DM Domain Research Articles

Male-biased expression of X-chromosomal DM domain-less Dmrt8 genes in the mouse

The vertebrate DMRT gene family encodes putative transcription factors related to the sexual regulators Doublesex ( Drosophila melanogaster) and MAB-3 ( Caenorhabditis elegans). They share a highly conserved DNA binding motif, the DM domain. In human and mouse seven DMRT genes ( DMRT1– DMRT7) have been analyzed. DMRT8, a gene related to DMRT7, is located on the X chromosome in placental mammals. While DMRT8 is single copy in most mammals, three copies are present in mouse, rat, and rabbit. Despite the loss of the DM domain, DMRT8 genes have been maintained in the mammalian lineage, suggesting a DM domain-independent function. In adult mouse, two Dmrt8 genes are expressed exclusively in testis. Dmrt8. 1 mRNA was detected in Sertoli cells by in situ hybridization. In embryos, Dmrt8. 2 shows a dynamic expression restricted to male and female gonads and might therefore be involved in sexual development in the mouse.

Targeted disruption of the DM domain containing transcription factor Dmrt2 reveals an essential role in somite patterning

Dmrt2 is expressed in the dermomyotome of developing vertebrate somites. To determine the role of Dmrt2 during mouse embryonic development, we generated a null mutation of Dmrt2 via homologous recombination in embryonic stem cells. Dmrt2 heterozygous mice derived from these cells are phenotypically normal. However, Dmrt2 homozygotes die soon after birth. The cause of death is likely due to abnormal rib and sternal development, leading to an inability to breathe. Loss of Dmrt2 leads to embryonic somite patterning defects, first evidenced at embryonic day (E) 10.5 and more pronounced by E11.5. Notably, both the dermomyotome and myotome fail to adopt a normal epithelial morphology in the absence of Dmrt2. Accompanying these morphological defects are alterations in the expression patterns of dermomyotomal and myotomal transcription factors including Pax3, Paraxis, Myf5, myogenin, Mrf4 and MyoD. Despite these defects, embryos harvested from E13.5 onwards exhibited relatively normal muscle pattern and mass, suggesting that early myotomal defects are corrected by a Dmrt2-independent mechanism. Taken together, our results define an essential function for Dmrt2 in somite development and provide evidence that DM domain genes have been co-opted into other critical developmental pathways distinct from that of sex determination or differentiation.

Characterisation of a DM domain-containing transcription factor from Trichostrongylus vitrinus (Nematoda: Strongylida)

The sequence and expression profile of Tv-mab-23, a gene encoding a DM domain-containing protein from the parasitic nematode Trichostrongylus vitrinus, were investigated. The gene, containing an open reading frame (ORF) of 537 bp, encoded a predicted protein which had an overall amino acid identity of 45% to the MAB-23 molecule of the free-living nematode Caenorhabditis elegans. High levels of conservation were particularly predominant at the amino terminus of the proteins, with 86% identity over the first 58 amino acid residues in a region containing the highly conserved zinc module of the DM domain and conserved regions of the recognition helix of DM domain-containing transcription factors. Tv-mab-23 was expressed in a stage-specific manner in T. vitrinus. The highest levels of expression in fourth stage larvae coincide with the period in which profound gender-specific alterations in physiology occur in both the parasitic and free-living nematodes.

Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Male- and female-specific splicing isoforms of DSX share a novel DNA-binding domain, designated the DM motif. Broadly conserved among metazoan sex-determining factors, the DM domain contains a nonclassical zinc module and binds in the DNA minor groove. Here, we characterize the DM motif by site-directed and random mutagenesis using a yeast one-hybrid (Y1H) system and extend this analysis by chemogenetic complementation in vitro. The Y1H system is based on a sex-specific Drosophila enhancer element and validated through studies of intersexual dsx mutations. We demonstrate that the eight motif-specific histidines and cysteines engaged in zinc coordination are each critical and cannot be interchanged; folding also requires conserved aliphatic side chains in the hydrophobic core. Mutations that impair DNA binding tend to occur at conserved positions, whereas neutral substitutions occur at nonconserved sites. Evidence for a specific salt bridge between a conserved lysine and the DNA backbone is obtained through the synthesis of nonstandard protein and DNA analogs. Together, these results provide molecular links between the structure of the DM domain and its function in the regulation of sexual dimorphism.

