Two C. elegans DM domain proteins, DMD-3 and MAB-3, function in late stages of male somatic gonad development.

To bring about sexual dimorphism in form, information from the sex determination pathway must trigger sex-specific modifications in developmental programs. DM-domain encoding genes have been found to be involved in sex determination in a multitude of animals, often at the level of male somatic gonad formation. Here we report our findings that the DM-domain transcription factors MAB-3 and DMD-3 function together in multiple steps during the late stages of C. elegans male somatic gonad development. Both mab-3 and dmd-3 are expressed in the linker cell and hindgut of L4 males and dmd-3 is also expressed in presumptive vas deferens cells. Furthermore, dmd-3, but not mab-3, expression in the linker cell is downstream of nhr-67, a nuclear hormone receptor that was previously shown to control late stages of linker cell migration. In mab-3; dmd-3 double mutant males, the last stage of linker cell migration is partially defective, resulting in aberrant linker cell shapes and often a failure of the linker cell to complete its migration to the hindgut. When mab-3; dmd-3 double mutant linker cells do complete their migration, they fail to be engulfed by the hindgut, indicating that dmd-3 and mab-3 activity are essential for this process. Furthermore, linker cell death and clearance are delayed in mab-3; dmd-3 double mutants, resulting in the linker cell persisting into adulthood. Finally, DMD-3 and MAB-3 function to activate expression of the bZIP transcription factor encoding gene zip-5 and downregulate the expression of the zinc metalloprotease ZMP-1 in the linker cell. Taken together, these results demonstrate a requirement for DM-domain transcription factors in controlling C. elegans male gonad formation, supporting the notion that the earliest DM-domain genes were involved in male somatic gonad development in the last common ancestor of the bilaterians.

DMRT1: An Ancient Sexual Regulator Required for Human Gonadogenesis.

Transcriptional regulators related to the invertebrate sexual regulators doublesex and mab-3 occur throughout metazoans and control sex in most animal groups. Seven of these DMRT genes are found in mammals, and mouse genetics has shown that one, Dmrt1, plays a crucial role in testis differentiation, both in germ cells and somatic cells. Deletions and, more recently, point mutations affecting human DMRT1 have demonstrated that its heterozygosity is associated with 46,XY complete gonadal dysgenesis. Most of our detailed knowledge of DMRT1 function in the testis, the focus of this review, derives from mouse studies, which have revealed that DMRT1 is essential for male somatic and germ cell differentiation and maintenance of male somatic cell fate after differentiation. Moreover, ectopic DMRT1 can reprogram differentiated female granulosa cells into male Sertoli-like cells. The ability of DMRT1 to control sexual cell fate likely derives from at least 3 properties. First, DMRT1 functionally collaborates with another key male sex regulator, SOX9, and possibly other proteins to maintain and reprogram sexual cell fate. Second, and related, DMRT1 appears to function as a pioneer transcription factor, binding "closed" inaccessible chromatin and promoting its opening to allow binding by other regulators including SOX9. Third, DMRT1 binds DNA by a highly unusual form of interaction and can bind with different stoichiometries.

Molecular characterization, computational analysis and expression profiling of Dmrt1 gene in Indian major carp, Labeo rohita (Hamilton 1822).

Sexual dimorphism of fish morphology, physiology and behavior is diverse and complex in nature. Doublesex and mab-3 related transcription factor (Dmrt) is a large protein family whose function is sexual development and differentiation in vertebrates. Here, we report a full-length cDNA sequence of Labeo rohita (rohu) Dmrt1 of 907 bp length having 798 bp of open reading frame encoding 265 amino acids. The molecular weight of rohu DMRT1 protein was found to be 28.74 KDa and isoelectric point was 7.53. DMRT1 protein contains 23 positively and 24 negatively charged amino acids with a GRAVY score of -0.618. A characteristic DM domain was found in DMRT1 protein, which is a novel DNA-binding domain. Phylogenetic analysis showed maximum similarity with Cyprinus carpio when compared with DMRT1 of other vertebrates. Molecular docking study identified active sites to be targeted for drug designing. Rohu DMRT1 was observed to interact with other proteins such as FOXL2, CYP19a1a, AMH and SOX9a. Differential expression study revealed higher expression in testis tissue implying its role in male sex differentiation and testicular development. The information generated in the present work could facilitate further research to resolve the issues related to gonadal maturation and reproduction of commercially important aquaculture species.

