The absence of mitochondrial thioredoxin 2 causes massive apoptosis, exencephaly, and early embryonic lethality in homozygous mice

Abstract

Thioredoxin 2 (Trx-2) is a small redox protein containing the thioredoxin active site Trp-Cys-Gly-Pro-Cys that is localized to the mitochondria by a mitochondrial leader sequence and encoded by a nuclear gene (Trx-2). Trx-2 plays an important role in cell viability and the regulation of apoptosis in vitro. To investigate the role of Trx-2 in mouse development, we studied the phenotype of mice that have the Trx-2 gene silenced by mutational insertion. Homozygous mutant embryos do not survive to birth and die after implantation at Theiler stage 15/16. The homozygous mutant embryos display an open anterior neural tube and show massively increased apoptosis at 10.5 days postcoitus and are not present by 12.5 days postcoitus. The timing of the embryonic lethality coincides with the maturation of the mitochondria, since they begin oxidative phosphorylation during this stage of embryogenesis. In addition, embryonic fibroblasts cultured from homozygous Trx-2-null embryos were not viable. Heterozygous mice are fertile and have no discernible phenotype visible by external observation, despite having decreased Trx-2 mRNA and protein. These results show that the mitochondrial redox protein Trx-2 is required for normal development of the mouse embryo and for actively respiring cells.

FIG. 1.

Disruption of mouse Trx-2. (A) Diagram showing the three exons of the Trx-2 locus (with an arrow indicating the location of active-site cysteines) (not drawn to scale), the retroviral insertion of LTR-SA-BGEO-pA-PGK-neo-SD-LTR2 (hatched boxes) 351 bp upstream of exon 1, and the location of genotyping PCR primers A and B. (B) Genotyping of mouse DNA by two PCRs: one PCR for the mutant allele (lanes A) and one PCR for the wild-type allele (lanes B). Trx-2^+/− mice are positive for both A and B, Trx2^+/+ (wild-type) mice are positive only for B, and Trx-2^−/− mice are positive only for A. The top bands are the specific PCR products, and the bottom bands present in all of the lanes are the primer dimer.

FIG. 2.

Anterior neural tube defect and increased apoptosis in homozygous Trx-2^−/− mice at 9.5 and 10.5 dpc (representative embryos and images). Aberrant anterior neural tube closure in Trx-2^−/− embryo, indicated by arrows, was compared to the wild-type embryo by hemotoxylin and eosin staining of paraffin-embedded embryos at 9.5 dpc (A and B) and at 10.5 dpc (E and F). In panels C and D are shown increased anti-cleaved caspase-3 antibody-positive cells from Trx-2^−/− embryos compared to wild-type embryos by immunohistochemical staining of paraffin-embedded 9.5-dpc embryos (adjacent slices of the embryos shown in panels A and B). In panels G and H, massive apoptosis in 10.5-dpc Trx-2^−/− embryos can be seen compared to the wild-type embryos as visualized by anti-caspase-3 antibody staining of paraffin-embedded embryos (adjacent slices of the embryos shown in E and F). Heavy staining in the uterine tissue is not specific to cleaved caspase-3 and is due to endogenous mouse immunoglobulin G interactions with the secondary antibody.

FIG. 3.

Loss of Trx-2 protein in Trx-2^−/− embryos. A Western blot analysis of Trx-2 protein in 30 μg of tissue homogenate from 9.5-dpc embryos is shown.

FIG. 4.

Decrease of Trx-2 transcript/Trx-2 protein in heterozygous mice. (A) Trx-2 expression as shown by Northern blot analysis of wild-type and heterozygous Trx-2^+/− mice on 2 μg of liver mRNA probed with Trx-2 and GAPDH cDNA. (B) Trx-2 protein expression in the brains, livers, hearts, lungs, and kidneys of wild-type Trx-2^+/+ and heterozygous mice Trx-2^+/− by Western blot of 20 μg of tissue homogenate probed with α-Trx-2 rabbit polyclonal antibody.

FIG. 5.

Effects of decreased Trx-2 protein expression on the mitochondrial thioredoxin system, MnSOD, and catalase. (A) Western blot analysis of 20 μg of liver mitochondrial lysates from Trx-2^+/+ and Trx-2^+/− mice. The same blot was stripped and reprobed with antibodies to TRX-2 (14 kDa), TRXR-2 (56 kDa), PRDX-3 (22 kDa), MnSOD (24 kDa), and catalase (65 kDa). (B) Western blot analysis of TrxR-2 protein in 30 μg of tissue homogenate from 9.5-dpc embryos. (C) TrxR-2 gene expression in Trx-2^+/+ and Trx-2^+/− mice analyzed by Northern blot with TrxR-2 and GAPDH cDNA.