Somatic sex determination

C. elegans occurs in two natural sexes, the XX hermaphrodite and the XO male, which differ extensively in anatomy, physiology, and behavior. All somatic differences between the sexes result from the differential activity of a "global" sex determination regulatory pathway. This pathway also controls X chromosome dosage compensation, which is coordinated with sex determination by the action of the three SDC proteins. The SDC proteins control somatic and germline sex by transcriptional repression of the her-1 gene. HER-1 is a secreted protein that controls a regulatory module consisting of a transmembrane receptor, TRA-2, three intracellular FEM proteins, and the zinc finger transcription factor TRA-1. The molecular workings of this regulatory module are still being elucidated. Similarity of TRA-2 to patched receptors and of TRA-1 to GLI proteins suggests that parts of the global pathway originally derived from a Hedgehog signaling pathway. TRA-1 controls all aspects of somatic sexual differentiation, presumably by regulating a variety of tissue- and cell-specific downstream targets, including the cell death regulator EGL-1 and the male sexual regulator MAB-3. Sex determination evolves rapidly, and conservation of sexual regulators between phyla has been elusive. An apparent exception involves DM domain proteins, including MAB-3, which control sexual differentiation in nematodes, arthropods, and vertebrates. Important issues needing more study include the detailed molecular mechanisms of the global pathway, the identities of additional sexual regulators acting in the global pathway and downstream of TRA-1, and the evolutionary history of the sex determination pathway. Recently developed genetic and genomic technologies and comparative studies in divergent species have begun to address these issues.

Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes

DMRT1, a gene containing the Doublesex/Mab-3 DNA-binding motif (DM domain), encodes a transcription factor that regulates early differentiation of Sertoli cells in the testes of vertebrates. Here, we describe the pattern of expression of six DMRT genes ( DMRT1, 2a, 2b, 3, 4, and 5) during gonadal development in the Japanese pufferfish, Takifugu rubripes, for the first time. Conventional histological analysis showed that a primordial gonad is present 2 weeks after hatching and that sexual differentiation had occurred by 6 weeks after hatching, as determined by the formation of cavities in the ovaries. RT-PCR analysis showed that the DMRT1 transcript was abundant in the testes, but not in the ovaries, of 3-month-old fish. DMRT3 transcript was also detected in the testes, but not in the ovaries, indicating that the patterns of gonadal expression of DMRT3 are very similar to DMRT1. The other four DMRT genes examined did not exhibit sex-specific expression patterns. In situ hybridization analysis revealed that, after gonadal differentiation was complete, DMRT1 was expressed in the Sertoli cells that were in proximity to proliferating spermatogonia. These results indicate that DMRT1 involved in gonadal development rather than sex differentiation and that its expression correlates with the proliferation of spermatogonia in T. rubripes. The expression profile of DMRT1 in T. rubripes was similar to that in the teleost species Medaka.

The DM Domain Protein MAB-3 Promotes Sex-Specific Neurogenesis in C. elegans by Regulating bHLH Proteins

Sexual dimorphism in the nervous system is required for sexual behavior and reproduction in many metazoan species. However, little is known of how sex determination pathways impose sex specificity on nervous system development. In C. elegans, the conserved sexual regulator MAB-3 controls several aspects of male development, including formation of V rays, male-specific sense organs required for mating. Here we show that MAB-3 promotes expression of the proneural protein LIN-32 in V ray precursors by transcriptional repression of ref-1, a member of the Hes family of neurogenic factors. Mutations in ref-1 restore lin-32::gfp expression and normal V ray development to mab-3 mutants, suggesting that ref-1 is the primary target of MAB-3 in the V ray lineage. Proteins related to MAB-3 (DM domain proteins) control sexual differentiation in diverse metazoans. We therefore suggest that regulation of Hes genes by DM domain proteins may be a general mechanism for specifying sex-specific neurons.

Gene structure, multiple alternative splicing, and expression in gonads of zebrafish Dmrt1

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla although the existence of two sexes is nearly universal in the animal kingdom. The only molecular similarity in sex determination found so far between phyla is among the fly doublesex, worm mab-3, and vertebrate Dmrt1/ DMY, which contain a zinc-finger-like DNA-binding motif, DM domain. Here we report that three isoforms of the zebrafish Dmrt1 were generated in gonads by multiple alternative splicing, which encoded predicted proteins with 267, 246, and 132 amino acids, respectively. By cDNA cloning and genomic structure analysis, we found that there were seven exons of Dmrt1, which were alternatively spliced to generate the Dmrt1 isoforms. Northern blotting analysis revealed that expression of zebrafish Dmrt1 was higher in testis than ovary. Real time fluorescent quantitative RT-PCR indicated that expression of isoform a of Dmrt1 was dominantly higher than those of Dmrt1 b and c. Furthermore, in situ hybridization to gonads sections showed that Dmrt1 was expressed in developing germ cells of both testis and ovary, suggesting that the Dmrt1 gene is not only associated with testis development, but also, may be important in ovary differentiation of zebrafish.

