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• W2006351736 abstract "Article15 February 2002free access The centrosomal protein TACC3 is essential for hematopoietic stem cell function and genetically interfaces with p53-regulated apoptosis Roland P. Piekorz Roland P. Piekorz Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Angelika Hoffmeyer Angelika Hoffmeyer Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Christopher D. Duntsch Christopher D. Duntsch Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, University of Tennessee Health Science Center, Memphis, TN, 38105 USA Search for more papers by this author Catriona McKay Catriona McKay Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Present address: Blood Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Search for more papers by this author Hideaki Nakajima Hideaki Nakajima Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Present address: Blood Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Search for more papers by this author Veronika Sexl Veronika Sexl Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Present address: Department of Pharmacology, University of Vienna, A-1090 Vienna, Austria Search for more papers by this author Linda Snyder Linda Snyder Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Jerold Rehg Jerold Rehg Memphis, TN, 38063 USA Search for more papers by this author James N. Ihle Corresponding Author James N. Ihle Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, University of Tennessee Health Science Center, Memphis, TN, 38105 USA Search for more papers by this author Roland P. Piekorz Roland P. Piekorz Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Angelika Hoffmeyer Angelika Hoffmeyer Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Christopher D. Duntsch Christopher D. Duntsch Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, University of Tennessee Health Science Center, Memphis, TN, 38105 USA Search for more papers by this author Catriona McKay Catriona McKay Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Present address: Blood Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Search for more papers by this author Hideaki Nakajima Hideaki Nakajima Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Present address: Blood Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Search for more papers by this author Veronika Sexl Veronika Sexl Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Present address: Department of Pharmacology, University of Vienna, A-1090 Vienna, Austria Search for more papers by this author Linda Snyder Linda Snyder Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Search for more papers by this author Jerold Rehg Jerold Rehg Memphis, TN, 38063 USA Search for more papers by this author James N. Ihle Corresponding Author James N. Ihle Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA Department of Biochemistry, University of Tennessee Health Science Center, Memphis, TN, 38105 USA Search for more papers by this author Author Information Roland P. Piekorz1,2, Angelika Hoffmeyer1,2, Christopher D. Duntsch2,3, Catriona McKay1,2,4, Hideaki Nakajima2,4, Veronika Sexl2,5, Linda Snyder1,2, Jerold Rehg6 and James N. Ihle 1,2,3 1Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN, 38105 USA 2Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, 38105 USA 3Department of Biochemistry, University of Tennessee Health Science Center, Memphis, TN, 38105 USA 4Present address: Blood Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan 5Present address: Department of Pharmacology, University of Vienna, A-1090 Vienna, Austria 6Memphis, TN, 38063 USA ‡A.Hoffmeyer, C.D.Duntsch, C.McKay and H.Nakajima contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:653-664https://doi.org/10.1093/emboj/21.4.653 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TACC3 is a centrosomal/mitotic spindle-associated protein that is highly expressed in a cell cycle-dependent manner in hematopoietic lineage cells. During embryonic development, TACC3 is expressed in a variety of tissues in addition to the hematopoietic lineages. TACC3 deficiency causes an embryonic lethality at mid- to late gestation involving several lineages of cells. Hematopoietic stem cells, while capable of terminal differentiation, are unable to be expanded in vitro or in vivo in reconstitution approaches. Although gross alterations in centrosome numbers and chromosomal segregation are not observed, TACC3 deficiency is associated with a high rate of apoptosis and expression of