Volume 5, Issue 6, Pages 993-1002 (June 2000) DNA Ligase IV Deficiency in Mice Leads to Defective Neurogenesis and Embryonic Lethality via the p53 Pathway  Karen M Frank, Norman E Sharpless, Yijie Gao, JoAnn M Sekiguchi, David O Ferguson, Chengming Zhu, John P Manis, James Horner, Ronald A DePinho, Frederick W Alt  Molecular Cell  Volume 5, Issue 6, Pages 993-1002 (June 2000) DOI: 10.1016/S1097-2765(00)80264-6

Figure 1 Lig4 Null Mice in a p53-Deficient Background Have a Profound Growth Deficiency and a Profoundly Decreased Survival Potential (A) Weights of mice with increasing age. All animals included are male. Number of mice included for each genotype are indicated in parentheses. The last three points for Lig4−/− p53+/− are the average weights of two mice. Genotypes are indicated for each panel. (B) Percentage of mice of each genotype surviving at each age. Of the 16 Lig4−/− p53−/− mice analyzed, 15 succumbed to disseminated lymphoma with enlarged lymph nodes, spleens, and thymuses. (C) Photograph of 4-week-old sibling littermates illustrating the profound size difference between Lig4−/− mice in a p53 heterozygous or null background compared to controls. Genotypes are indicated. Molecular Cell 2000 5, 993-1002DOI: (10.1016/S1097-2765(00)80264-6)

Figure 2 Rescue of Neuronal Apoptosis Correlates with Rescue of Embryonic Lethality Histological analysis of CNS (cortex) from progeny of Lig4-deficient mice crossed with p53- or INK4a/ARF-deficient mice. Coronal sections were stained with hematoxylin and eosin. Apoptosis was identified by the presence of pyknotic nuclei (dense hematoxylin stain, indicated by arrows). Representative sections are shown. (A and B) Lig4+/+ INK4aΔ2/3−/−, E13.5. (C and D) Lig4−/− INK4aΔ2/3−/−, E13.5. (E and F) Lig4−/− p53+/−, E13.5. (G and H) Lig4−/− p53−/−, E14.5. (I and J) Lig4−/−, E13.5. (A, C, E, G, and I) 400× original magnification. (B, D, F, H, and J) 1000× original magnification. VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. Molecular Cell 2000 5, 993-1002DOI: (10.1016/S1097-2765(00)80264-6)

Figure 3 Analysis of Lymphocyte Development and Pro-B Cell Lymphomas in Lig4−/− p53−/− Mice (A) Flow cytometric analysis of thymocytes from 3-week-old mice showing CD4 and CD8 profile. The percentage of CD4+ CD8+ cells is shown. Genotypes and cellularity of each thymus are indicated above each panel. DP, CD4+ CD8+ double positive. (B) Flow cytometric analysis of bone marrow lymphocytes from 3-week-old mice showing B220 and IgM profile. The percentage of B220+ IgM+ cells is shown. Genotypes are the same as indicated above (A). (C) Flow cytometric analysis of lymphocytes from E15 fetal liver cultures showing B220 and IgM profile. The percentage of B220+ IgM+ cells is shown. Genotypes are indicated above each panel. (A–C) Data shown are representative of at least three separate experiments. (D) Southern analyses of lymphoma DNA. EcoRI-digested DNA from Rag2−/− bone marrow (RBM), Rag2−/− spleen (RS), Rag2−/− thymus (NT), control spleen (NS), and lymphomas from Lig4−/− p53−/− mice probed with JH (top panel), HS3a (second panel), c-Myc (third panel), or DNA-PKcs (loading control, bottom panel) probes. Bold numbers, relative gene dosage based on setting RS control to 2. PCR: relative levels of c-Myc dosage as revealed by quantitative fluorogenic PCR. Asterisks indicate that rearrangements of the IgH J locus in control spleen (NS) and Lig4−/− p53−/− pro-B cell lymphomas result in the appearance of submolar bands of various sizes, some of which might be below the range of the depicted gels for Southern detection or deleted beyond the range of the JH probe; the varying levels of residual germline JH band (6.5 kb) in different tumor samples likely reflects varying degrees of normal tissue contribution to the sample. Quantitation for the JH gene dosage was only performed on bands that clearly represented amplification above the germline level in sample RS. A repeat experiment in which the lower molecular bands below 2 kB are visible demonstrated amplification of the JH locus in all samples. Doublets revealed by the HS3a probe in some samples represent a polymorphic difference between the 129 and C57B/6 alleles. Molecular Cell 2000 5, 993-1002DOI: (10.1016/S1097-2765(00)80264-6)

Figure 4 In Vitro Growth Characteristics of MEFs Doubly Null for Lig4 and p53 (A) Growth curves of Lig4−/− p53−/− MEFs and controls. An average of two experiments is shown. Genotypes are indicated on the graph for (A–C). (B) 3T9 Assay of Lig4−/− p53−/− MEFs and controls. E13.5 MEFs were serially passaged as described in Experimental Procedures. Results represent two lines per genotype, with replicate plates per line. (C) Dose response to ionizing radiation. Cells were seeded, irradiated, and counted as described in Experimental Procedures. At later time points, some Lig4−/− p53−/− cells were able to proliferate. This graph is representative of three experiments. Molecular Cell 2000 5, 993-1002DOI: (10.1016/S1097-2765(00)80264-6)

Figure 5 In Vitro Growth Characteristics of MEFs Doubly Null for Lig4 and INK4a/ARF (A) 3T9 Assay of Lig4−/− INK4aΔ2/3−/− MEFs and controls. Genotypes are indicated in graph. Results represent two lines per genotype, with replicate plates per line. (B) p53 Western: INK4a/ARF null cells that were either wild type, heterozygous, or null for Lig4 were seeded at equal density 24 hr prior to harvesting protein. UVB treated samples were irradiated 12 hr prior to lysis. These data are representative of multiple experiments performed on two or more lines per genotype (ten cultures examined). (C) Dose response to ionizing radiation. INK4a/ARF null MEFs that were either wild type, heterozygous, or null for Lig4 were seeded, irradiated, and counted as described in Experimental Procedures. Genotypes are indicated in graph. This graph is representative of two experiments. Molecular Cell 2000 5, 993-1002DOI: (10.1016/S1097-2765(00)80264-6)