Sex-specific expression of an evolutionarily conserved male regulatory gene, DMRT1, in birds

Based on its Z-sex-chromosomal location and its structural homology to male sexual regulatory factors in humans (DMRT1 and DMRT2), Drosophila (Dsx), and Caenorhabditis elegans (Mab-3), chicken DMRT1 is an excellent candidate for a testis-determining factor in birds. The data we present provide further strong support for this hypothesis. By whole mount in situ hybridization chicken DMRT1 is expressed at higher levels in the male than in the female genital ridges during early stages of embryogenesis. Its expression becomes testis-specific after onset of sexual differentiation. Northern blot and RT PCR analysis showed that in adult birds DMRT1 is expressed exclusively in the testis. We propose that two gene dosages are required for testis formation in ZZ males, whereas expression from a single Z chromosome in ZW females leads to female sexual differentiation.     

Sex determination in the chicken embryo.

The chicken embryo represents a suitable model for studying vertebrate sex determination and gonadal sex differentiation. While the basic mechanism of sex determination in birds is still unknown, gonadal morphogenesis is very similar to that in mammals, and most of the genes implicated in mammalian sex determination have avian homologues. However, in the chicken embryo, these genes show some interesting differences in structure or expression patterns to their mammalian counterparts, broadening our understanding of their functions. The novel candidate testis-determining gene in mammals, DMRT1, is also present in the chicken, and is expressed specifically in the embryonic gonads. In chicken embryos, DMRT1 is more highly expressed in the gonads and Müllerian ducts of male embryos than in those of females. Meanwhile, expression of the orphan nuclear receptor, Steroidogenic Factor 1 (SF1) is up-regulated during ovarian differentiation in the chicken embryo. This contrasts with the expression pattern of SF1 in mouse embryos, in which expression is down-regulated during female differentiation. Another orphan receptor initially implicated in mammalian sex determination, DAX1, is poorly conserved in the chicken. A chicken DAX1 homologue isolated from a urogenital ridge library lacked the unusual DNA-binding motif seen in mammals. Chicken DAX1 is autosomal, and is expressed in the embryonic gonads, showing somewhat higher expression in female compared to male gonads, as in mammals. However, expression is not down-regulated at the onset of testicular differentiation in chicken embryos, as occurs in mice. These comparative data shed light on vertebrate sex determination in general.     

DMRT1 in a ratite bird: evidence for a role in sex determination and discovery of a putative regulatory element

Unlike mammals, birds have a ZZ male/ZW female sex-determining system. In most birds, the Z is large and gene rich, whereas the W is small and heterochromatic, but the ancient group of ratite birds are characterized by sex chromosomes that are virtually homomorphic. Any gene differentially present on the ratite Z and W is therefore a strong candidate for a sex-determining role. We have cloned part of the candidate bird sex-determining gene DMRT1 from the emu, a ratite bird, and have shown that it is expressed during the stages of development corresponding to gonadal differentiation in the chicken. The gene maps to the distal region of the Z short arm and is absent from the large W chromosome. Because most sequences on the emu W chromosome are shared with the Z, the Z-specific location constitutes strong evidence that differential dosage of DMRT1 is involved in sex determination in all birds. The sequence of emu DMRT1 has 88% homology with chicken DMRT1 and 65% with human DMRT1. Unexpectedly, an unexpressed 270-bp region in intron 3 of emu DMRT1 showed 90% homology with a sequence in the corresponding intron of human DMRT1. This extraordinarily high conservation across 300 million years of evolution suggests an important function, perhaps involved in control of DMRT1 expression and vertebrate sex determination.     

奥利亚罗非鱼DMRT1和DMRT4抗体制备及组织表达谱分析

DMRT1和DMRT4是DMRT基因家族的成员,该家族成员与果蝇的性别决定基因和线虫性别决定基因一样,所编码的蛋白质都包含一个具有DNA结合能力的保守基序,即DM结构域,并以锌指结构与特异DNA序列相结合,在性别决定和分化发育中起调控作用。采用RT-PCR方法分别从奥利亚罗非鱼卵巢和精巢中扩增克隆出DMRT1和DMRT4全长cDNA片段,构建表达载体,在大肠杆菌中表达了BMP-DMRT4和BMP-DMRT1蛋白。经Xa切割、Amylose-sepharose柱层析纯化后作为抗原免疫新西兰白兔制备了DMRT1和DMRT4多克隆抗体,并进行纯化。对纯化多抗进行Western blot分析,结果表明获得了高特异性的DMRT1和DMRT4抗体。为了观察DMRT1和DMRT4在组织中的表达谱,首先,我们通过实时荧光定量RT-PCR检测雌雄奥利亚罗非鱼多种组织mRNA的表达,仅在卵巢和脑中检测到DMRT4,在精巢中检测到DMRT1;其次,制备了多种组织匀浆蛋白,使用纯化的抗体进行Western blot分析,仅分别在卵巢和精巢中检测到DMRT4和DMRT1蛋白的表达;制备多种奥利亚罗非鱼组织切片,使用纯化的DMRT4和DMRT1多抗进行免疫组织化学分析,发现DMRT4仅在卵巢表达,而DMRT1仅在精巢表达。这些结果有助于阐明DMRT4和DMRT1的功能及在鱼类性别调控中的作用。     

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.     

