (darkly staining, BrdU-positive cells present in the intermediate [I] zone) are seen in the Rb mutant sections (panels E and H), compared to wild type (panels D and G). Double mutant sections show some reduction in ectopic S phase activity compared to Rb mutant, although notably above wild-type levels. Increased S phase activity is also present in the ventricular (V) zone of Rb mutant and double mutant embryos, but this has not been quantitated in this study. Magnification, panels A–F = 10×; panels G–I = 40×. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 4 E2f-1-Dependent Up-Regulation of p53 Activity in Rb Mutant Brain Electrophoretic mobility shift assay for p53 DNA binding activity in brains of wild-type (WT), Rb mutant (Rb−/−), and double mutant (Rb−/−; E2f-1−/−) embryos at E13.5. Specific p53 DNA binding activity is increased in the Rb mutant sample relative to the wild-type and double mutant samples, which are comparable. In all samples, binding is abolished in the presence of 100-fold (100 ng) unlabeled p53 consensus site oligonucleotide competitor DNA. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 5 Effects of E2f-1 Mutation on Apoptosis and S Phase Entry in the Rb-Deficient PNS Sections of the sensory ganglia from E13.5 wild-type, Rb mutant, and double mutant embryos are shown, stained for apoptotic cells (TUNEL method, A–C) or cells in S phase (BrdU incorporation, D–F). Note that the level of apoptosis (indicated by darkly staining cells) in the double mutant section (C) is comparable to that in the Rb mutant (B); low, background levels of apoptosis are shown in the wild-type ganglion (A). Increased levels of S phase activity (darkly staining, BrdU-positive cells) are seen in the Rb mutant sections (E), compared to wild-type (D). The double mutant ganglion (F) shows some reduction in S phase activity compared to Rb mutant, although significantly above wild-type levels. Magnification, panels A–F = 40×. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 6 Appearance and Erythroid Profile of Double Mutant Embryos at E13.5 (A–C) Embryos of different genotypes were recovered at E13.5 and photographed in saline. In contast to the obvious pale appearance of the Rb mutant embryo (B), the double mutant embryo (C) is superficially indistinguishable from wild-type (A). Note that blood flow can be assessed in the crania of wild-type and double mutant embryos (arrows) but not in the Rb mutant. (D–F) Peripheral blood smears from E13.5 embryos of different genotypes stained with Wright-Giemsa. Blood smears from wild-type embryos (D) have approximately equal numbers of primitive, nucleated erythrocytes (black arrows) and definitive, enucleated erythrocytes (red arrows). The blood smear from the Rb mutant embryo (E) shows the documented, severe reduction in enuceated erythrocytes, while in the blood smear from the double mutant embryo, enucleated cells are readily detected. See text for quantitation. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 7 Late Gestation Double Mutant Embryos Exhibit a Variety of Defects in Erythropoiesis, Skeletal Muscle, and Lung (A and B) Viable wild-type (A) and double mutant (B) embryos were recovered at E17 and photographed in saline. Note that the double mutant embryo is paler in apperance, indicative of an ongoing erythropoietic defect. (C and D) Peripheral blood smears from E17 wild-type (C) and double mutant (D) embryos stained with Wright-Giemsa. Note the abnormally high proportion of nucleated erythrocytes (red arrow) in the double mutant blood smear and the presence of erythrocytes with abnormal cell shape (black arrows), both indications of inefficient erythropoiesis or response to anemia. (E and F) Histological sections of axial skeletal muscle from E17 wild-type (E) and double mutant (F) embryos stained with hematoxylin and eosin (H & E). Note the marked reduction in muscle mass and the presence of cells with enlarged nuclei in the double mutant embryo. (G and H) H & E-stained sections of lung tissue from E17 wild-type (G) and double mutant embryo (H). Well formed terminal bronchioles are readily detected in wild-type lung at this stage (arrows) but these structures are absent or malformed in the double mutant. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)

Figure 8 Model of Dual Function of E2F-1 in the Induction of S Phase Entry and Apoptosis Data presented here support a model in which E2F-1 acts in inappropriate S phase entry and apoptosis in certain cells of the Rb-deficient mouse through distinct pathways. The model illustrates that other pRB targets are also implicated in normal regulation of S phase entry. The induction of p19ARF has not been demonstrated in this system; this and other speculative components of the model are indicated with question marks. The model also suggests that induction of apoptosis in response to viral oncoproteins that bind to pRB may also require E2F-1 function. See text for details. Molecular Cell 1998 2, 293-304DOI: (10.1016/S1097-2765(00)80274-9)