doublesex: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions and Regulation of Splicing | Developmental Biology | Effects of Mutation | References

Gene name - doublesex

Synonyms -

Cytological map position - 84E1-2

Function - transcription factor

Keywords - somatic sex determination

Symbol - dsx

FlyBase ID:FBgn0000504

Genetic map position - 3-48.1

Classification - novel zinc finger domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Kimura, K., Sato, C., Koganezawa, M and Yamamoto, D. (2015). Drosophila ovipositor extension in mating behavior and egg deposition involves distinct sets of brain interneurons. PLoS One 10: e0126445. PubMed ID: 25955600
Summary:
Oviposition is a female-specific behavior that directly affects fecundity, and therefore fitness. If a fertilized female encounters another male that she has evaluated to be of better quality than her previous mate, it would be beneficial for her to remate with this male rather than depositing her eggs. Females who decided not to remate exhibited rejection behavior toward a courting male and engaged in oviposition. Although recent studies of Drosophila melanogaster identified sensory neurons and putative second-order ascending interneurons that mediate uterine afferents affecting female reproductive behavior, little is known about the brain circuitry that selectively activates rejection versus oviposition behaviors. This study identified the sexually dimorphic pC2l and female-specific pMN2 neurons, two distinct classes of doublesex (dsx)-expressing neurons that can initiate ovipositor extension associated with rejection and oviposition behavior, respectively. pC2l interneurons, which induced ovipositor extrusion for rejection in females, had homologues that controlled courtship behavior in males. Activation of these two classes of neurons appeared to be mutually exclusive and each governed hierarchical control of the motor program in the VNC either for rejection or oviposition, contributing centrally to the switching on or off of the alternative motor programs.
Price, D.C., Egizi, A. and Fonseca, D.M. (2015). The ubiquity and ancestry of insect doublesex. Sci Rep 5: 13068. PubMed ID: 26278009
Summary:
The doublesex (dsx) gene functions as a molecular switch at the base of the insect sex determination cascade, and triggers male or female somatic sexual differentiation in Drosophila. To understand the evolution of this integral gene relative to other arthropods, this study tested for the presence of dsx within public EST and genome sequencing projects representative of all 32 hexapod orders. dsx was found to be ubiquitous, with putative orthologs recovered from 30 orders. Additionally, both alternatively spliced and putative paralogous dsx transcripts were recovered from several orders of hexapods, including basal lineages, indicating the likely presence of these characteristics in the hexapod common ancestor. Of note, other arthropods such as chelicerates and crustaceans express two dsx genes, both of which are shown to lack alternative splicing. Furthermore, a large degree of length heterogeneity was discovered in the common region of dsx coding sequences within and among orders, possibly resulting from lineage-specific selective pressures inherent to each taxon. This work serves as a valuable resource for understanding the evolution of sex determination in insects. 

Shirangi, T. R., Wong, A. M., Truman, J. W. and Stern, D. L. (2016). Doublesex regulates the connectivity of a neural circuit controlling Drosophila male courtship song. Dev Cell 37: 533-544. PubMed ID: 27326931
Summary:
It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. This study reports the discovery of male-specific thoracic interneurons-the TN1A neurons-that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron, hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the hg1 motoneuron is regulated by the sexual differentiation gene doublesex. doublesex was shown to be required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the hg1 motoneuron. These findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.
Rezaval, C., Pattnaik, S., Pavlou, H. J., Nojima, T., Bruggemeier, B., D'Souza, L. A., Dweck, H. K. and Goodwin, S. F. (2016). Activation of latent courtship circuitry in the brain of Drosophila females induces male-like behaviors. Curr Biol 26: 2508-2515. PubMed ID: 27568592
Summary:
Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors. Fruit fly males exhibit an elaborate courtship display toward a potential mate. Females never actively court males, but their response to the male's display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru). Activation of male-specific dsx/fru+ P1 neurons in the brain initiates the male's courtship display, suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx+ neurons play a pivotal role in sexual receptivity and post-mating behaviors. This study manipulated the function of dsx+ neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, it was found that activation of female dsx+ neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. Specific dsx+ neurons critical for driving male courtship were uncovered and pheromones were identified that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, this study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.
Wagamitsu, S., Takase, D., Aoki, F. and Suzuki, M. G. (2017). Identification of the Doublesex protein binding sites that activate expression of lozenge in the female genital disc in Drosophila melanogaster. Mech Dev [Epub ahead of print]. PubMed ID: 28087460
Summary:
Normal sexual differentiation in the genital organs is essential for the animal species that use sexual reproduction. Although it is known that doublesex (dsx) is required for the sexual development of the genitalia in various insect species, the direct target genes responsible for the sexual differentiation of the genitalia have not been identified. The lozenge (lz) gene is expressed in the female genital disc and is essential for developments of spermathecae and accessory glands in Drosophila melanogaster. The female-specific isoform of DSX (DSXF) is required for activating lz expression in the female genital disc. However, it still remains unclear whether the DSXF directly activates the transcription of lz in the female genital disc. This study found two sequences (lz-DBS1 and lz-DBS2) within lz locus that showed high homology to the DSX binding motif identified previously. Competition assays using recombinant DSX DNA-binding domain (DSX-DBD) protein verified that the DSX-DBD protein bound to lz-DBS1 and lz-DBS2 in a sequence-specific manner with lower affinity than to the known DSX binding site in the bric-a-brac 1 (bab1) gene. Reporter gene analyses revealed that a 2.5-kbp lz genomic fragment containing lz-DBS1 and lz-DBS2 drove reporter gene (EGFP) expression in a manner similar to endogenous lz expression in the female genital disc. Mutations in lz-DBS1 alone significantly reduced the area of EGFP-expressing region, while EGFP expression in the female genital disc was abolished when both sites were mutated. These results demonstrated that DSX directly activates female-specific lz expression in the genital disc through lz-DBS1 and lz-DBS2.
BIOLOGICAL OVERVIEW

What does it take to be a sexually functional fly? In terms of biochemical genetics, an intact developmental hierarchy for sex determination is of prime importance. The Sex lethal gene is at the top of this hierarchy (see Schematic of the sex determination hierarchy in Control of male sexual behavior in Drosophila by the sex determination pathway, Billeter, 2006). In females, the Sex lethal splice factor assures production of functional transformer and transformer 2 mRNA. The coding for these proteins assures the production of the female version of Doublesex protein. In males, the Sex lethal splice factor is not made; consequently, the Transformer message is not properly processed, and Transformer proteins are not produced. This causes an alternative splicing of Doublesex mRNA to an mRNA that codes for a male version of Doublesex protein.

Doublesex acts as a transcription factor. As with most hierarchies, the agent at the bottom (in this case Doublesex) does all the work. Mutants in the dsx gene develop as intersexes. Since intersexuality results from the absence of a functional dsx product, it is thought that the male version of DSX represses genes that are involved in determining female fate, and the female version is involved in repressing male fate.

Rather than acting alone, Doublesex acts in concert with other regulatory genes (Steinmann-Zwicky, 1990). For example, doublesex acts in concert with engrailed, Polycomb and extra sex combs to form the sex comb, a structure differentiated in the anterior compartment of the basitarsus on the prothoracic leg of males but not females. The best known molecular example of transcription factor interaction involved in differential regulation of a gene in the two sexes is the role of doublesex in the fat body specific transcription of the two yolk protein genes (An, 1995).

There are two interesting questions that are unresolved. First, does Doublesex act only as a repressor? Second, does Doublesex do all the work of somatic sex determination? The answer to both these questions is no. There is some evidence, that not all Doublesex roles can be ascribed to gene repression. Heat shock-induced expression of the male cDNA in either sex results in three novel phenotypes: transformation of bristles on all legs toward a sex comb-like morphology, pigmentation of dorsal spinules and ventral setae in third-instar larvae, and lethality. These results were not predicted by DSX function as a repressor, and provide evidence that the role of the male DSX protein includes activation of some aspects of male differentiation as well as repression of female differentiation (Jursnich, 1993).

Doublesex does not seem to be the only factor involved in somatic sex determination. doublesex does not control a neuronally-determined feature of sex-specific anatomy--a muscle in the male's abdomen, whose normal development is, however, dependent on the action of fruitless. An assessment of the effects of doublesex mutation on general reproductive actions and on a particular component of the courtship sequence (male "singing" behavior) leads to the suggestion that there is a previously unrecognized branch within the sex-determination hierarchy that controls the differentiation of the male- and female- specific phenotypes of Drosophila. This new branch separates from the doublesex-related one immediately before the action of that gene (just after transformer and transformer-2) and appears to control as least some aspects of neuronally determined sexual differentiation of males (Taylor, 1994).