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.     

鸟类性别决定候选基因在性反转鸡胚中的表达

DMRT1、PKCIW和FET1是鸟类性别决定过程中重要的候选基因。以芳香化酶抑制剂处理的鸡胚为实验材料, 对这3个基因的表达变化进行了研究。结果表明, 在整个性别决定关键时期(E4.5 ~ E10.5), DMRT1在雄性的表达量显著高于雌性, 并且在ZW性反转鸡胚中表达大幅上升, 表明DMRT1的上调表达是与睾丸形成相关的。PKCIW基因在雌性特异表达并在性反转鸡胚表达上升, 这可能与其特殊作用模式有关, 即使性反转鸡胚PKCIW代偿性的表达升高, 却也未能阻止睾丸的形成。此外, FET1为雌性特异表达, 但在性反转鸡胚中表达无变化。综上, 实验结果支持了DMRT1是鸟类睾丸发育决定因子的假说。     

Sex determination: insights from the chicken

Not all vertebrates share the familiar system of XX:XY sex determination seen in mammals. In the chicken and other birds, sex is determined by a ZZ:ZW sex chromosome system. Gonadal development in the chicken has provided insights into the molecular genetics of vertebrate sex determination and how it has evolved. Such comparative studies show that vertebrate sex-determining pathways comprise both conserved and divergent elements. The chicken embryo resembles lower vertebrates in that estrogens play a central role in gonadal sex differentiation. However, several genes shown to be critical for mammalian sex determination are also expressed in the chicken, but their expression patterns differ, indicating functional plasticity. While the genetic trigger for sex determination in birds remains unknown, some promising candidate genes have recently emerged. The Z-linked gene, DMRT1, supports the Z-dosage model of avian sex determination. Two novel W-linked genes, ASW and FET1, represent candidate female determinants.     

黑鲷DMRT1基因cDNA的克隆、组织表达谱及在性别逆转前后性腺中的表达

利用RT—PCR和3′-RACE的方法从黑鲷精巢中克隆丁DMRT1基因cDNA的部分序列。组织特异性表达分析表明DMRT1基因只在精巢中表达,半定量RT—PCR检测显示DMRT1基因在性别逆转前、性别逆转期和性别逆转后的精巢中的表达量无显著差异,在鱼类成体的精巢中,DMRT1的表达量可能不会因精巢结构或生理状态的改变而发生改变,而始终维持一定量的表达。     

鲫Dmrt3基因的克隆和表达分析

DMRT家族是一个与性别决定相关的转录因子家族。为了研究家族成员之一的Dmrt3在我国重要养殖鱼类鲫胚胎发育、性别分化中的功能以及在育种中的作用,我们克隆了鲫Dmrt3基因的cDNA全长,并对Dmrt3基因在发育早期和不同组织中的表达进行了分析。结果显示:鲫Dmrt3基因cDNA全长为2182 bp,其中5￠端非编码区408 bp,3'端非编码区427 bp,开放阅读框1347 bp,编码448个氨基酸。蛋白结构预测显示DMRT3除了正常的DM结构域外,还有DMA结构域,在进化上与DMRT4和DMRT5的亲缘关系更近。巢式RT-PCR分析结果表明Dmrt3直到尾芽期才开始有微量表达,表达量在15体节期有明显增加但仍然处在一个较低的水平;在成体组织中只在精巢中检测到表达。这种表达时空模式提示Dmrt3可能在早期器官发生和雄性性腺发育调控中起作用。对Dmrt3启动子CpG岛的甲基化分析表明所检测的组织和配子中并不发生甲基化,说明这种雌雄特异性和组织特异性差异表达并不是通过对该基因启动子的差异甲基化修饰来调控的。此外,我们还发现了鲫Dmrt3的一个由逆转录产物形成的假基因pDmrt3。这些结果为进一步研究鲫Dmrt3在性别分化中的作用和评估其在鲫性别控制育种中的价值,以及分析DMRT家族的进化关系提供了基础资料。