Independent evolution for sex determination and differentiation in the DMRT family in animals.

Some DMRT family genes including arthropod dsx, nematode mab-3, and vertebrate dmrt1 are involved in sex determination and/or differentiation in bilaterian animals. Although there have been some reports about evolutionary analyses of the family by using its phylogenetic trees, it is still undecided as to whether these three sex determination-related genes share orthologous relationships or not. To clarify this question, we analyzed evolutional relationships among the family members in various bilaterians by using not only phylogenetic tree analysis, but also synteny analysis. We found that only four genes, dmrt2a/2b, dmrt3, dmrt4/ 5 and dmrt93B were commonly present in invertebrate bilateria. The syntenies of dmrt2a/2b-dmrt3 and dmrt4/5-dmrt93B are conserved before and after two rounds of whole genome duplication in the ancestral vertebrate. Importantly, this indicates that dmrt1 must have appeared in the common vertebrate ancestor. In addition, dmrt1, dsx, or mab-3 formed each different cluster at a distance in our phylogenetic tree. From these findings, we concluded that the three sex determination-related genes, dmrt1, dsx, and mab-3 have no orthologous relationships, and suggested independent evolution for sex determination and differentiation in the DMRT gene family. Our results may supply clues about why sex-determining systems have diverged during animal evolution.

Transcriptional variants of Dmrt1 and expression of four Dmrt genes in the blunt snout bream, Megalobrama amblycephala.

Doublesex and Mab3 related transcription factor (DMRT), characterized by a conserved DM domain, function as sex-related transcription factors and also play critical roles in ontogenesis. In this study, 4 Dmrt genes in the blunt snout bream, Megalobrama amblycephala, were identified, characterized and their mRNA expression in different adult organs, during embryogenesis and gonadal development in larvae were determined by quantitative real time PCR. There are 4 Dmrt1 isoforms in the M. amblycephala genome, which were expressed highly in the testis and weakly in the ovary. The complete cDNAs of the M. amblycephala Dmrt2a, Dmrt2b and Dmrt3 were predicted to encode 510, 328 and 449 amino acids, respectively. The M. amblycephala Dmrt2a mRNA peaked at 11hpf (hour post fertilizing) during early embryonic stages, while Dmrt2b was highly expressed during late embryonic stages. Both the M. amblycephala Dmrt2a and Dmrt2b were expressed highly in the gill and exhibited a sexually dimorphic expression pattern. The M. amblycephala Dmrt3 was expressed highly in the gill, muscle and brain, at 40dph (day post hatching) during early development and at stage V in the testis during gonadal development. All fish Dmrts except Dmrt5 were found in the M. amblycephala genome. The observed expression patterns of these Dmrts in developing embryos and larvae, as well as different adult organs indicate conserved sexual or extragonadal functions of the Dmrts through evolution.

Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1.

Genes related to Dmrt1, which encodes a DNA-binding DM domain transcription factor, act as triggers for primary sex determination in a broad range of metazoan species. However, this role is fulfilled in mammals by Sry, a newly evolved gene on the Y chromosome, such that Dmrt1 has become dispensable for primary sex determination and instead maintains Sertoli cell phenotype in postnatal testes. Here, we report that enforced expression of Dmrt1 in XX mouse fetal gonads using a Wt1-BAC transgene system is sufficient to drive testicular differentiation and male secondary sex development. XX transgenic fetal gonads showed typical testicular size and vasculature. Key ovarian markers, including Wnt4 and Foxl2, were repressed. Sertoli cells expressing the hallmark testis-determining gene Sox9 were formed, although they did not assemble into normal testis cords. Other bipotential lineages differentiated into testicular cell types, including steroidogenic fetal Leydig cells and non-meiotic germ cells. As a consequence, male internal and external reproductive organs developed postnatally, with an absence of female reproductive tissues. These results reveal that Dmrt1 has retained its ability to act as the primary testis-determining trigger in mammals, even though this function is no longer normally required. Thus, Dmrt1 provides a common thread in the evolution of sex determination mechanisms in metazoans.