Other complex sex traits are just beginning to be understood. For example, Drosophila seminal fluid reduces the competitive ability of sperm from other males, thereby increasing male fitness (Rice, 1996). For example, a Drosophila seminal fluid protein has been identified that stimulates egg laying in females for 1 day after mating (Herndon, 1995). Such competitive traits in males lead to a reduction in female survivorship. Is Doublesex involved in regulating male competitive traits?

Sex traits regulated in the germ line will not be regulated by Doublesex, since a different hierarchy of gene regulation effects germ line sex characteristics than effects autosomal sex characteristics (See Sex lethal). In fact, the convoluted network of gene regulation results in an absence of direct functioning of the sex hierarchy in some gene pathways where it might be expected to act. For example, DSX does not regulate Yolk protein gene expression in follicle cells of the ovary. The responsible regulator is an ovary-specific GATA factor, dGATAb, known as Serpent (Lossky, 1995).

A genomic analysis of Drosophila somatic sexual differentiation and its regulation

The female Dsx protein (DsxF) has been ectopically expressed from a constitutive promoter and its regulatory activities have been examined, independent of other upstream factors involved in female sex determination. DsxF functions as a positive regulator of female differentiation and a negative regulator of male differentiation. As predicted by the DNA-binding properties of the Dsx protein, DsxF and DsxM compete with each other for the regulation of target genes. In addition to directing sex-specific differentiation, DsxF plays an important role in sexual behavior. Wild-type males ectopically expressing DsxF are actively courted by other males. This acquisition of feminine sex appeal is likely due to the induction of female pheromones by DsxF. More extreme behavioral abnormalities are observed when DsxF is ectopically expressed in dsx- XY animals; these animals are not only courted by, but also copulate with, wild-type males. Evidence is also provided that intersex is required for the feminizing activities of DsxF and that it is not regulated by the sex-specific splicing cascade (Waterbury, 1999).

Using constitutively expressed dsxF transgene, it was asked if DsxF could interfere with the formation of sex combs on the basitarsus of the foreleg in males. Females do not have sex combs; instead, they have a traverse row of bristles. Males and females homozygous for loss-of-function dsx alleles have bristles that are not aligned as a traverse row and do not resemble sex comb teeth. DsxM has been shown to have a positive role in sex-comb formation; intermediate sex combs form when DsxM is expressed in females carrying a dsxDom allele and ectopic sex combs form on all six legs in males or females when DsxM is expressed ubiquitously under hsp70 control. However, Dsx F does not appear to affect sex-comb formation in otherwise wild-type males when dsxF is ectopically expressed using an actin-dsxF or an hs70-dsxF transgene. These results led to the idea that DsxF does not play a role in the formation of sex combs (Waterbury, 1999).

A single copy of the hsp83-dsxF transgene has no readily apparent effects on sex-comb formation in otherwise wild-type males. To look more closely for a competitive balance between DsxF and DsxM on sex comb formation, endogenous copies of dsx were removed. By reducing the level of endogenous dsx, DsxF is able to influence sex comb formation, resulting in phenotypically intersexual or intermediate sex combs, with the teeth becoming more bristle-like. Removal of all endogenous dsx results in complete loss of sex combs and transformation to female bristles. These results suggest that DsxF acts to negatively regulate sex comb formation in females and indicates that a competition exists between DsxF and DsxM when both protein forms are present. This is the first example of a negative role for DsxF (Waterbury, 1999).

To date, the only known direct target of Dsx binding is the fat body enhancer (FBE), which lies directly between the two yolk protein genes, yp-1 and yp-2. Both forms of Dsx bind to the same sites within the FBE with opposite regulatory effects on yp-1 transcription: DsxF activates and DsxM represses. Yp-1 is expressed in both the fat body and the ovaries of females; however, Dsx only regulates expression within the fat body. DsxF is not absolutely essential for yp-1 expression in the fat body. In the complete absence of dsx activity, XX and XY animals express low levels of yp-1. One copy of the hsp83-dsxF transgene in otherwise wild-type males is sufficient to activate yp-1 expression in the fat body. Thus DsxF acts to positively regulate yp-1 expression in the fat body. The level of yp-1 expression induced in transgene males is only ~2.5-fold less than that expressed in the fat body of wild-type females (Waterbury, 1999).

According to the competition model for Dsx binding to the FBE and the phenotypic effects observed on sex combs described earlier, one would expect to see an increase in yp-1 expression as the level of negatively competing DsxM is reduced. To test this, the dose of endogenous dsx was varied and the amount of yp-1 mRNA transcript was measured. As predicted, the levels of yp-1 mRNA increase as endogenous dsx is reduced or completely removed. Although mRNA expression levels change as a result of dsx gene dosage, Dsx is not the only factor responsible for yp gene regulation (Waterbury, 1999).

A test was also performed to see whether the hsp83-dsxF transgene could drive expression of an hsp70-lacZ reporter construct through an upstream minimal fat body enhancer element, o. The o enhancer contains a Dsx protein-binding site, an overlapping Aef1 transcription factor-binding site, and an overlapping, potential bZip protein-binding site. Four tandem copies of the o element upstream of the LacZ reporter are sufficient for expression in wild-type females but not in wild-type males or XX;dsx- flies. Ectopic expression of DsxF in dsx+ males induces lac-Z expression from the o element:hsp70-lacZ reporter. Thus, a reporter containing only the minimal Dsx enhancer responds like the endogenous yp1 gene to the feminizing activities of the hsp83-dsxF transgene (Waterbury, 1999).

It has been hypothesized that intersex (ix) acts in parallel with or downstream of dsx in females. This hypothesis is based principally on the similarity of dsx and ix mutant phenotypes in females. Mutant females homozygous for ix have an intersexual phenotype that closely resembles that of dsx mutant animals. Additionally, expression of yp-1 mRNA is greatly reduced in ix- females and the level of mRNA is comparable to that seen in dsx- females. In contrast, and unlike dsx- males, males homozygous for ix- have no observable phenotype and do not express detectable levels of yp-1 mRNA. The latter result indicates that DsxM can repress yp-1 transcription in the absence of Ix protein. Given that DsxF can induce yp-1 expression in males, an examination was carried out to see if this induction is dependent upon ix. Males carrying the dsxF transgene, but homozygous for ix- and wild type for dsx are phenotypically wild type and fertile. However, without ix, DsxF is no longer able to induce expression of yp-1. Similarly, in males, induction of LacZ expression from the o element hsp70-lacZ reporter by the dsxF transgene is also dependent upon the ix gene. These findings suggest that DsxF and Ix function synergistically to activate full transcription of yp-1 in the fat body. They also indicate that Ix is either constitutively expressed in males or is under the direct control of the DsxF protein (Waterbury, 1999).

DsxF plays an important role in sexual behavior. The courtship behavior of wild-type Drosophila males has been well characterized and involves a series of choreographed routines. It begins with an orientation of the male toward the female, followed by wing extension and vibration to produce stimulatory songs, tapping, licking of the female genitalia, mounting, abdomen curling, and finally copulation. The genetic regulatory circuits controlling these different sexual behaviors appear to be more complicated than those involved in directing the differentiation of either male- or female-specific adult cuticular structures. At least three genes are known to contribute to sexual behavior: dsx, dissatisfaction (dsf), and fruitless (fru). Mutations in all three genes alter male sexual behavior and/or neurogenesis, while female behavior and/or neuronal development are affected only by dsx and dsf mutations. As with dsx, fru is alternatively spliced in females by the Sxl - tra/tra-2 splicing cascade and thus fru is independent of dsx. Although genetic studies have suggested that dsf is also under the control of the Sxl - tra/tra-2 splicing cascade, recent cloning and additional analysis of dsf have suggested that it represents a tra/tra-2 independent pathway (Waterbury, 1999).