the p53 target gene, p21Waf1/Cip1. Hematopoietic stem cell functions, as well as deficiencies in other cell lineages, can be rescued by combining the TACC3 deficiency with p53 deficiency. The results support the concept that TACC3 is a critical component of the centrosome/mitotic spindle apparatus and its absence triggers p53-mediated apoptosis. Introduction The centrosome is essential for the organization of the bipolar mitotic spindle during cell division. The functions of centrosome/spindle apparatus-associated proteins can be envisioned to be structural, e.g. γ-tubulin, forming ring complexes that are associated with centrosomes (Moritz and Agard, 2001) and mediate the assembly and maintenance of the spindle apparatus; functional, e.g. motor proteins like dynein or CENP-E (Compton, 2000) providing the reactions necessary for chromosomal positioning, attachment and segregation; or regulatory, e.g. the signaling proteins Mad2 and Bub1 as components of the spindle assembly checkpoint, which senses unattached chromosomes and therefore the integrity of the mitotic process (Shah and Cleveland, 2000). During the mitotic phase, a large number of regulatory proteins become associated with the centrosome/spindle apparatus including the tumor suppressor gene products Rb (Thomas et al., 1996), BRCA1 (Hsu and White, 1998) and p53 (Morris et al., 2000). Also associated with the centrosome/spindle apparatus during mitosis are members of a recently identified and evolutionarily conserved protein family, referred to as the TACC (transforming acidic coiled-coil-containing) family. These proteins are characterized by a unique C-terminal coiled-coil domain of ∼200 amino acids, but lack significant homology outside of this domain (Gergely et al., 2000a). The mammalian TACC family consists of three genes. TACC1, the first family member identified, was discovered as a gene amplified in breast cancer (Still et al., 1999a). A related gene (AZU-1/TACC2) was identified by homology and its expression was found to be reduced in breast cancer (Chen et al., 2000). ECTACC, a splice variant of the human TACC2 gene, shows increased expression in endothelial cells upon erythropoietin (Epo) treatment and is predominantly found in heart and skeletal muscle (Pu et al., 2001). Lastly, the TACC3 gene was identified in a yeast two-hybrid screen for genes encoding ARNT-interacting proteins (Sadek et al., 2000), by gene homology to TACC1 (Still et al., 1999b), as an Epo-induced gene in erythroid progenitors (McKeveney et al., 2001) or, in our case, as a Stat5-interacting gene product in yeast two-hybrid screens (our unpublished data). In addition to the mammalian genes, a Drosophila TACC (dTACC) was identified as a centrosomal protein that interacts with microtubules and plays an essential role in normal spindle function during embryogenesis (Gergely et al., 2000a). A 10-fold reduction in dTACC expression leads to mitotic defects and death during early embryogenesis and female sterility in adults. Localization of the protein to the mitotic spindle requires the C-terminal coiled-coil domain and this domain is sufficient to target heterologous proteins to the centrosomal complex (Gergely et al., 2000a). Recent studies have indicated that the localization may be mediated by interaction with another centrosomal/spindle apparatus-associated and evolutionarily conserved protein, Msps/XMAP215/ch-TOG (Cullen and Ohkura, 2001; Lee et al., 2001). Since Msps/XMAP215/ch-TOG associates with microtubuli and localizes preferentially at the centrosome, these studies provided a mechanism linking the dTACC–Msps complex to the stability of centrosomal microtubuli in Drosophila embryos. Lastly, in Xenopus, the TACC-related gene is termed Maskin based on its isolation in a complex containing CPEB (cytoplasmic polyadenylation element binding factor) and eIF-4E-associated protein during oocyte maturation (Stebbins-Boaz et al., 1999). A role for Maskin in translational regulation has been proposed since this complex localizes certain mRNAs, such as cyclin B1, to the mitotic apparatus/centrosome and thereby allows the regulation of mRNA translation with cell cycle progression by the integrity of the mitotic machinery (Groisman et al., 2000). The functions of the mammalian TACC proteins are largely unknown. The identification of TACC1 as a highly amplified gene in breast cancer tissues suggests a role in malignant transformation (Still et al., 1999a). In contrast, a TACC2 splice variant is expressed at low levels in tumorigenic cell lines and its