Molecular cloning, expression ofSox5 and its down-regulation ofDmrt1 transcription in Zebrafish

The doublesex and mab-3-related transcription factor 1 (Dmrt1) is the founding member of a family of DM domain genes, involved in gonadogenesis. Here we report the cloning of zebrafish Sox5 and use real time PCR to show that its expression increases while expression of Dmrt1 decreases during embryogenesis. Characterization of the proximal promoter of zebrafish Dmrt1 revealed two positive and two negative regulatory regions and a Sox5-binding site. Co-transfection of dmrt1 (with or without the Sox-binding site), driving an EGFP reporter and Sox5 showed further that Sox5 bound the Dmrt1 promoter and inhibited Dmrt1 expression. This antagonistic partnership between Dmrt1 and Sox5 suggests a potential transcriptional regulatory mechanism for Dmrt1 in early embryogenesis.

Molecular cloning, characterization, and expression in brain and gonad of Dmrt5 of zebrafish

The DM domain gene family has at least eight members with conserved DNA-binding DM domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of Caenorhabditis elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. There is also few structural and functional analysis concerning DM domain genes of the model fish, zebrafish. We report here molecular cloning, sequence, and expression of zebrafish Dmrt5, which consists of two exons, and encodes a 440-amino acid protein with conserved DMA and DMB domains in addition to DM domain. Phylogenetic analysis shows that zebrafish Dmrt5 fits within the Dmrt5 clade of fish and mammals. Zebrafish Dmrt5 was expressed in early gastrula period, subsequently increased to a high level in late stage of gastrula period (bud stage) and lower until the hatch period. In situ hybridization analysis showed its expression in developing central nervous system of embryos, especially in mid-brain and mid-hind brain boundary. In adult, its expression was restricted in brain and developing germ cells, especially in spermatogonia, spermatocytes, spermatids, and sperm cells, and in developing oocytes, including early perinucleolus stage oocyte, late yolk vesicle stage oocyte, and oil drop stage oocyte. These data suggest that zebrafish Dmrt5 have potentially important roles in gonadal development and may have contributed to the functional endocrine axis.

Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes

The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20-30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation.

Developmentally regulated and non-sex-specific expression of autosomal dmrt genes in embryos of the Medaka fish ( Oryzias latipes)

The dmrt 2 2 For simplicity, the fish terminology has been used for all vertebrate dmrt genes throughout the manuscript (for details see Volff et al., 2003b). gene family of vertebrates comprises several transcription factors that share a highly conserved DNA-binding domain, the DM domain. Like some of their invertebrate counterparts, e.g. Drosophila doublesex ( dsx) and the Caenorhabditis elegans Mab3, several are implicated in sex determination and differentiation. Thus far, dmrt genes represent the only factors involved in sexual development that are conserved across phyla. In the teleost Medaka ( Oryzias latipes), a duplicated copy of dmrt1, designated dmrt1bY or dmy, has recently been postulated to be the master regulator of male development in this species. Here, we have analyzed the expression of four additional Medaka dmrt genes during embryonic and larval development. In contrast to other vertebrates, the autosomally located dmrt1a gene of Medaka is not expressed at detectable levels during embryogenesis. On the other hand, dmrt2, dmrt3 and dmrt4 show highly restricted and non-overlapping expression patterns during embryogenesis. While dmrt2 is expressed in early somites, dmrt3 transcripts are found in dorsal interneurons and dmrt4 is expressed in the developing olfactory system. Other than in mouse, they do not show any sex specific expression and no transcription could be detected in the early developing gonads. However, all four analyzed dmrt genes share expression in the differentiating gonad of larvae and in adult testis.

