Abstract
DM domain transcription factors play important roles in sexual development in a wide variety of species from invertebrate to humans. Among seven mammalian family members of DM domain transcription factors, DMRT1 has been studied in mouse and human for its conserved role in male gonadal identity. Chromosomal deletion of 9p24.3, the region in which DMRT1 is located, is associated with 46,XY gonadal dysgenesis. Dmrt1 knockout (KO) mice also showed male-to-female gonadal reprogramming. However, the phenotype of Dmrt1 KO mouse appears only after birth while 46,XY gonadal dysgenesis occurs during the developmental phase, and the cause behind this difference remained unknown. We hypothesized that in human the function of other DMRT genes clustered with DMRT1, namely DMRT3, might also be impaired by the chromosomal deletion, which leads to the gonadal dysgenesis phenotype. Thus, simultaneous loss of multiple DM domain genes in mice could have a more severe impact on gonadal development. To address this issue, we generated double KO mice for Dmrt1 and Dmrt3 via the CRISPR/Cas9 system. Comparing adult and neonatal testes of single and double KO mice, we found that loss of Dmrt1 or Dmrt3, or both, does not have apparent effect on male gonadal formation during embryonic development. Our study demonstrated that the discrepancy between human with 9p24.3 deletion and Dmrt1 KO mouse could not be explained by the simultaneous loss of Dmrt3 gene. CRISPR/Cas9 is a versatile and straightforward approach to elucidate the questions that were otherwise difficult to address with conventional methods.
Highlights
DM domain transcription factors are evolutionarily conserved among metazoan species and are involved in the gonadal development in a wide range of species [1,2,3,4,5]
The injected embryos were transferred to the pseudo-pregnant female mice and the obtained pups were examined for the genotypes of both Dmrt1 and Dmrt3 loci
Since it was impossible to discriminate whether the mutations in Dmrt1 locus and Dmrt3 locus were on the single chromosome or on two homologous chromosomes at this point, we mated the mice with simultaneous mutations with wild-type (WT) C57BL/6 mice and examined the genotype of F1 generation
Summary
DM domain transcription factors are evolutionarily conserved among metazoan species and are involved in the gonadal development in a wide range of species [1,2,3,4,5]. Human with chromosomal deletion of 9p24.3, the region in which DMRT1 is located, are often associated with 46,XY gonadal dysgenesis [10,11]. Dmrt deficiency caused the degeneration and feminization of male gonad, partly recapitulating the etiology of human patients of 9p24.3 deletion. The embryonic development of male gonad is essentially normal in those mice, which shows stark difference from the etiology of human 9p24.3 deletion in which prenatal feminization is observed. Despite the phenotypic variation among the patients with 9p24.3 deletion, at least some patients with clear 46,XY gonadal dysgenesis phenotype retain normal DMRT1 exon sequences in their normal chromosome 9, implying haploinsufficiency of the DMRT1 gene in humans [11,12]. Heterozygous Dmrt mutant mice do not show abnormality [2]
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