overexpression reduces the malignant phenotype leading to the suggestion that TACC2 might function as a tumor suppressor gene (Chen et al., 2000). Finally, TACC3 has been proposed to play a role in hypoxic responses based on its association with ARNT, a gene essential for hypoxic responses (Sadek et al., 2000). Irrespective, the characteristic localization of all mammalian members at the centrosome/mitotic spindle indicates a role of these proteins in chromosomal segregation and cell division (Gergely et al., 2000b). To explore the function of the mammalian TACC3 gene, we have characterized the lineage specificity of its expression, explored the cell cycle dependence for its expression in lymphocytes and examined its subcellular localization. To further establish the physiological relevance of the gene we have developed mutant strains of mice lacking the gene. Together our studies demonstrate that TACC3 is highly tissue specific in its normal pattern of expression and within the lymphoid lineages it is specifically expressed during the S/G2/M phases of the cell cycle at which time it is associated predominantly with the mitotic spindle and centrosomal regions. The absence of TACC3 results in an embryonic lethality, demonstrating the essential role of TACC3 protein for cell expansion during embryonic development and in hematopoiesis. The defects caused by TACC3 deficiency are obviated on a p53-deficient background, supporting the concept that the TACC3 protein is a critical sensor of the mitotic apparatus that couples to a p53-dependent pathway to regulate cell cycle progression. Results Lineage and cell cycle-dependent expression of TACC3 As illustrated in Figure 1A, relatively few adult tissues express TACC3 mRNA with the exception of lung and high levels of expression in testis. However, TACC3 is expressed at high levels in all hematopoietic sites including bone marrow, fetal liver, thymus and spleen. This pattern of expression is quite distinct from that of TACC2 (Figure 1A) or TACC1 (not shown), which are not expressed in hematopoietic tissues. In particular, TACC2 is more widely expressed, with the highest levels of transcripts being present in heart and muscle. Two mRNAs of 4 and 10 kb are seen in most tissues although the ratios vary. Muscle and heart predominantly express the 10 kb transcript while the 4 kb transcript is more ubiquitously expressed. These results are similar to the pattern of expression of the TACC2 mRNA in human tissues (Pu et al., 2001). Figure 1.Lineage-dependent expression of the TACC3 gene. (A) The distribution of TACC3 and TACC2 transcripts in tissues from adult mice (lanes 1–12) and during embryogenesis (days 7–17 of development; lanes 13–16) was determined by northern blot analysis [lanes 1–8 and 13–16, poly(A)+ mRNA; 9–12, total RNA]. (B) Preactivated T cells growing in IL-2 (gc) were starved overnight (st.) and restimulated with IL-2 for the indicated periods of time. TACC3 mRNA levels were analyzed by northern blotting. The percentage of cells in S phase of the cell cycle is indicated (nd, not determined). Hybridization for GAPDH [lanes 1–8 and 13–16 in (A)] or staining of 28S rRNA with ethidium bromide [lanes 9–12 in A, lanes in (B)] was used to control RNA loading. Download figure Download PowerPoint In T lymphocytes, TACC3 expression is cell cycle regulated (Figure 1B). In these experiments, T cells were expanded in vitro with anti-CD3 and interleukin-2 (IL-2) starved of cytokines overnight and restimulated with IL-2. As illustrated, starved T cells (st) have little TACC3 gene transcripts but expression is induced following stimulation with IL-2. The highest levels of expression occurred at 18 h following stimulation at which time 60% of the cells were through the S-phase. Comparable results were obtained with naïve T cells or B cells stimulated with anti-CD3 and IL-2 or anti-IgM and IL-4, respectively (data not shown). Therefore, TACC3 is a cell cycle-regulated, non-immediate early gene in the response of lymphocytes to cytokines. During embryonic development, TACC3 gene transcripts are detected starting at E7, strongly upregulated at E11, and expression is maintained throughout the remainder of embryogenesis (Figure 1A). At E18.5, expression was detected by in situ hybridization in a variety of developing organs/tissues including the neuroepithelium of the forebrain, epithelial cells of the gut as well as epithelial cells of other organs (data not shown). The highest levels of TACC3 expression were found in the fetal liver throughout the last half