The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency

Abstract

Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. Here, we show that in mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas, whereas these tumors do not form in Dmrt1 mutant C57BL/6J mice. Conditional gene targeting indicates that Dmrt1 is required in fetal germ cells but not in Sertoli cells to prevent teratoma formation. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes, whose missexpression may underlie teratoma formation. DMRT1 indirectly activates the GDNF coreceptor Ret, and it directly represses the pluripotency regulator Sox2. Analysis of human germ cell tumors reveals similar gene expression changes correlated to DMRT1 levels. Dmrt1 behaves genetically as a dose-sensitive tumor suppressor gene in 129Sv mice, and natural variation in Dmrt1 activity can confer teratoma susceptibility. This work reveals a genetic link between testicular dysgenesis, pluripotency regulation, and teratoma susceptibility that is highly sensitive to genetic background and to gene dosage.

Fig. 1.

129Sv Dmrt1^−/− mice have a high incidence of teratomas. (A and B) Hematoxylin and eosin (H&E) staining of testis sections from adult B6 mice. (A) Wild-type; (B) Dmrt1^−/−; (C) testes from 129Sv adults. Left: Wild-type; middle: Dmrt1^−/− dysgenic; right: Dmrt1^−/− teratoma. (D) Teratoma incidence in 129Sv Dmrt1 mutant mice, tumors per testis. (E) H&E staining of embryonal carcinoma cells at day of birth in 129Sv Dmrt1^−/−. (F) H&E staining of 4-week-old teratoma from 129Sv Dmrt1^−/−. (Scale bars, 10 μm.)

Fig. 2.

Reduced Ret expression in Dmrt1 mutant testes (A) Heat map showing 11 mRNAs reduced >2-fold, and eight mRNAs elevated >2-fold in Dmrt1^−/− compared to wild-type. (B) qRT-PCR of Ret mRNA at E13.5, normalized to Hprt. Error bars: SD from testes of six animals of each genotype (**, P < 0.005). (C–H) Double staining for the germ cell marker TRA98 (C and F) and RET (D and G) in wild-type (C–E) and Dmrt1^−/− (F–H) testis sections. (Scale bars, 20 μm.)

Fig. 3.

DMRT1 controls mitotic proliferation in 129Sv germ cells. (A–F) Double staining for the germ cell marker TRA98 (A and D) and active cell cycle marker Ki67 (B and E) in wild-type (A–C) and Dmrt1^−/− (D–F) testis sections. Green cells in merged images (C and F) are mitotically active somatic cells, whereas yellow cells are mitotically active germ cells. (Scale bars, 50 μm.) (G) Percentage Ki67 positive germ cells in wild-type vs. Dmrt1^−/− (**, P < 0.005).

Fig. 4.

DMRT1 represses pluripotency regulators (A–F) Expression of pluripotency regulators and E-cadherin (D–F). Clusters of OCT3/4, NANOG, and SOX2 expressing cells present in Dmrt1^−/− testes at birth but not in Dmrt1^+/− controls. Cells expressing elevated E-cadherin are within and adjacent to clusters of cells expressing pluripotency regulators. (Scale bars, 50 μm.) (G) qRT-PCR of Sox2, Nanog, Oct3/4, and E-cadherin mRNAs at E15.5 in wild-type vs. Dmrt1^−/− using Hprt as a normalizer. Error bars: SD from three animals of each genotype. (*, P < 0.01, **, P < 0.005). (H) ChIP-qPCR of Sox2 and Btg2 regulatory regions, comparing enrichment in chromatin immunoprecipitated with DMRT1 relative to input chromatin. Numbering below gene names indicates region covered by amplicons tested, relative to start of transcription for each gene. The negative control promoter Btg2 was chosen because it is expressed at similar levels in germ cells and Sertoli cells and does not change expression in Dmrt1 mutant testes. Error bars: SD of duplicate qPCR of sample.

Fig. 5.

Dmrt1 dose sensitivity in teratoma formation. Percentage of testes with teratomas is indicated for each genotype.