Evolution of DMY, a Newly Emergent Male Sex-Determination Gene of Medaka Fish

Abstract The Japanese medaka fish Oryzias latipes has an XX/XY sex-determination system. The Y-linked sex-determination gene DMY is a duplicate of the autosomal gene DMRT1, which encodes a DM-domain-containing transcriptional factor. DMY appears to have originated recently within Oryzias, allowing a detailed evolutionary study of the initial steps that led to the new gene and new sex-determination system. Here I analyze the publicly available DMRT1 and DMY gene sequences of Oryzias species and report the following findings. First, the synonymous substitution rate in DMY is 1.73 times that in DMRT1, consistent with the male-driven evolution hypothesis. Second, the ratio of the rate of nonsynonymous nucleotide substitution (dN) to that of synonymous substitution (dS) is significantly higher in DMY than in DMRT1. Third, in DMRT1, the dN/dS ratio for the DM domain is lower than that for non-DM regions, as expected from the functional importance of the DM domain. But in DMY, the opposite is observed and the DM domain is likely under positive Darwinian selection. Fourth, only one characteristic amino acid distinguishes all DMY sequences from all DMRT1 sequences, suggesting that a single amino acid change may be largely responsible for the establishment of DMY as the male sex-determination gene in medaka fish.

Sex determination in fish: Lessons from the sex-determining gene of the teleost medaka, Oryzias latipes.

Although sex determination systems in animals are diverse, sex-determining genes have been identified only in mammals and some invertebrates. Recently, DMY (DM domain gene on the Y chromosome) has been found in the sex-determining region on the Y chromosome of the teleost medaka fish, Oryzias latipes. Functional and expression analyses of DMY show it to be the leading candidate for the male-determining master gene of the medaka. Although some work is required to define DMY as the master sex-determining gene, medaka is expected to be a good experimental animal for investigating the precise mechanisms involved in primary sex determination in non-mammalian vertebrates. In this article, the process of identification of DMY and is summarized and the origins of DMY and sexual development of the medaka's gonads are reviewed. In addition, putative functions of DMY are discussed.

Evolutionary dynamics of the DM domain gene family in metazoans.

The DM domain gene family encodes putative transcription factors related to the sexual regulators Doublesex from Drosophila melanogaster and MAB-3 from Caenorhabditis elegans. While some DM domain proteins are involved in sexual development in very distant metazoan phyla and one in somite development, the function of the great majority of them remains unclear. DM domain genes underwent frequent independent events of gene duplication during the course of evolution and the number of DM domain genes differs between phyla. Variation is even observed within the vertebrate lineage, where some genes present in mammals are absent from fish and vice versa. Of particular interest is the very recent duplication of the DM domain gene dtmrt1 that apparently led to the formation of the master male-determining gene in the medaka fish but not in more divergent fish species. Hence, the DM domain gene family undergoes an important evolutionary turnover probably associated in some cases with novel expression patterns and possibly with new functions. Here we examine the current classification of vertebrate DM domain dmrt genes based on structural features, and propose a simpler nomenclature for dmrt genes.

Sexually dimorphic expression of multiple doublesex-related genes in the embryonic mouse gonad

The only molecular similarity shown so far for sexual regulatory genes among different phyla involves doublesex ( dsx) of Drosophila, mab-3 and mab-23 of Caenorhabditis elegans, and Dmrt1 of vertebrates. These genes encode DM domain transcription factors (DM= dsx and mab-3) and are required for sexual differentiation. In the case of dsx and mab-3, the two genes control analogous aspects of sexual development, bind similar DNA sequences, and are capable of functional substitution in vivo. All three phyla have multiple DM domain genes, but it is unknown how many of these are involved in sexual development. Mammals, for example, have at least seven DM domain genes, but embryonic expression has only been examined in detail for Dmrt1( dsx- and mab-3 related transcription factor 1). We have identified additional murine DM domain genes and have examined their expression in the mouse embryo, with emphasis on the developing gonad. At least three murine DM domain genes in addition to Dmrt1 are expressed in the embryonic gonad: Dmrt4 is expressed at similar levels in gonads of both sexes; Dmrt3 is more highly expressed in males; and Dmrt7 is more highly expressed in females. Expression of three other genes is low or absent in the embryonic gonad. Two of these, Dmrt5 and Dmrt6, are expressed primarily in the brain, and the third, Dmrt2, is expressed in presomitic mesoderm and developing somites. Our data suggest that multiple DM domain genes may be involved in mammalian sexual development, and that they may function in both testis and ovary development.