dsx- males have a lower measured courtship index toward females than wild-type males, exhibiting a reduced frequency of wing extension and song singing, and are defective in the production of the sine song. dsf- males, in contrast, actively court with nearly normal courtship routines; however, they fail to discriminate between the sexes and court males with the same avidity as females. They are also slow to copulate, due to defects in abdominal neuronal development that affect abdominal curling. Mutations in fru cause a number of defects in male courtship. fru- males court with greatly reduced vigor compared to wild-type males, and the later courtship routines, such as singing and copulation, are abnormal or missing. Finally, fru- males court males and females with equal avidity. It was asked whether the dsxF transgene has any effects on male courtship behavior. As a (partial) control for these experiments, the effect of another transgene, hsp83-traF, was examined on male courtship behavior. This transgene expresses female Tra protein, and together with Tra-2, should direct the female-specific expression not only of dsx, but also of fru. The hsp83-traF transgene is expected to more strongly feminize XY animals than hsp83-dsxF; however, the feminization of XY animals by the hsp83-traF is not complete in all tissues, and male-specific structures, such as the Muscle of Lawrence, are still observed. When hsp83-traF pseudofemales were placed in individual chambers with another male or female, they showed little interest in courting. When they did court, they did not discriminate between males and females and only very early courtship routines were observed, such as orientation, tapping, and brief wing vibration (Waterbury, 1999).

dsxF transgene males are fertile and can and will mate with females. The measured courtship index of dsxF transgene males demonstrates that they court virgin females with as much interest as wild-type males. The duration of copulation of dsxF transgene males was also measured and there is a significant, slight reduction in the time of copulation compared to wild-type males A test was performed to see whether the dsxF transgene males would discriminate between females and males. Unlike dsf-, fru-, or hsp83-traF males, dsxF transgene males do not court wild-type males. However, wild-type males court transgene males and transgene males court each other with significant courtship indices. Hsp83-traF pseudofemales also elicited high levels of courtship from wild-type males. Given that dsxF transgene males do not court wild-type males, the observed behavioral abnormalities are unlikely to be due to an inability to discriminate between the sexes. Rather it is suspected that the dsxF transgene males produce female attractants that are responsible for eliciting courtship behaviors by other males (Waterbury, 1999).

While the dsxF transgene had no apparent effect on the courtship behavior of (otherwise) wild-type males toward females, courtship behavior could be altered by reducing the dose of dsx gene. To distinguish XY dsxF males from XX females in this experiment, the XY animals were marked with the Y chromosome-linked eye marker Bs. The defect caused by Bs has been shown to cause a twofold reduction in male courtship. Because males heterozygous for dsx- court as wild-type males do, this twofold reduction in the courtship index is likely due to the impaired visual system of the Bs animals. The courtship of transgenic XY animals either heterozygous or homozygous for dsx- was examined. Heterozygous transgenic males, court less frequently and less aggressively than controls, and when they do court, it is not sustained for long periods of time. Even more severe defects in courtship behavior were evident for dsxF pseudofemales when compared to CI of dsx nulls. They show little interest in females and perform only early mating behaviors (orientation, tapping, wing extension and vibration). The courtship index of these pseudofemales is comparable, although significantly less than that of the hsp83-traF transgene males (Waterbury, 1999).

Because the dsxF pseudofemales exhibit reduced male courtship behavior, it was asked whether these pseudofemales would respond like wild-type females to courtship by wild-type males. While dsxF pseudofemales actively reject courting wild-type males, they do allow themselves to be mated. Unlike wild-type females, however, the dsxF pseudofemales continue to move around the chamber during copulation and flick their wings in an apparent attempt to dislodge the male. This difference in activity during copulation is evident in the relative frequency of line crossing by wild-type females and dsxF pseudofemales. In addition dsxF pseudofemales take two- to three-fold longer to mate than wild-type females (Waterbury, 1999).

A plausible explanation for the high levels of courtship elicited from wild-type males by dsxF transgene males is that ectopic expression of DsxF protein induces the expression of female pheromones. Pheromones are produced by oenocytes located directly beneath the adult abdominal cuticle and consist of several long chain hydrocarbons. Females and males each generate their own characteristic aphrodisiac and antiaphrodisiac pheromones. Two long-chained compounds characterized as male attractants, 7,11-heptacosadiene (7,11-27:2 or 7,11-HCD) and 7,11-nonacosadiene (7,11-29:2 or 7,11-NCD), are produced by females. Females also produce two minor compounds: 27:0 and 7-27:1. Males lack these female-specific compounds and instead produce compounds thought to be antiaphrodisiacs, such as 5-tricosene (5-23:1 or 5-T) and 7-tricosene (7-23:1 or 7-T). Although 5-T is only present in rather small quantities in wild-type males, it has significant inhibitory effects on male courtship. Wild-type females produce only trace amounts of 5-T. The antiaphrodisiac 7-T is shown to be present in both sexes; however, males produce much higher levels than females (Waterbury, 1999).

dsx is known to have a role in the production of these pheromones but that role has not been fully defined. To a first approximation, the pheromone profile of homozygous dsx- females resembles that of wild-type males. dsx mutant females have little or no 7,11-NCD or 7,11-HCD, and instead produce reduced levels of the two minor female-specific hydrocarbons (27:0 and 7-27:1) and high levels of the male hydrocarbons (7-T and 5-T). The pheromone profile of dsx- XY animals is similar to that of wild-type males: the levels of 7-T and 5-T remain high; however, unlike wild-type males, dsx- males have small but detectable amounts of the female aphrodisiac 7,11-NCD, and produce the two minor female-specific hydrocarbons, 27:0 and 7-27:1, at levels comparable to dsx- females. Introduction of one copy of the dsxF transgene into otherwise wild-type males is sufficient to dramatically alter the pheromone profile of these XY animals. The results are most straightforward for the two major female-specific aphrodisiacs, though similar changes are observed for the minor female-specific hydrocarbons, 27:0 and 7-27:1. In contrast to wild-type males, dsxF transgene males produce significant amounts of the female-specific dienes, 7-HCD (168.5 ng/fly) and 7-NCD (42.0 ng/fly). When one copy of the endogenous dsx gene is removed, the levels of 7,11-NCD increase while the amount of 7,11-HCD drops slightly. Essentially the same female pheromone profile is observed when both endogenous alleles are removed. These results suggest that DsxF has a positive effect on the production of the female-characteristic compounds 7,11-HCD and 7,11-NCD and would account for the lack of either diene in wild-type males and dsx- females (Waterbury, 1999).

Production of male-characteristic pheromones is also altered in dsxF transgene males. One copy of the dsxF transgene is sufficient to reduce the levels of the potent male antiaphrodisiac 5-T to trace amounts (10.0 ng/fly), a level similar to that detected in control females (10.0 ng/fly). This is much less than that found in XX and XY dsx mutants (31.5 and 52.0 ng/fly, respectively. As observed for the female-specific compounds 7,11-HCD and 7,11-NCD, this effect on 5-T production is largely independent of endogenous dsx. Together with the observation that relatively high levels of 5-T are found in XX and XY dsx mutants, these results suggest that reduction of 5-T synthesis caused by DsxF cannot be overcome by DsxM. Production of the antiaphrodisiac 7-T is also reduced by DsxF. The amount of 7-T decreases nearly 10-fold from 836.0 ng/fly in wild-type males to 93.0 ng/fly in dsxF transgene males. This amount is less than that detected in XX or XY dsx mutants (411.5 and 588.5 ng/fly, respectively) and close to that measured in wild-type females (103.0 ng/fly) (Waterbury, 1999).

Three lines of evidence argue that the changes observed in hydrocarbon profiles are a consequence of dsxF expression. (1) Similar results were obtained in all dsxF transgenic lines examined; (2) males transgenic for a control mini-white construct have a male-characteristic hydrocarbon profile. (3) The pheromone profile of males transgenic for hsp83-traF resembles that of wild-type females. However, traF feminizes the pheromone profile solely by directing expression of dsx in the female mode. In addition to the long-chained hydrocarbons synthesized by oenocytes under the adult cuticle, another male-specific compound, cis-vaccenyl acetate (cVA), is produced by the ejaculatory bulb in males and transferred to females during mating. XX and XY dsx mutants both produce cVA . Unlike production of 5-T and 7-T, production of cVA does not seem to be strongly affected by DsxF in the presence of DsxM, although quantitated amounts of cVA do decrease from 160.0 ng/fly in control males to 45.0 and 107.0 ng/fly in dsxF;+/+ and dsxF; dsx1/+ males, respectively. However, when all endogenous dsx is removed, no cVA can be detected in XY flies carrying the transgene. Because XX and XY dsx1/Df flies produce cVA, it is possible that cVA production is negatively regulated by DsxF but only efficiently in the complete absence of DsxM (Waterbury, 1999).