of embryogenesis and in the developing fetal thymus (data not shown). In contrast, only low levels of the two TACC2 transcripts are observed during embryonic development. The results support the conclusion that TACC3 is highly expressed within the hematopoietic lineages as well as in developing epithelial layers of various organs. Localization of TACC3 to the centrosome/mitotic apparatus To establish the subcellular localization of the murine TACC3 protein, exponentially growing mouse embryonic fibroblasts were stained with purified anti-mouse TACC3 antibodies. As illustrated in Figure 2, in mitotic cells, TACC3 protein was associated predominantly with the centrosomal region and mitotic spindle and colocalized with α-tubulin on spindle microtubules and γ-tubulin in the centrosomal region (data not shown). In interphase cells, TACC3 localizes to the cytoplasm and perinuclear region (data not shown). These results are comparable to recent studies examining the subcellular localization of the human TACC3 protein (Gergely et al., 2000b). Figure 2.Centrosomal localization of TACC3 during mitosis. Immunofluorescence detection of endogenous TACC3 protein (A, C and E) and DNA (B, D and F) in mouse embryo fibroblasts during different phases of mitosis (metaphase, A and B; anaphase, C and D; and telophase, E and F). Download figure Download PowerPoint TACC3 deficiency causes growth retardation and embryonic lethality The physiological functions of TACC3 were assessed by deriving TACC3-deficient mice. The targeting vector (Figure 3A) was designed to disrupt the third exon and would be predicted to alter splicing in such a manner as to generate a null mutation. Heterozygous TACC3-deficient mice were phenotypically normal and were bred to obtain homozygously null mice. As detailed below, homozygous TACC3 deficiency was associated with embryonic lethality at mid- to late gestation. Analysis of RNA and protein from fetal livers, or total embryo extracts, of homozygous mutant mice is illustrated in Figure 3C and D. No transcripts were detected that hybridized with a TACC3 cDNA probe, indicating that the genomic modifications resulted in a null mutation. Consistent with the RNA data, immunoprecipitation and western blotting with an antiserum against an N-terminal peptide that would be retained in an alternatively spliced RNA capable of encoding an internally deleted TACC3 protein, failed to detect any immunoreactive protein. Together the results demonstrate that the mutated locus creates a TACC3 protein null mutant strain. Figure 3.Targeted disruption of the TACC3 gene. (A) Structure and targeting strategy of the TACC3 locus. Empty boxes indicate exons 1–5 of the TACC3 locus. The locations of the 3′ external probe and PCR primers for genotyping are indicated. ATG denotes the first coding exon. Restriction enzyme sites are as follows: A, AflII; E, EcoRI; H, HindIII. (B) Screening PCR of F1 mice and TACC3-targeted embryos for the presence of the disrupted allele. (C) Analysis of TACC3 mRNA and protein expression (D) in TACC3-deficient embryos. Total RNA was isolated from fetal liver cells, whereas protein lysates were obtained from whole embryos and analyzed by IP/western blotting. Download figure Download PowerPoint The distributions of genotypes of embryos from breedings of heterozygous mutant mice at various stages of embryonic development are tabulated in Table I. At mid-gestation (E9.5–11.5) the frequency of homozygously null embryos is close to the expected distribution. However, the frequency subsequently decreases to ∼40% of that expected from normal Mendelian distribution. This frequency is maintained until late in embryogenesis when a second period of embryonic lethality occurs such that at birth the frequency of homozygous null mutants is only 5% of that expected and none have been born alive among >55 litters that were closely monitored. Morphologically, TACC3-deficient embryos were readily detected by a striking growth retardation (Figure 4) throughout the second half of embryogenesis. Approximately two-thirds of the homozygous null embryos displayed a severe facial cleft. Histological sections from homozygous null embryos revealed normal organ structure, although most organs were smaller and underdeveloped relative to the controls (data not shown). Figure 4.Embryonic lethality and growth retardation due to TACC3 deficiency. Embryos were dissected at days 12.5 (A and B), 13.5 (C and D) and 17 (E and F) p.c. from the embryo sacs and were photographed. The left panels show the morphology of