Volume 6, Issue 2, Pages 293-306 (August 2000) Loss of E2F4 Activity Leads to Abnormal Development of Multiple Cellular Lineages  Rachel E. Rempel, M.Teresa Saenz-Robles, Robert Storms, Scott Morham, Seiichi Ishida, Amber Engel, Laszlo Jakoi, Mona F. Melhem, James M. Pipas, Clay Smith, Joseph R. Nevins  Molecular Cell  Volume 6, Issue 2, Pages 293-306 (August 2000) DOI: 10.1016/S1097-2765(00)00030-7

Figure 1 Disruption of the E2F4 Gene (A) Depiction of the mouse E2F4 gene, the targeting vector, and the allele resulting from homologous recombination. Exons are depicted as boxes. Indicated by asterisks is the AT-AC intron, a minor class of intron, that is found in the E2F4 gene as well as the genes encoding other E2F family members (Wu and Krainer 1997). The targeting vector was designed to replace coding exons 1–3 with the neoR gene in an antisense orientation and includes the negative selectable marker HSV-thymidine kinase. A 5′ 0.7 kb EcoRI-SacI genomic fragment was used to screen for recombinant alleles. The new EcoRI site introduced by the neoR gene at the targeted allele results in the generation of a 3.7 kb EcoRI fragment, easily distinguished from the 10.5 kb wild-type allele. (B) Southern analysis of DNA extracted from the targeted embryonic stem cell clone and from the tails of progeny of an E2F4+/− × E2F4+/− cross. Mouse genomic DNA was digested with the restriction enzyme EcoRI and analyzed by Southern blotting using the genomic probe described in (A). (C) Western analysis for E2F4 in wild-type, E2F4+/−, and E2F4−/− MEFs. Whole cell extracts (25 μg) from cycling MEFs were processed for Western blot analysis and probed with a polyclonal anti-E2F4 antibody raised against the C-terminal 20 amino acids of E2F4. (D) Photograph depicting an E2F4−/− mouse on the right alongside a wild-type sibling at 2 weeks of age. Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)

Figure 2 Effects of Loss of E2F4 Function on Cell Proliferation (A) Gel mobility shift analysis of E2F complexes in nuclear extracts prepared from quiescent (top panels) and serum-stimulated (bottom panels) cultures of wild-type, E2F4+/−, and E2F4−/− MEFs. The identity of complexes is demonstrated by the positions of the complexes in the gel as well as antibody supershifts (right panels). NS refers to nonspecific DNA-protein complexes. (B) Gel mobility shift analysis of E2F complexes in nuclear extracts prepared from E2F4+/+ and E2F4−/− thymocytes. (C) Kinetics of growth regulation in wild-type, E2F4+/− and E2F4−/− MEFs. Left panel: kinetics of S phase induction in MEFs serum starved for 48 hr and then stimulated with serum. At the indicated times, BrdU (10 μM) was added and cells were incubated 2 hr prior to fixation and immunostaining with BrdU-specific antibodies. Cells were visualized by fluorescent microscopy and the percentage of cells staining positively for BrdU determined. Right panel: response of MEFs to serum withdrawal. Cycling MEFs were brought into quiescence by replacement of the culture medium with DMEM containing 0.2% serum. BrdU was added at 6 hr intervals, and the cells were fixed, immunostained, and BrdU incorporation assessed. Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)

Figure 3 Analysis of E2F4 Expression Western analysis of E2F4 protein expression in adult mouse tissues. Protein lysates (100 μg) from selected tissues were processed for Western blot analysis and probed with a polyclonal antibody to E2F4. Nonspecific bands (NS) represent protein species that were not blocked by preincubation of the α-E2F4 antibody with the immunizing peptide. Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)

Figure 4 Loss of E2F4 Activity Leads to Abnormalities in Hematopoietic Development (A) Peripheral blood stained with Wright-Giemsa from 3-day-old E2F4+/+ and E2F4−/− mice. (B) Bone marrow from 3-day-old E2F4+/+ and E2F4−/− mice. Bone marrow was collected from the long bones, red blood cells were lysed, and the white blood cells were cytocentrifuged onto slides and stained with Wright-Giemsa for the analysis of cell morphology. Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)

Figure 5 Quantitative Analysis of Hematopoietic Lineage Defects in E2F4 Mice (A) Light scatter profile of nucleated cells from the bone marrow of a 3-day-old E2F4−/− mouse compared to bone marrow from 3-day-old E2F4+/+ and E2F4+/− mice. The R1 region indicates the total population of cells analyzed. The R2 region indicates where granulocytes/monocytes are located. The R3 region indicates where erythroid precursors fall on the scatter profile. (B) FACS analysis of bone marrow cells stained with antibodies to the erythroid surface marker TER-119 and counter stained with anti-CD3e that recognizes T lymphocytes. (C) FACS analysis of bone marrow cells stained with antibodies to the mature monocyte/granulocyte surface marker Mac-1, and the mature granulocyte surface marker Gr-1. (D) FACS analysis of bone marrow cells stained with an antibody to the B lymphocyte marker B220 and counter stained with anti-CD3e. (E) FACS analysis of thymocytes from 6-day-old E2F4+/+, E2F4+/−, and E2F4−/− mice stained with CD4 and CD8 antibodies. Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)

Figure 6 Analysis of Apoptosis in Hematopoietic Lineage Cells (A) FACS analysis of total bone marrow and Gr-1/Mac-1 cell populations derived from the indicated genotypes and stained with 7-AAD and annexinV-PE. Bone marrow samples isolated from E2F4+/+ (n = 18) mice and E2F4−/− (n = 5) mice (3 to 6 days old) were stained with annexin V-PE, 7-AAD, and anti-Gr-1 and anti-Mac-1 antibodies. Cells out of the total marrow expressing high levels of Gr-1 and Mac-1 were gated to assess apoptosis in the myeloid subpopulation. (B) Quantitation of the cell populations that are positive for annexinV staining. Total apoptotic cells, subdivided into annexin positive, 7-AAD negative, and annexin positive, 7-AAD positive groups were graphed and error bars indicated. Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)

Figure 7 Loss of E2F4 Activity Leads to Abnormalities in the Intestinal Epithelium (A) Intestinal sections from 3-day-old mouse siblings, left panel E2F4+/− and right panel E2F4−/− (5 μM sections, hematoxylin and eosin stained). (B) Proximal intestinal sections from 18-day-old mouse siblings, left panel E2F4+/− and right panel E2F4−/− (5 μM sections, hematoxylin and eosin stained). (C) Distal intestinal (ileum) sections from 18-day-old mouse siblings, left panel E2F4+/− and right panel E2F4−/− (5 μM sections, hematoxylin and eosin stained). (D) Distal intestinal (ileum) sections stained for mucin from 4-day-old mouse siblings, left panel E2F4+/− and right panel E2F4−/− (5 μM sections, Alcian Blue stained). Molecular Cell 2000 6, 293-306DOI: (10.1016/S1097-2765(00)00030-7)