Given that ix appears to have a role in yolk protein production in dsxF transgene males, a test was performed to see if there is a similar dependence on ix for pheromone production. Males mutant for ix have a hydrocarbon profile similar to wild-type males and dsx- males, indicating that no role for ix can be assigned in males under these conditions. This result is in agreement with all previous results regarding the lack of phenotypic effects of ix in males. In contrast, females mutant for ix produce a hydrocarbon profile very different from that of wild-type females. Similar to the profile of dsx- females, 7,11-HCD and 7,11-NCD were not detectable in ix- females, suggesting that ix is required for the production of these two female-specific compounds. This result is in contrast to that described earlier for yp-1 expression in the fat body where neither ix nor dsx alone is required for basal yp-1 expression, but both are required for full expression. Removal of ix also results in an increase in male-characteristic compounds, such as 5-T and 7-T. Once again, this result is similar to that observed in dsx- females. These results suggest that DsxF and Ix function together to promote the production of female-specific pheromones. This conclusion is further supported by the pheromone profile of dsxF transgene males mutant for ix. Where one copy of the dsxF transgene in males can induce production of the female-specific dienes 7,11-HCD and 7,11-NCD, neither of these compounds can be detected in dsxF transgene males mutant for ix. A similar dependence on ix is seen in production of the male-characteristic compounds 5-T and 7-T. These results suggest that DsxF and Ix function together to prevent the production of male-characteristic pheromones (Waterbury, 1999).

Previous studies have shown that ix is required for normal female development, but is dispensable in males. From these earlier studies alone, it is unclear whether ix is regulated directly by the Sxl - tra splicing cascade or by dsx, or constitutively expressed in both sexes. Because ix is required for the induction of both yp mRNA synthesis and female pheromones in dsxF transgene males, it would appear that ix expression is not directly dependent upon the Sxl - tra splicing cascade. While the possibility that DsxF induces ix expression in XY animals cannot be excluded, the results presented in this study would be most easily explained by nonsex-specific constitutive expression of ix. How does ix function in female sexual differentiation? With respect to yp expression and probably also pheromone production, the results argue that ix is an essential cofactor for DsxF, enabling DsxF to function as a positive regulator. Two different mechanisms could account for the effects of ix mutations on yp expression in normal females and in dsxF transgene males. ix could correspond to the unknown bZip transcription factor that is postulated to bind adjacent to Dsx in the minimal o element enhancer. An alternative and seemingly more likely mechanism is that Ix physically interacts with and/or modifies DsxF to potentiate its positive regulatory activities. A potentiating function, perhaps mediated through interactions with the female-specific domain of the Dsx protein, would also account for the role of ix in facilitating the repression of male-specific developmental pathways by DsxF. These questions will ultimately be resolved by the cloning and characterization of the ix gene (Waterbury, 1999).

A genomic analysis of Drosophila somatic sexual differentiation illustrating a rich diversity of dsx function

In virtually all animals, males and females are morphologically, physiologically and behaviorally distinct. Using cDNA microarrays representing one-third of Drosophila genes to identify genes that are expressed sex-differentially in somatic tissues, an expression analysis was carried out on adult males and females that: (1) were wild type; (2) lacked a germline; or (3) were mutant for sex-determination regulatory genes. Statistical analysis identified 63 genes sex-differentially expressed in the soma, 20 of which (thus far) have been confirmed by RNA blots. In situ hybridization experiments with 11 of these genes showed they were sex-differentially expressed only in internal genital organs. The nature of the products these genes encode provides insight into the molecular physiology of these reproductive tissues. Analysis of the regulation of these genes revealed that their adult expression patterns are specified by the sex hierarchy during development, and that doublesex probably functions in diverse ways to set their activities (Arbeitman, 2004).

When does sex hierarchy regulation of the 11 selected genes occur? There are two known mechanisms by which sex-differential gene expression in adults is generated: (1) the sex hierarchy actively regulates gene expression in adults, as is the case for Yolk protein 1 (Yp1); or (2) the hierarchy functions earlier in development to specify which sex-specific adult tissues will be formed but does not regulate gene expression in those tissues in the adult. Temperature-sensitive tra2 alleles were used; this allowed switching between the male and female mode of splicing dsx. In chromosomally XX tra-2ts animals, female development occurs at the permissive temperature (16°C), whereas male development occurs at the non-permissive temperature (29°C). Animals were raised at one temperature, collected 0-24 hours after eclosion and maintained at their original temperature for one more day. Then half of each group was switched to the other temperature (16°C to 29°C, or 29°C to 16°C). All animals were maintained for three more days, and RNA was then extracted. Under these conditions, expression of Yp1 (the positive control, responded to temperature shifts as expected; the Yp1 transcript was reduced when animals were switched from 16°C to 29°C and induced when animals were switched from 29°C to 16°C. By contrast, expression of the 11 other genes analyzed did not change substantially over the three days following the temperature shifts. Thus, sex-differential expression of all 11 genes is the consequence of the developmental action of the sex hierarchy and is independent of the hierarchy during adult stages (Arbeitman, 2004).

Since all 11 of the genes analyzed in these tra-2ts experiments are expressed within the sex-specific genital organs, these findings have implications for how the sex hierarchy functions in the construction of sex-specific organs. In the male accessory gland and ejaculatory duct, the sex hierarchy has been shown to function during the late larval/early pupal period to specify the adult expression patterns of several genes. The results for CG18284, CG17022 and CG17843, which are expressed exclusively in the male accessory glands, and for CG8708, which is expressed in the ejaculatory duct, are consistent with these findings in that the four genes are regulated by the action of the sex hierarchy prior to adulthood. For three other internal genital organs, the ejaculatory bulb, spermathecae and parovaria, there was no prior knowledge about when the hierarchy functioned. The data indicate that the adult sex-specific patterns of expression of at least some genes in these tissues (CG2858 in ejaculatory bulb, and CG17012 in spermathecae and parovaria) are also determined by the prior developmental action of the hierarchy. Taken together, these parallel findings with respect to five of the major organs of the internal genitalia are consistent with the notion that the sex hierarchy functions developmentally in all these organs to set up the eventual patterns of adult gene expression (Arbeitman, 2004).

As suggested by the microarray data, the 11 genes expressed in internal genital organs are almost certainly regulated by dsx rather than by fru, since dsx is known to regulate development of these tissues and FRUM is not expressed within the internal genitalia. The DSXM and DSXF proteins are known to have both positive and negative roles. Therefore, attempts were made to understand the manner in which the DSX proteins regulate these genes. For the two genes expressed in multiple tissues in adults, in situ hybridizations to XX and XY dsx null individuals were carried out to assess the mode of dsx regulation in individual organs. For the five genes that are expressed in one organ of the internal genitalia and nowhere else in adults, microarray data from dsx null individuals was used. The results of these analyses are suggestive of multiple modes of dsx regulation (Arbeitman, 2004).

To determine how dsx regulated the female gene CG17012 in spermathecae, in situ hybridization was carried out on wholemounts of internal genitalia of dsx null and wild-type individuals. Spermathecae are not recognizable in all dsx mutant individuals, so just those individuals with spermathecae were evaluated. Reduced expression, as compared with wild-type females, was seen in spermathecae of dsx null individuals. The diminished CG17012 expression in dsx mutants suggests that DSXF positively regulates its sex-specific expression. The microarray data for CG17012 are consistent with this conclusion and do not reveal any effect of DSXM on CG17012 expression. However, another possible explanation for reduced expression in dsx null individuals is that dsx mutants are unable to mate. There is evidence for changes in female behaviors mediated (perhaps at the transcriptional level) by seminal fluid and sperm transferred during copulation, including a reduction in receptivity to courtship and enhanced egg laying (Arbeitman, 2004).

To determine whether copulation, seminal fluid or sperm affect expression of CG17012, whole-mount in situ hybridization was performed on tissue from the following 5-day-old adults: (1) wild-type females; (2) virgin wild-type females that were separated from males prior to eclosion; and (3) tud females mated to tud males, which do not produce sperm but transfer other seminal fluid components. Comparable high levels of CG17012 expression were observed in the spermathecae and parovaria of all three types of females, suggesting lower expression in dsx mutants is not a consequence of their failure to mate, but rather that female-specific expression is the result of positive regulation by DSXF, and that DSXM plays no role in its regulation. This pattern of dsx regulation has not been previously reported (Arbeitman, 2004).