wild-type embryos (A, C and E), while litter-mate mutant embryos on the right panels are characterized by a reduced size (B, D and F). The knockout embryo at E13.5 (D) displays a facial cleft. Download figure Download PowerPoint Table 1. Genotypic distribution of embryos and mice from TACC3 heterozygous intercrosses Stage No. of litters Total No. TACC3(+/+) TACC3(+/−) TACC3(−/−) (−/−) % 9.5–10.5 7 68 24 28 16 23.5 10.5–11.5 6 45 8 28 9 20 12–12.5 10 74 25 41 8 10.8 13–13.5 24 165 53 93 19 11.5 14–15.5 42 302 104 170 28 9.3 16–17.5 31 189 53 113 23 12.2 18–19 31 215 73 130 12 5.6 Newborns 55 297 88 209 (4)a (1.3) Embryos were collected between days 9.5 and 19.0 of pregnancy from TACC3(+/−) intercrosses. The frequency of mutant embryos is indicated in the table as a percentage of the total number of embryos analyzed. a New borns found on day 1 were dead. Hematopoietic deficiencies of TACC3 null embryos The high levels of expression of TACC3 in fetal liver prompted us to examine TACC3-deficient hematopoietic cells in detail. As illustrated in Table II, there was a profound deficiency in hematopoietic stem cell colony forming activity although the total number of fetal liver cells was only one-half to one-quarter of that of controls. In general, the frequency of colony forming cells in TACC3-deficient fetal livers was 1–5% of that of controls including cells responding to stem cell factor (SCF), IL-3 or mixtures of these cytokines with IL-6 and Epo. Among the various lineages, the least affected progenitors were those committed to the erythroid lineage (CFU-E) and responsive to Epo as a single cytokine. However, the more primitive progenitors of the erythroid lineage (BFU-E) were greatly reduced with TACC3 deficiency. In addition to a numerical reduction, the sizes of the individual colonies, in all conditions, were significantly smaller than controls (data not shown). In the lymphoid lineage, TACC3 deficiency resulted in a striking reduction in thymus size and in the number of fetal thymocytes, although the residual cells expressed both CD4 and CD8 (Figure 5A and B). Together the data indicate that hematopoietic stem cell differentiation is unaffected although the ability to proliferate and to expand is dramatically reduced. Figure 5.Delayed generation of thymocytes in TACC3-deficient embryos. (A) Histological appearance of thymi from wild-type and TACC3-deficient embryos (E18.5). (B) Fetal thymocytes from wild-type and TACC3-deficient embryos were analyzed for the percentage of CD4/CD8 double positive and single positive cells at the stages of development indicated. Download figure Download PowerPoint Table 2. Colony-forming ability of hematopoietic progenitors from fetal livers of wild-type and TACC3-deficient embryos Colony type Cytokine(s) Genotype TACC3(−/−) versus TACC3(+/+) (%) TACC3(+/+) TACC3(+/−) TACC3(−/−) CFU-Mix SCF 88 ± 34.5 (n = 4) 94 ± 48 (n = 2) 1.4 ± 1.4 (n = 4) 1.6 CFU-Mix (BFU-E) IL-3, IL-6, SCF, EPO 9 ± 5 (n = 5) 5 ± 3 (n = 6) 0.3 ± 0.3 (n = 6) 3.3 CFU-Mix (others) IL-3, IL-6, SCF, EPO 39 ± 12 (n = 5) 36 ± 7 (n = 6) 2.2 ± 1.8 (n = 6) 5.6 BFU-E IL-3, EPO 48 ± 21 (n = 5) 32 ± 13 (n = 6) 0.3 ± 0.3 (n = 6) <1 CFU-E EPO 888 ± 251 (n = 3) 900 ± 43 (n = 2) 168 ± 41 (n = 2) 18.9 CFU-Mix IL-3 261 ± 99 (n = 3) 241 ± 113 (n = 4) 14 ± 8 (n = 4) 5.3 CFU-GM GM-CSF 83 ± 29 (n = 3) 49 ± 9 (n = 4) 3 ± 3 (n = 4) 3.6 CFU-M M-CSF 240 ± 90 (n = 3) 224 ± 78 (n = 2) 10.3 ± 9.5 (n = 2) 4.5 CFU-Meg TPO 54 ± 6.7 (n = 2) n.d. 0.0 ± 0.0 (n = 2) 0.0 CFU-Eo IL-5 44 ± 17 (n = 3) 28 ± 2.8 (n = 2) 0.3 ± 0.3 (n = 2) <1 Data represent the mean ± SD of numbers of colonies/105 fetal liver cells. n.d., not determined. To further assess hematopoietic stem cell function, reconstitution experiments were used. Fetal liver cells from TACC3-deficient embryos were unable to reconstitute the myeloid lineages in lethally irradiated wild-type mice at either 2.5 × 106 or 2.5 × 107 transferred fetal liver cells/recipient (data not shown). The latter cell dose is ∼20-fold higher than that required for 80% reconstitution of lethally irradiated mice with wild-type cells. Fetal liver cells from TACC3-deficient embryos were also unable to reconstitute the lymphoid lineage in sublethally irradiated mice that had greatly reduced lymphoid lineage cells due to Jak3 deficiency (data not shown). The results indicate that TACC3 deficiency resulted in a cell intrinsic defect in hematopoietic stem cell function. The possibilities existed that the decreased proliferative capacity of TACC3-deficient cells was associated with a decreased