The sex determining gene of medaka: a Y-specific DM domain gene (DMY) is required for male development

Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. To clone positionally the sex-determining region of the medaka, Oryzias latipes, we generated a Y congenic strain to highlight the genetic differences between the X and Y chromosomes from inbred strains of medaka. We used recombinant breakpoint analysis and deletion analysis of the Y chromosome of a congenic XY female to restrict the sex-determining region to 250-kb stretch of the Y chromosome. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only one gene was Y specific. The full-length cDNA sequence of this gene encodes a putative protein of 267 amino acids, including the highly conserved DM domain. We thus named it DMY. To establish a role for DMY during sexual differentiation, we screened wild medaka populations for naturally occurring DMY mutants. Two XY females with distinct mutations in DMY were found in separate populations. The first heritable mutant – a single insertion in exon 3 and the subsequent truncation of DMY – resulted in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. Furthermore, during normal development, DMY is expressed only in somatic cells of XY gonads. These findings strongly suggest that the sex-specific DMY is required for normal testicular development and is a prime candidate for the medaka sex-determining gene.

Restricted expression of DMRT3 in chicken and mouse embryos

Vertebrate DM domain genes encode a novel group of proteins related to the Drosophila doublesex and Caenorhabditis elegans mab-3 transcription factors. It is shown here that the recently identified gene, DMRT3, has a restricted embryonic expression profile that is conserved in chicken and mouse embryos. DMRT3 is expressed primarily in the forebrain, neural tube and nasal placode of both species. In the chicken, DMRT3 is also expressed in newly forming tail somites at early developmental stages and, later, in the Müllerian ducts of the urogenital system.

Conservation of the function of DMRT1 regulatory sequences in mammalian sex differentiation.

Among genes involved in sex determination and differentiation, DMRT1 is the only one characterized to date containing a domain (the DM domain) that is conserved between phyla. To study DMRT1 transcriptional regulation within mammalian phyla, we generated transgenic mice that express green fluorescent protein (GFP) or Cre-recombinase (Cre) under the control of 2.6 kb of pig DMRT1 5' flanking sequences (pDMRT1p-GFP and pDMRT1p-Cre, respectively). Within the pDMRT1p-GFP positive mice, GFP expression was observed in the XY genital ridge by embryonic day 11.5 (e11.5) and remained detectable during testis embryonic development to birth. GFP expression was restricted within testis cords as soon as cords were detectable. No fluorescence was observed in developing ovaries, although more sensitive RT-PCR analysis revealed transgene expression in embryonic ovaries from e13.5 to e15.5. RT-PCR performed on fluorescent activated cell sorter (FACS)-purified GFP cells from e14.5, e17.5, and e19.5 developing testis showed that GFP expression was restricted to cells expressing the endogenous mouse Dmrt1. GFP cells also expressed Mis and Oct4, showing that the transgene is expressed in both Sertoli cell and germ cell compartments. In postnatal testis, transgene expression was detectable by GFP fluorescence from P0 to P21 in mice heterozygous for the transgene and through adulthood in mice homozygous for the transgene. In pDMRT1p-Cre positive mice, Cre expression was detected within the genital ridges of both XY and XX embryos. We conclude that DMRT1 regulatory mechanisms during sexual differentiation are functionally conserved across mammalian evolution. The transgenic mouse lines described should provide useful marker systems for studies involving Dmrt1 gene expression during sex differentiation.

Molecular cloning and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus

The DMRT genes constitute a family of genes, which possess a common motif called the DM domain. DMRT1 is considered to be involved in sex determination and/or sex differentiation, but not much information exists about the function of the other gene family members. We cloned DMRT genes of two important model fish species, the medaka, Oryzias latipes, and the platyfish, Xiphophorus maculatus. Based on sequence similarity and genomic structure with known DMRT genes, the gene from the medaka was identified as OlaDMRT4, and those from the platyfish as XmaDMRT2 and XmaDMRT4. OlaDMRT4 was assigned to the linkage group 18 (LG18) of the medaka by linkage analysis and fluorescence in situ hybridization. The earlier cloned medaka DMRT1, 2 and 3 genes form a cluster on LG9. Therefore, OlaDMRT4 does not belong to the DMRT gene cluster. In adult medaka fish, OlaDMRT4 is expressed in the brain, eyes, gill, kidney, as well as testis and ovary. During development, OlaDMRT4 exists as maternal transcripts, and is expressed until early larval stages. This pattern of expression differs from the other known medaka DMRT genes. Surprisingly it is also not the same as its putative tilapia ortholog ( DMO). These differences in expression suggest that DMRT4 might fulfill divergent functions in different species.