For the three genes (CG17843, CG17022 and CG18284) expressed exclusively in the male accessory glands, the microarray results suggest that their expression is not dependent on DSXM, as comparable high expression levels are seen in XY wild-type and XY dsx null individuals. Rather, male-specific expression appears to be the consequence of the negative action of DSXF in females; their expression is higher in XX dsx null individuals than in wild-type females. These observations are consistent with the previous finding that male-specific development of the accessory glands is the consequence of DSXF acting in females to prevent accessory gland formation. Interestingly, expression of these three genes is significantly higher in XY dsx null than XX dsx null, but is not significantly different between wild-type males and fru males, suggesting an additional sex-differential complexity to their expression (Arbeitman, 2004).

paired (prd) is also expressed in the male accessory gland, and is of interest as its expression is required during both development and adulthood for accessory gland formation and physiology. The role of dsx in prd expression was examined by means of in situ hybridization of frozen sections of XX and XY dsx null individuals, and XX and XY wild-type controls. Comparable levels of prd expression were observed in the accessory glands of XY wild-type animals and both XX and XY intersexual animals, with no visible expression in XX wild-type animals. These observations suggest that male-specific expression of prd is not a consequence of positive regulation by DSXM, but rather of negative action by DSXF to prevent the formation of male accessory glands, as was observed for the other accessory gland genes above (Arbeitman, 2004).

Finally, for CG2858 and CG8708, expressed exclusively in the ejaculatory bulb and ejaculatory duct, respectively, the microarray data suggest another mode of dsx regulation. For both of these genes, expression appears to be lower in XY dsx null animals than in the three genotypes expressing DSXM (XY wild type, XX tra and XX dsxD pseudomales), suggesting that DSXM positively regulates their expression. Expression also appears higher in XX dsx null compared with XX wild-type animals, suggesting negative regulation by DSXF in the formation of these tissues in females. This pattern of regulation has not been previously reported for a gene under the control of dsx, although it is the exact converse of how the Yp genes are regulated (Arbeitman, 2004).

Taken together these findings reveal a rich diversity of dsx function. In the accessory gland the sole role of dsx revealed to date is the action of DSXF to prevent the formation of the organ in females, whereas in the spermathecae, ejaculatory duct and ejaculatory bulb dsx appears to have two types of functions: (1) the hierarchy must be acting, via dsx, to direct these tissues to an alternative developmental fate in the inappropriate sex; (2) as shown in this study, dsx may also function in the appropriate sex in these three organs prior to adulthood, and probably during the late larval/early pupal period, to establish the potential for the appropriate patterns of gene expression (Arbeitman, 2004).

The sex determination hierarchy in Drosophila is well understood at the molecular-genetic level, but the genes that are sex-differentially regulated by the hierarchy have only begun to be identified. This study has examined sex-differential gene expression in adults, the stage of the Drosophila life cycle that displays the most striking differences between the sexes. This study adds substantially to knowledge of the types of genes expressed sex-differentially in somatic tissues, provides molecular entry points for elucidating the functions of reproductive organs of both sexes, and expands understanding of the timing and mode of gene regulation by the sex hierarchy (Arbeitman, 2004).


GENE STRUCTURE

There are two transcripts, with common N-terminal sequences. The first the exons are shared by male and female transcripts, while the female transcript is coded for by a unique fourth exon. The male transcript is coded for by a different fourth and fifth exon. The female specific fourth exon is found within the third male intron. The presence of two bands of the same mobility in both the primer extension and S1 nuclease experiments, using either male or female RNA, shows that the DSX transcripts begin at two positions separated by 38 nucleotides, and that the same pair of 5' ends is used in both males and females (Burtis, 1989).

Genomic length - 45 kb

Transcript lengths - 3.9 kb for female transcript, 3.9 and 2.9 kb for male transcripts

Bases in 5' UTR - 1022

Exons - 4 for the female, 5 for the male transcripts

Bases in 3' UTR - 918 for female, 1098 for male


PROTEIN STRUCTURE

Amino Acids - 427 for female protein and 549 for male protein

Structural Domains

A 13 nucleotide sequence occurs six times in the first half of the female-specific exon (Burtis, 1989). The two Doublesex proteins share a common and novel zinc finger-related DNA binding domain distinct from any reported class of zinc binding proteins. Combined N-terminal and C-terminal deletion experiments confirm that for male DSX protein there exists only one domain capable of binding to the fat body enhancer of the Yp1-Yp2 intergenic region, and that this domain is located in the common region shared by the male and female DSX polypeptides. These results indicate that the same binding domain is essential for the activities of the DSX polypeptides in both males and females (Erdman, 1993).

Regulation of sexual dimorphism: mutational and chemogenetic analysis of the doublesex DM domain

Doublesex 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. The DM motif has been characterized by site-directed and random mutagenesis using a yeast one-hybrid (Y1H) system and this analysis has been extended 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. 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 (Zhang, 2006).

The DM domain defines a newly recognized family of minor-groove transcription factors. Such proteins participate in metazoan sex-determining pathways and general developmental patterning, a classical target of the Drosophila sex-determining hierarchy. Although the sex-specific isoforms of Dsx have long been regarded as terminal differentiation factors, recent genetic evidence suggests that DsxF and DsxM also function to integrate sex-specific and positional information in morphogenesis. Of particular interest are potential interactions between homeotic proteins (such as Abdominal A and Abdominal B) in the joint regulation of segmental identity. Because the homeodomain contains a helix-turn-helix (HTH) motif that binds within the DNA major groove, it is possible that coordinate gene regulation is effected through the approximation of major- and minor-groove DNA-binding sites within a sex-specific multiprotein-DNA complex. Juxtaposition of major- and minor-groove DNA control sites has been observed in the fbe control site: Dsx target sites overlap a putative bZIP site, which in principle may allow or preclude simultaneous occupancy. The helical arms of bZIP transcription factors (like the homeodomain HTH) bind within the major groove (Zhang, 2006).

The DM domain contains a novel Zn module and nascent helical tail. The helical propensity of the tail, intrinsic to its sequence, depends on specific DNA binding for its realization. Such induced fit is unrelated to metal ion binding. How these elements bind DNA is not well understood. As a first step toward their functional characterization, attempts were made to delimit domain boundaries by deletion analysis and identify key side chains by mutagenesis. To this end, a rapid and efficient genetic screen in S. cerevisiae was constructed based on a DsxF-regulated Y1H system. Its design recapitulates the physiological regulation of yp1 gene expression by specific Dsx target sites in the fat body enhancer (fbe). Control studies of intersexual dsx mutations and nonconsensus base-pair substitutions in the fbe sites established the validity of this model. As expected, DM-regulated expression of the Y1H reporter gene requires intact metal-binding sites in the Zn module. Functional boundaries of the DM domain span Dsx residues 31 to 105 in accord with its evolutionary consensus and previous DNA-binding studies. Surprisingly, although deletion of the C-terminal segment (residues 98 to 105) leads to a 10-fold decrease in reporter gene activation, none of the 18 substitutions in this segment are deleterious, and the segment itself may functionally be replaced by polyalanine. These results suggest that a critical parameter is provided by the length of the distal tail but not its sequence. Since the tail folds on DNA binding to form one or more alpha-helical segments, it is imagined that a minimum C-terminal length is required for segmental helical stability. Similar findings have been described previously in studies of adaptive RNA binding by helical arginine-rich ARM peptides (Zhang, 2006).

Contributions of individual side chains were probed by a combination of site-directed and random mutagenesis. Site-directed mutations established a correlation with previous effects of alanine substitutions in the tail as assayed by gel mobility-shift assay, verified the dispensability of N-terminal residues 1 to 31, and demonstrated that cysteines and histidines are not interchangeable in the Zn module. Random mutagenesis provided an overview of allowed and disallowed substitutions. The majority of substitutions do not perturb reporter gene activation; these are distributed throughout the DM domain. Indeed, some residues tolerate diverse substitutions, indicating that such side chains are not required for either folding or for DNA binding. Loss-of-function mutations are by contrast confined to a limited region of the DM domain, defining sites that are critical to folding or predicted to interact with DNA (Zhang, 2006).

The core of the DM Zn module contains conserved aliphatic and aromatic side chains in addition to the immediate metal-binding ligands. Substitutions of internal side chains I54 and L56 result in white colonies, indicating that formation of a hydrophobic core is essential. Because the side chain of L56 packs against C44, H59, and K60, loss-of-function substitutions by Ala, Pro, or Gln would be expected to introduce, respectively, a destabilizing cavity (L56A), conformational perturbation (L56P), or an unfavorable buried polar group (L56Q). The side chain of I54 likewise packs against C44, L52, and H59, wherein substitutions would introduce a similar spectrum of perturbations. Substitutions at N49 are also deleterious. This internal polar side chain packs between the two metal-binding sites, enabling the side chain carboxamide to participate in a network of hydrogen bonds. The asymmetric distribution of partial charges near the S-Zn2+ bonds may be stabilized by this network. In addition, N49 has long-range contacts with the aliphatic portions of K60 and R79 (which may in turn contact DNA). Many sites at the surface of the DM domain or in the tail are tolerant of substitutions (residues P41, P42, K53, T55, R61, K64, R66, Y67, T69, E71, K72, R74, L75, V82, M83, L85, and Q86). Such findings are reminiscent of a pioneering structure-based analysis of allowed and disallowed sequences in the HTH domain of phage lambda repressor (Zhang, 2006).