rate of proliferation and/or an increased rate of apoptosis. To begin to distinguish between these possibilities, the frequency of apoptotic cells was examined at various stages of development. As illustrated in Figure 6, a dramatic increase in TdT-mediated dUTP nick end labeling (TUNEL)-positive cells was evident in the forebrain, gut and fetal liver as well as in other tissues (data not shown). To determine whether the increased apoptosis might be associated with the activation of p53, the expression of p21waf1/cip1 was examined in fetal liver cells. Consistent with a potential role for p53 in the increased apoptosis, high levels of p21Waf1/Cip1 protein were present in extracts of fetal livers from TACC3-deficient embryos (E16–E18.5) but not in extracts from wild-type fetal livers (Figure 6G). Figure 6.Increased apoptosis and expression of p21Waf1/Cip1 in fetal liver cells from TACC3-deficient embryos. Sagittal sections of E18.5 wild-type (A, C and E) and knockout embryos (B, D and F) were analyzed for the presence of apoptotic/TUNEL-positive (green) cells. Selected organs and structures shown include the forebrain cortex (A and B), gut (C and D) and fetal liver (E and F). (G) Protein levels of the cell cycle inhibitor p21Waf1/Cip1 in fetal liver cell extracts from wild-type and knockout embryos (E17.5). Download figure Download PowerPoint p53 deficiency rescues hematopoietic stem cell function of TACC3-deficient cells The above results were consistent with the concept that the absence of TACC3 was inducing a p53-mediated apoptosis. To explore this possibility, the TACC3 deficiency was crossed onto a p53 deficiency. In order to exclude background modifier genes on the p53 background, primarily progeny from a TACC3+/−; p53+/− F1 intercross were examined, including mice that were genotypically TACC3+/+; p53−/−. As illustrated in Figure 7A, p53 deficiency had a significant effect on the embryonic lethality. For example, during late embryogenesis, the ratio of the actual to the expected number of viable embryos increased from 0.49 to 1.1 (E16–E17.5) and from 0.22 to 0.92 (E18–E19) when TACC3-deficient embryos were heterozygously deficient for p53. Equally striking, TACC3-deficient embryos that were homozygously deficient for p53 were somewhat more susceptible to embryonic lethality than p53 wild-type embryos (reduction to 0.17 for E16–E17.5). The results suggest that the interaction of p53 with TACC3 is complex, with a reduction of p53 being beneficial while the complete absence increases lethality. Perhaps the most striking result, however, was the ability to obtain viable mice at a low frequency that were genotypically either heterozygous or homozygous for p53 deficiency and were homozygously TACC3 deficient (Figure 7B and C). This contrasts with the lack of any viable mice from over 55 litters examined to date of TACC3-deficient and p53+/+ embryos from the F1 intercross. Figure 7.Impact of p53 on the lethality of TACC3-deficient embryos. (A) The ratios of actual versus expected Mendelian frequencies were determined for TACC3-deficient embryos and mice dependent on the p53 background. Numbers of observed versus expected individuals alive: days 12.5–15.5 of embryonic development, 47/117 for p53(+/+), 10/11 for p53(+/−) and 7/11 for p53(−/−); days 16–17.5 of embryonic development, 23/47 for p53(+/+), 34/31 for p53(+/−) and 5/29 for p53(−/−); days 18–19 of embryonic development, 12/54 for p53(+/+), 12/13 for p53(+/−) and 0/12 for p53(−/−); mice, at least 4 weeks of age, 0/150 for p53(+/+), 8/107 for p53(+/−) and 3/67 for p53(−/−). TACC3-deficient mice that survive on a p53 heterozygous (B) or p53-deficient (C) background are characterized by the occurrence of kinky or twisted tails. Download figure Download PowerPoint Viable offspring from the above cross were further characterized. Characteristically, all the mice had a kinky tail (Figure 7B and C) and males and females were sterile. Peripheral blood patterns, analyzed by FACS with lineage specific markers, were found to be normal (data not shown). Moreover, the ability of T cells to proliferate in response to anti-CD3 and IL-2 was normal (data not shown). Lastly, bone marrow colony forming activity was normal. Among the three TACC3 and p53 double deficient mice that were allowed to age, one developed a sarcoma, one developed both a sarcoma and a thymoma while the third animal developed a peripheral T-cell lymphoma, all at ∼5 months of age. The results indicate that TA" @default.
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