A knockout mouse approach reveals that TCTP functions as an essential factor for cell proliferation and survival in a tissue- or cell type-specific manner

Abstract

Translationally controlled Tumor Protein (TCTP) is an evolutionally highly conserved protein which has been implicated in many cellular functions that are related to cell growth, death, and even the allergic response of the host. To address the physiological roles of TCTP, we generated TCTP knockout mice by targeted gene disruption. Heterozygous mutants appeared to be developmentally normal. However, homozygous mutants (TCTP(-/-)) were embryonic lethal. TCTP(-/-) embryos were smaller in size than the control littermates at all postimplantation stages examined. Although TCTP is widely expressed in both extraembryonic and embryonic tissues, the most prominent defect of the TCTP(-/-) embryo at embryonic stage day 5.5 (E5.5) was in its epiblast, which had a reduced number of cells compared with wild-type controls. The knockout embryos also suffered a higher incidence of apoptosis in epiblast starting about E6.5 and subsequently died around E9.5-10.5 with a severely disorganized structure. Last, we demonstrated that TCTP(-/-) and control mouse embryonic fibroblasts manifested similar proliferation activities and apoptotic sensitivities to various death stimuli. Taken together, our results suggest that despite that TCTP is widely expressed in many tissues or cell types, it appears to regulate cell proliferation and survival in a tissue- or cell type-specific manner.

Figure 1.

Targeted disruption of the TCTP gene. (A) The structures of the wild-type, targeting vector, and recombinant alleles are shown together with some relevant restriction sites (E, EcoRI; H, HindIII; K, KpnI; N, NdeI). The 5′ and 3′ probes and the predicted length of EcoRI or NdeI restriction fragments in Southern blot analysis are as indicated. (B) Southern blot analysis of the recombinant ES cell clones harboring the “targeted allele.” Genomic DNA extracted from ES cell clones (lanes 1 and 2, nonrecombinants; lanes 3 and 4, clones 248 and 280) was digested with NdeI and probed with the 5′probe. (C) Same as in B except that the genomic DNA was digested with EcoRI and probed with the 3′ probe. The predicted signals for the wild-type (wt) and targeted allele (mt) are as indicated. (D) Representative genotypic analysis of E9.5 embryos harboring the wt (+) or deleted allele (dl or “−”) of the TCTP gene from a TCTP+/− intercross. Genotyping was performed by PCR using primers P1 and P5 for the wild-type (wt, 450 base pairs) and P1 and P4 for the deleted allele (dl, 250 base pairs). (E) Immunoblotting analysis of representative E9.5 embryos with the indicated genotypes using antibodies specific to TCTP or β-actin.

Figure 2.

Morphological analysis of normal and TCTP null embryos. Photomicrographs of littermates dissected at various stages (E6.5–E9.5) as indicated. al, allantois; epc, ectoplacental cone; h, head folds; j, junction between the embryonic and extraembryonic regions. Scale bar in all panels, 100 μm.

Figure 3.

Brachyury and Shh expression in normal and TCTP-null embryos. Whole-mount in situ hybridization of wild-type and TCTP-null embryos at various stages as indicated was carried out using riboprobes specific to Brachyury (A and B) or Shh (C). (C) Left, E8.5 TCTP^+/− embryo; right, E9.5 TCTP^−/− embryo. am, axial mesendoderm; nt, notochord; n, node; ps, primitive streak; endo, endoderm. Scale bar in all panels, 100 μm.

Figure 4.

Histological analysis of E5.5 control and TCTP-null embryos “genotyped” by immunostaining. Sagittal sections of E5.5 embryos derived from heterozygous intercrosses were first stained with TCTP-specific antibody and visualized by confocal microscopy as described in Materials and Methods. After immunostaining (right panels of B and D), the same section was stained with hematoxylin and eosin (HE; left panels of B and D). (A and C) HE staining of the most central 4-μm sections of the same embryos as that shown in B and D, respectively. TCTP-expressing N (A and B) and nonexpressing M littermates (C and D) are as indicated. ve, visceral endoderm; epi, epiblast.