A trend is observed wherein mutations that block transcriptional activation occur at conserved sites, whereas neutral substitutions occur at nonconserved sites. N43 and K53, for example, are well defined on the surface of the Zn module but not conserved. Substitutions N43I, N43Y, K53N, K53M, and K53E are well tolerated. Key exceptions to this trend are noteworthy. (1) Arg is conserved at positions 46 and 48 (positions 12 and 14 of the DM consensus). Indeed, R46 is the site of an intersexual mutation in mab-3 (C. elegans). Uncharged substitutions at these sites are nonetheless well tolerated. (2) Two glycines (G51 and G58) are invariant at sites adjoining metal-binding ligands (H50 and H59, respectively). In the solution structure, the glycines exhibit positive phi angles and so occupy regions of the Ramachandran plot ordinarily unfavorable to L-amino acids. Nonetheless, at each site, Ala is well tolerated, whereas other substitutions are disallowed. Modeling suggests that the variant side chains would project into solvent and not disrupt core packing. Tolerance of some substitutions suggests that positive phi angles at positions 51 and 58 are not necessary for metal-dependent folding; alternatively, it is possible that some L-amino acids can adopt unfavorable positive phi angles with only a modest free-energy penalty. Intolerance of other substitutions may indicate that neighboring surfaces are close to the DNA or DNA contact sites. (3) DM sequences contain a conserved aromatic side chain at position 65. In the Dsx structure, F65 packs between metal-binding sites. Surprisingly, the aromatic side chain may functionally be substituted by Ala, Cys, Lys, or Val, indicating that a broad range of packing schemes is well tolerated. This feature contrasts with the importance of a central aromatic side chain in the classical Zn finger. It would be of future interest to purify an F65A variant to assess its structure, stability, and DNA-binding properties (Zhang, 2006).

Of special interest are sites on the protein surface at which substitutions are not tolerated. Because such substitutions would not be expected to impair metal ion binding or protein folding, these sites define putative DNA contacts. Candidates are provided by several tail residues and K60, which defines a basic patch on the surface of the Zn module. K60 is highly conserved among metazoan DM sequences. Whereas substitutions at K57 (K57M, K57A, and K57N) result in light-blue colonies, several substitutions at K60 (K60E, K60Q, K60M, and K60H) yield white colonies. The inactivity of the K60H and K60Q variants is noteworthy in light of the hydrogen-bonding capability of His and Gln. Interestingly, the K60R variant exhibits wild-type activity, suggesting that the positive charge of K60 contributes to specific DNA binding. The importance of K60 is consistent with the 10-fold decrement in specific DNA binding that was previously observed in gel mobility shift assay studies of a norleucine analog. Further evidence that K60 contacts the DNA backbone is provided by methylphosphonate interference experiments. It is possible that K57 also contacts the DNA but that this contact contributes only modestly to the binding free energy (Zhang, 2006).

Understanding the molecular-genetic function of dsx in Drosophila development will in the future require biochemical reconstitution of sex-specific transcriptional preinitiation complexes. A central feature of such complexes will be the DM-DNA interface. The present study has utilized prior molecular-genetic characterization of a Dsx-responsive enhancer element to construct a Y1H system for mutational analysis of the DM domain. The results suggest sites of protein-DNA interaction and provide insight into the structural requirements of zinc-dependent protein folding. It is anticipated that these data will provide a foundation for crystallographic studies of DM-DNA complexes. Integration of structural and mutational studies promises to provide insight into the evolution and function of DM transcription factors (Zhang, 2006).


EVOLUTIONARY HOMOLOGS

Sex determination across evolution - connecting the dots: Evolution of sex determination mechanisms

The variety of primary sex determination cues was appreciated long before the advent of molecular genetics. The two broadest categories are genetic sex determination (GSD), in which the sex of offspring is set by a sex chromosome or an autosomal gene, and environmental sex determination (ESD), in which sex is determined by temperature (as with turtles), local sex ratio (as with some tropical fish), or population density (as with mermithid nematodes). Though little is known about the molecular mechanisms of ESD, within the GSD systems many different mechanisms have been uncovered. Dual sex chromosome systems, in which either the female (ZW/ZZ) or the male (XX/XY) is heterogametic, are common, as are systems set by the ratio of the number of X chromosomes to sets of autosomes (X:A). There are also systems in which heterozygosity at a single locus is required for female development (known as complementary sex determination), as well as systems involving sex determination via multiple genes with additive effects (Haag, 2005; see full text of article).

Molecular genetic investigations of GSD in model systems such as Drosophila, Caenorhabditis, and mice have revealed a clear lack of conservation, underscoring the diversity. For example, although the primary sex determination signal in both D. melanogaster and C. elegans is the X:A ratio, the fruit fly pathway consists of a cell-autonomous cascade of regulated mRNA splicing, while that of the nematode follows a Hedgehog-like intercellular signaling pathway. GSD in mammals depends (with some interesting exceptions upon a Y-specific dominant gene (Sry) encoding a transcription factor. In the face of such impressive differences, perhaps the assumption of homology should be questioned: could it be that sex determination in different taxa has arisen independently over and over again in evolution? Until 1998, this seemed like a good bet (Haag, 2005).

The discovery of the homology of the key sex-determining genes doublesex in Drosophila and mab-3 in C. elegans provided the first evidence for a common evolutionary basis of sex determination in animals. Soon, related doublesex-mab-3 (DM)-family genes with roles in male sexual development were discovered in vertebrates and even cnidarians. Here at last was a smoking gun that could link the diverse metazoan sex determination systems. But as satisfying as the result was, it immediately gave birth to another mystery: if the enormous diversity of sex determination systems are all derived from a common ancestor, how could they possibly have been modified so radically? After all, sexual differentiation and reproduction are hardly unimportant developmental processes (Haag, 2005).

To understand how such diversity came to be, differences between closely related species must be examined. This approach allows the discovery and interpretation of small-scale sex determination changes before they are obscured by subsequent changes. The processes discovered in this way might then be reasonably extrapolated to explain the seemingly unrelated systems of more deeply diverged taxa. Work in dipterans has revealed three evolutionary phenomena that characterize shorter-term sex determination evolution (Haag, 2005).

The first of these is the often astounding rate of molecular evolution at the level of nucleotide and aminoacid sequences. Although some sex-determining genes are well conserved, many show unprecedented substitution rates. An extreme example is the central integrator of the X:A ratio in Caenorhabditis, xol-1. The xol-1 orthologues of the closely related nematodes C. elegans and C. briggsae are a mere 22% identical, even though genes surrounding xol-1 are much better conserved. Remarkably, the 3′ neighbor of xol-1, the immunoglobulin dim-1, is only 5 kb away and is essentially identical between species (Haag, 2005).

A second phenomenon, best exemplified by dipteran insects, is the modification of genetic control pathways through the gain or loss of key pathway components. In Drosophila, the first gene to respond to the X:A ratio is Sxl, whose transcription is regulated by both autosomal and X-linked factors very early in development. When X: A = 1 (i.e., in female embryos), Sxl transcription occurs and produces Sxl protein. Later in development, transcription from a second promoter occurs in both sexes, but these transcripts cannot be productively spliced without the earlier burst of Sxl expression. As a result, only females sustain Sxl expression, and in turn only females can productively splice the mRNA of tra, its downstream target. Productive splicing of tra is required to produce the female-specific form of dsx, a founding member of the DM family mentioned above (Haag, 2005).

In a series of groundbreaking papers, Saccone and colleagues investigated the pathway in the more distantly related heterogametic Mediterranean fruit fly Ceratitis capitata. The first surprise was that although a highly conserved Sxl homologue exists in Ceratitis, it does not undergo sex-specific regulation similar to that of Drosophila, which suggests that it does not play a key switch role (Saccone, 1998). Similar results have also been found for the housefly, Musca domestica, indicating that the role of Sxl in sex determination may be restricted to Drosophila and its closest relatives. In contrast, tra and dsx are key sex regulators in all dipterans examined thus far (Haag, 2005).