Identification of three doublesex genes in the monogonont rotifer Brachionus koreanus and their transcriptional responses to environmental stressor-triggered population growth retardation.

Doublesex and Mab-3-related transcription factor (Dmrt) gene family members have rarely been identified or characterized in aquatic invertebrates. In this study, we identified and characterized three DMdomain-containing genes - Dmrt11E, Dmrt93B, and Dmrt99B - in the monogonont rotifer, Brachionus koreanus. DMdomains of the proteins encoded by the B.koreanus Dmrt (Bk-Dmrt) genes had high similarities to DM domains of other invertebrate species. To understand the potential effects of environmental stressors on the transcriptional expression of Dmrt genes in rotifers, we exposed B.koreanus to a wide range of UV-B radiation and different concentrations of benzo[a]pyrene (B[a]P) over different time courses. Transcript levels of all Bk-Dmrt genes decreased significantly in response to relatively high doses of UV-B irradiation, and were also downregulated in response to exposure to UV-B radiation over time. Transcript levels of all Bk-Dmrt genes were downregulated in response to B[a]P exposure for 24h. This decrease in expression of all Bk-Dmrt genes was concomitant with the growth retardation induced by UV-B and B[a]P exposure. We concluded that both environmental stressors have detrimental effects on transcriptional regulation of all Bk-Dmrt genes, especially relatively high doses of these stressors, leading to growth retardation. However, further studies are required to better understand the potential role of Dmrt genes in environmental stressor-triggered growth retardation in the rotifer B.koreanus.

Nuclear factor-Y (NF-Y) regulates transcription of mouse Dmrt7 gene by binding to tandem CCAAT boxes in its proximal promoter.

Dmrt7, a member of the Dmrt family of genes, is required for spermatogenesis. However, promoter functions of the gene Dmrt7 remain unknown. We have cloned and characterized the proximal promoter region of the mouse Dmrt7 gene. Functional analysis of the 5' flanking region by sequential deletion mutations revealed crucial positive elements between -60 and +1, in which two highly conserved and tandem CCAAT boxes: the CCAAT box1 (-48/-44) and the CCAAT box2 (-7/-3) are located. Site-directed mutagenesis studies demonstrated that both CCAAT boxes are indispensable to the promoter activity. Electrophoretic mobility shift assays (EMSAs) and gel-supershift assays indicated that transcription factor NF-Y binds to the promoter. Chromatin immunoprecipitation (ChIP) analysis demonstrated that NF-Y interacts in vivo with the promoter of the Dmrt7 gene in testis. Co-transfection and reporter analysis showed that over-expression of NF-Ys increased transcription of the Dmrt7-luc gene whereas expression of a dominant-negative NF-Ya decreased the transcription. This suggests that NF-Y can activate the Dmrt7 promoter. These results provide evidence of a transcription regulatory mechanism that controls Dmrt7 gene expression in mouse testis.

Isolation and sequencing of doublesex/male abnormal 3 (DM) related transcription factor (Dmrt) genes from the Asian toad Bufo gargarizans (Cantor, 1842)

The doublesex/male abnormal 3 (dsx/mab-3 or DM) domain gene family involved in sexual development encodes putative transcription factors including a DNA-binding homology motif, the DM domain. We used highly degenerate primers to clone and sequence seven distinct DM related transcription factor (Dmrt) genes from the Asian toad (Bufo gargarizans Cantor, 1842). A database search for the cloned sequences revealed the following percentage identity with the homologous Dmrt genes of the human: BgDmrt1 = 97 percent, BgDmrt2 = 97 percent, three isoforms of BgDmrt3 (BgDmrt3a = 93 percent, BgDmrt3b = 95 percent, BgDmrt3c = 100 percent) and two isoforms of BgDmrt5 (BgDmrt5 = 97 percent, BgDmrt5 = 91 percent). Based on DM domain amino acid sequence similarities we constructed a phylogenetic tree which grouped vertebrate and invertebrate Dmrt genes into seven distinct subfamilies. The DM domains of both human and the newly-discovered Bufo gargarizans genes contained two conserved zinc-chelating sites (CCHC and HCCC), except BgDmrt3b, which contained the CCRC and HCCC sites.