Figure 5.

Histological analysis of E6.5–E9.5 embryos derived from heterozygous intercrosses. Sagittal sections of TCTP-expressing (N) or nonexpressing (M) littermates at various stages as indicated. Arrows in A–D show the boundary between embryonic and extraembryonic regions. Embryos were “genotyped” by immunostaining using TCTP-specific antibodies as that described in the legend to Figure 4. ac, amniotic cavity; al, allantois; am, amnion; c, chorion; ce, cuboidal visceral endoderm; de, decidua; ec, ectoderm; ee, embryonic ectoderm; epc, ectoplacental cone; etc, ectoplacental cavity; ex, extraembryonic ectoderm; exc, exocoelomic cavity; h, head folds; m, mesoderm; pa, proamniotic cavity; SE, squamous visceral endoderm; so, somite. Arrowheads point to pyknotic cells. Scale bar, 50 μm (A, B, and F) and 100 μm (C and E–G).

Figure 6.

Increased apoptosis of TCTP-null mutants at the E6.5 but not E5.5 stage. Sections of TCTP(+) and TCTP(−) embryos at E5.5 (A and B) and E6.5 (C and D) stages were subjected to TUNEL analysis. Embryos were “genotyped” by immunostaining with TCTP-specific antibody. Apoptotic cells were stained brown. The graphs in E and F show mean percentages ± SEs of TUNEL-positive cells from three to four independent experiments. Only the difference between TCTP(+) and TCTP(−) embryos at the E6.5 stage was statistically significant; *p = 0.007; n = 3 (F). (D) An arrow points to one example of apoptotic cells. Scale bar, 25 μm (A and B) and 50 μm (C and D).

Figure 7.

Reduced expression of D- and E- but not B-type cyclins in TCTP^−/− embryos. (A) Protein extracts from E9.5 embryos with the indicated genotypes were analyzed by immunoblotting using antibodies specific to various proteins as indicated. Lane 1 was the extracts pooled from three embryos with mutant phenotype as shown in Figure 2D. Asterisk (*) indicates cyclin D3 cross-reacted with cyclin D2 antibody. (B) Quantitative RT-PCR was carried out using RNA isolated from E9.5 embryos with normal or abnormal morphology as described in Materials and Methods. Shown here is the relative mRNA level of various genes as indicated in abnormal versus normal embryos. The results (mean ± SD) were plotted from two independent experiments done in triplicate.

Figure 8.

Control and TCTP^−/− MEFs manifest similar proliferation and survival activities. (A) TCTP^f/f and TCTP^−/− MEFs grow similarly in culture. Control or TCTP^−/− MEFs were seeded in complete growth medium, and at various time points after seeding cells were counted by trypan blue exclusion assays. Inset in A shows the TCTP protein levels in cells used in this study. (B) Control and TCTP^−/− MEFs display similar cell size. Exponentially growing control or TCTP^−/− MEFs were stained by propidium iodide and analyzed by flow cytometry. The DNA content (PI) and the forward scatter height (FSC-H) distributions of cells at the G1 or G2/M phase of the cell cycle are as indicated. (C) Control and TCTP^−/− MEFs manifest similar apoptotic sensitivities to death inducers. Control or TCTP^−/− cells were treated with two different doses of various apoptotic stimuli as indicated. One day after treatment, cells that had undergone apoptosis were quantified by annexin V staining assay. Data shown here are mean ± SD of triplicates from experiments that were repeated three times with very similar results. Eto, etoposide; THG, thapsigargin; TUN, tunicamycin. (D) Similar activation of S6K1, Akt, and ERK after serum stimulation of control or TCTP^−/− MEFs. Cells to be analyzed were starved and restimulated with serum for various times as indicated. The same cell lysates were analyzed by two immunoblots (top and bottom) using antibodies specific to TCTP, β-actin, total, or active form of S6K1 (P-T389), Akt (P-S473), and ERK as indicated.