A further surprise came when the Ceratitis tra homologue was characterized. In the case of this gene, clear evidence for sex-specific regulation was found, and as with Drosophila, only females productively splice tra mRNA. However, this splicing difference can be explained nicely by a positive feedback, similar to that seen in Drosophila Sxl, in which Tra protein regulates its own splicing. It has been proposed that the dominant, male-specifying M factor on the Y chromosome inhibits this autoregulation. As a result, males cannot make functional Tra protein, and the male form of Dsx is produced. These experiments show not only how a pathway can evolve, but also, importantly, how X:A and heterogametic GSD systems can be interconverted by modifying the cue that regulates a conserved molecular switch gene (the splicing of tra mRNA) (Haag, 2005).

Finally, recent studies of Caenorhabditis nematodes have shed light on the genetic basis of the convergent evolution of sex determination related to mating system adaptations. An important factor in this area are new phylogenies of the genus, which consistently suggest the surprising possibility that the closely related hermaphroditic species C. elegans and C. briggsae acquired self-fertilization independently, from distinct gonochoristic (male/female) ancestors. Although this scenario is somewhat uncertain purely on parsimony grounds, recent work on the genetic control of the germline bisexuality that defines hermaphroditism has tipped the balance toward parallel evolution (Haag, 2005).

C. elegans fog-2, a gene required for spermatogenesis in hermaphrodites but not in males, has been cloned. It became clear that fog-2 is part of a large family of F-box genes and was produced by several recent rounds of gene duplication. The C. briggsae genome sequence suggested that while C. briggsae possesses a similarly large family of F-box proteins, the duplication event giving rise to fog-2 was specific to the C. elegans lineage. This work has been extended by the rigorous demonstration that fog-2 is indeed absent in C. briggsae. A short, C-terminal domain has been identified that makes FOG-2 uniquely able to perform its germline sex-determining function. This domain is probably derived from a frame-shifting mutation in an ancestral gene. Working with C. briggsae, evidence has been found of important species-specific regulation of germline sex determination. RNA interference and gene knockout approaches have shown that while C. elegans requires the male-promoting genes fem-2 and fem-3 to produce sperm in hermaphrodites, C. briggsae requires neither. Given that both genes have conserved roles in male somatic sex determination, this suggests that C. briggsae evolved hermaphroditism in a way that bypasses these genes (Haag, 2005).

The long-standing mystery of sex determination and its diversity began by comparisons between distantly related species. Recent work on closer relatives has uncovered processes that through a reasonable extrapolation enable the connection of these disparate dots into a fascinating picture of developmental evolution. Though the divergence is extreme, it is likely that a better understanding of the evolution of sex determination genes and pathways holds lessons about the evolution of development in general. The next major challenge will be to integrate the comparative developmental data with the ecological and population processes that are driving the evolution of sex determination. Only then will it be possible to say that the picture is complete (Haag, 2005).

The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). The Ceratitis homolog of Sxl does not appear to have a switch function: the gene is expressed in both sexes, irrespective of whether the male-determining Y is present or absent, which is inconsistent with a main sex-determining function. However, preliminary data suggest that the bottom-most component of the pathway, dsx, is not only present in Ceratitis (Ccdsx), but has conserved a role in sexual differentiation. The pre-mRNA of this gene is also alternatively spliced giving rise to sex-specific products that show a remarkable structural conservation when compared with the corresponding male and female products in Drosophila. Sequence analysis of Ccdsx revealed the presence of putative TRA/TRA-2-binding sites close to the regulated splice site, suggesting that the underlying mechanism of sex-specific splicing is conserved and under the control of proteins homologous to TRA and TRA-2. Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. It is proposed that this repression is achieved by M, the Y-linked male-determining factor (Pane, 2002).

The results show that Ceratitis and Drosophila sex-determining cascades share a conserved tra-->dsx genetic module to control sex determination and sexual differentiation as well as that tra sex-specific splicing regulation differs in the two species. In Drosophila, TRA protein, together with TRA-2, binds to the TRA/TRA-2 recognition sequences on the Drosophila dsx pre-mRNA and promotes the use of a nearby female-specific acceptor site. Cctra is needed to impose the female-specific splicing of Ccdsx, most probably by a similar mechanism as in Drosophila, invoking the existence of a Cctra2 homolog. This hypothesis is also supported by the finding of TRA/TRA-2 recognition sequences located in close vicinity to the female-specific acceptor site in Ccdsx pre-mRNA (Pane, 2002).

In Drosophila, tra female-specific splicing is promoted by SXL, which blocks the use of the non-sex-specific splice site present in the tra pre-mRNA. In Ceratitis, the presence of multiple TRA/TRA-2-binding elements within the Cctra male-specific exonic sequences strongly suggests that CcTRA and a hypothetical CcTRA-2 protein could bind to these sequences, thus mediating a direct autoregulation. The unusually strong phenotypic effects of the RNAi against this gene also support this model of Cctra regulation. The localization of the putative regulatory elements within the Cctra gene indicates a repression mode by which CcTRA in females prevents the recognition of male-specific splice sites. The mechanism by which Cctra seems to promote the female mode of processing of its own pre-mRNA by TRA/TRA-2-binding elements appears to be different also from the female-specific splicing of dsx. Rather than activating a splice site nearby the regulated exon, as in the case of dsx, inclusion of male-specific Cctra sequences is suppressed when CcTRA is present. Although this would be a novelty with respect to known Drosophila TRA/TRA-2 activities, it has been previously shown that the 'behavior' of these cis elements is context dependent and that changing the location of splicing enhancers can transform them into negative regulatory elements (Pane, 2002).

In Drosophila, the presence of the Y chromosome is necessary for male fertility but not for male development. By contrast, RNAi-treated Ceratitis embryos with a female XX karyotype can develop into fertile males, which indicates that transient repression of Cctra by RNAi is sufficient to implement fully normal male development. The cases of complete sexual transformation of genetic Ceratitis females (XX) into fertile males by RNAi demonstrate that the Y chromosome, except for the dominant male determiner M, does not supply any other contribution to both somatic and germline male development, as suggested by previous Y-chromosome deletion analysis. Other dipteran species, such as Musca domestica and Chrysomya rufifacies show a female and male germline sex determination that is completely dependent on the sexual fate of the soma. However, in Drosophila, the XX and XY germ cells seem to respond differently to sex determining somatic cues. Indeed the XY germ cells have also an autonomous stage-specific sex determination mechanism that probably integrates the somatic signal. In Ceratitis, Cctra could be required in XX somatic cells to let them induce the XX germ cells to differentiate as oogenic cells. Alternatively, Cctra could be required in XX germ cells to 'feminize' them. This case would be a novelty with respect to the known Drosophila transformer gene functions (Pane, 2002).

Since zygotes that carry a Y chromosome do not activate Cctra female-specific splicing and autoregulation, it is proposed that the Y-linked male-determining M factor prevents this activation. It is conceivable that Cctra is a direct target of the M factor. Presence of this M factor in the zygote may prevent the production of CcTRA protein. The Cctra positive feedback loop is a probable target for regulation, because of its sensitivity (already shown by RNAi). An important question to be addressed is how autoregulation of Cctra is initiated in XX embryos of C. capitata and how this is prevented in XY embryos. A possible explanation is suggested by the Cctra female-specific mRNAs encoding the full-length protein, which have been detected in unfertilized eggs. Depositing these Cctra transcripts in eggs may provide a source of activity that can be used later for 'female-specific' processing when Cctra is zygotically transcribed. Once zygotically activated in XX embryos, Cctra promotes its own female-specific splicing, maintaining the female sex determination and the female-specific splicing of the downstream Ccdsx gene. Taken together, these events induce the female differentiation. In the current model for sex determination of medfly, the M factor is directly involved in the Cctra sex-specific regulation. Thus, in the presence of M Cctra, autoregulation is blocked and the gene produces male-specific transcripts encoding short and possibly non-functional CcTRA peptides. The absence of CcTRA leads Ccdsx to produce male-specific transcripts by default, promoting male differentiation. The control of the M factor upon Cctra expression could be exerted at different levels. The male determiner M could, for example, act at the pre-translational level blocking the production of CcTRA protein from the maternal transcripts. M could act at the post-translational level antagonizing the formation of protein complexes necessary for the female splicing mode. Or M could act as a transient transcriptional repressor of Cctra to reduce the amount of active CcTRA below a threshold needed to maintain the feedback loop. The proposed autoregulatory model of Cctra may also explain the remarkable efficiency of sex reversal by Cctra RNAi: a transient silencing of Cctra by injecting dsRNA is sufficient to let the loop collapse. Furthermore, the sensitivity of this positive autoregulation could be an evolutionary widely conserved pre-requisite to permit a 'faster' recruitment/replacement of different upstream regulators and to easily evolve different sex determining primary signals, as observed in dipteran species (Pane, 2002).

Sex can even be determined by a maternal effect in dipteran species such as Sciara coprophila and Chrysomya rufifacies. The hypothesis of a Cctra maternal contribution to the activation of the zygotic Cctra gene has similarities to the model of sex determination proposed for Musca domestica. In the common housefly, the maternal product of the key switch gene F is needed to activate the zygotic function of F in females. Musca male development results whenever F cannot become active in the zygote. This happens when the male-determining M is present in the zygotic genome, or when maternal F is not functional because of either the presence of M or the mutational loss of function of F (Fman) in the germline. More interesting, embryonic RNAi against the Musca tra-2 homolog causes sex reversion of Musca XX adults into intersex and fertile males, although this gene is not sex-specifically expressed. These recent data in Musca and the results in Ceratitis support the idea that F of Musca functionally corresponds to the Ceratitis tra gene, which seems to autoregulate and maternally contribute to its own activation, rather than to the Drosophila tra gene (Pane, 2002).

These data show that a basic structure of sex determination is conserved in the two dipteran species, namely the flow of 'instructions' from tra to dsx. This confirms the model of 'bottom-up' evolution, suggesting that during evolution developmental cascades are built from bottom up and that the genes at the bottom are widely conserved, while further upstream new regulatory elements may be recruited. The results show that Ceratitis and Drosophila sex-determining cascade differ at the level of transformer as well as upstream of it. Indeed the gene has conserved its function during evolution, but it has female-specific positive autoregulation in Ceratitis, while in Drosophila it needs Sxl as upstream regulator to express its female determining function. More likely the sex-determining function of Sxl was co-opted after Drosophila and Ceratitis had separated more than 100 Myr ago. Furthermore, it is conceivable that the autoregulatory mechanism of Sxl could have been selected to overcome a mutation impairing the tra autoregulation. Hence, in both species the female pathway is maintained by a single gene positive-feedback mechanism through sex-specific alternative splicing. Single gene autoregulation by alternative splicing seems not to be infrequent in nature, especially in those genes encoding splicing regulators. Indeed, other genes encoding RNA-binding proteins are thought to autoregulate their expression by controlling the processing of their own pre-mRNAs. Such a single-gene network with positive regulation is capable of bistability. This suggests that the emergence of analogous positive autoregulation in different genes such as Drosophila Sxl and Ceratitis tra genes would have been selected, during evolution, to guarantee a similar ON/OFF-female/male bistable cell state (Pane, 2002).

Since Ceratitis capitata is a major agricultural pest in many areas of the world, the isolation of a key sex-determining gene such as Cctra will substantially aid the development of new strategies to optimize the efficacy of currently used male sterile techniques for pest control. It is expected that tra is also a key sex-determining gene in many other insect species. Hence, the isolation of corresponding tra genes will open new means to control not only agricultural pests but also medically relevant vectors of diseases such as Glossina palpalis and Anopheles gambiae (Pane, 2002).

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably non-functional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D.melanogaster XX; tra mutant adults. Though highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. It is suggested that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects (Pane, 2005).

One important finding that comes out of this study is that CcTRA is able to 'recognize' the TRA-TRA2 binding sites in vivo, though they are located in entirely divergent contexts, namely the Dmfru and Dmdsx genes of Drosophila. It is therefore tempting to speculate that the TRA-TRA2 binding sites are also target sequences for CcTRA activity in Ceratitis. In this species, TRA-TRA2 elements are present in exon 4 of the dsx homolog (Ccdsx) and in intron 1 of the Cctra gene. Ccdsx reveals a significant structural and sequence identity when compared to Dmdsx and shows a sex-specific expression pattern. The distribution of the cis elements in the Ccdsx gene is also similar to that of Dmdsx, since they are located in exon 4, which is sex-specifically regulated in both Ceratitis and Drosophila. Given that CcTRA can promote the proper female splicing of Dmdsx, it is conceivable that it also controls Ccdsx expression using a similar mechanism. In Ceratitis females, CcTRA is likely to bind the cis-elements in Ccdsx exon 4 and promote the fusion of exon 3 to exon 4. The resulting female mature transcripts encode the CcdsxF protein. By contrast, in males, where the CcTRA protein is absent, exon 4 is not included in the mature mRNA, with exon 3 being fused directly to exon 5. The mature mRNAs generated in males encode the CcdsxM isoform. Consistent with this model, when the Cctra gene is turned off by RNAi in XX individuals, Ccdsx expression pattern is switched from the female to the male mode of splicing. As in Drosophila, the Ccdsx isoforms are likely to control the development of sexually dimorphic traits in Ceratitis. Putative TRA-TRA2 elements are also surprisingly contained in intron 1 of the Cctra gene. This observation points to a role for the CcTRA protein in the processing of Cctra precursor mRNA. In Ceratitis, Cctra is sex-specifically expressed through post-transcriptional alternative splicing events. In females, the intron 1 is removed from the primary transcript and mature mRNAs that encode the full-length CcTRA protein are produced (Pane, 2005).

Differently, mature mRNAs generated in males retain portions of the intron 1 (i.e. male-specific exons), which contain stop codons and thus prematurely interrupt the translation of the CcTRA protein. The female splicing of the Cctra primary transcripts is dependent upon a functional Cctra gene. When Cctra is switched off by RNAi in early embryos, the emerging XX adults are males and express male variants from Cctra. These observations lead to the hypothesis that, in Ceratitis females, Cctra controls its own expression by means of a positive feedback loop. The results reported in this study further support this hypothesis and suggest that, in females, the CcTRA protein might bind the TRA-TRA2 binding sites in the Cctra pre-mRNA and promote female splicing events (Pane, 2005).

The binding of CcTRA to the cis-elements might prevent the usage of male splicing sites, thus leading the splicing machinery to use the criptic female sites. Consequently, the intron 1 is removed from Cctra precursor transcripts to produce the female mature mRNAs. An alternative possibility is represented by an activation mechanism in which CcTRA would enforce the usage of female splice sites. This model stems from the observation that the TRA-TRA2 elements are mainly located within the male-specific exons and therefore are included in male mature mRNAs. It is possible that, in females, 'male' transcripts are produced by the default mechanism and might behave as splicing intermediates and substrates for CcTRA activity. In this case, the binding of the CcTRA protein to the cis-regulatory elements would favor the use of the female splice sites and promote the removal of the male-specific exons. Both the repression and the activation mechanisms proposed would involve a new property for the TRA proteins as well as an intronic function for the TRA-TRA2 elements, which has not been described before. In females, Cctra mature mRNAs have a long open reading frame and represent the source of CcTRA protein to keep the feedback loop active and guarantee the memory of the female sexual state. In males, the M-factor is likely to impair the positive feedback loop at early stages, thus promoting the male developmental program. Interestingly, CcTRA activity in the Drosophila transgenic lines is dependent upon a functional endogenous Dmtra2 gene. CcTRA is not able to direct female splicing of dsx and fru pre-mRNAs in Drosophila when the DmTRA2 protein is absent. It is believed that, also in Ceratitis, female development involves the cooperation between CcTRA and a putative TRA2 homolog (CcTRA2), which is yet to be identified. Several observations further support this hypothesis. tra2 appears to be highly conserved in evolution and tra2 homologs have been described even in human. Recently a tra2 homolog was identified in the housefly Musca domestica, which diverged from Drosophila some 100 million years ago. Transient depletion of the tra2 function in Musca by RNAi triggers the sexual transformation of XX embryos, which normally become females, toward maleness. These observations all point to the existence of a conserved tra2 homolog in Medfly as strongly suggested by the sequence conservation of Tra/Tra-2 binding sites observed in the Ceratitis dsx homologue. The CcTRA2 protein might interact with CcTRA to control both the female-specific splicing of Ccdsx and the positive feedback loop established by the Cctra gene (Pane, 2005).

Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22

Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. This study identified a gene, Musca domestica male determiner (Mdmd), as the M-factor. Mdmd originated from a duplication of the spliceosomal factor gene CWC22 (nucampholin). Targeted Mdmd disruption results in complete sex reversal to fertile females because of a shift from male to female expression of the downstream genes transformer and doublesex The presence of Mdmd on different chromosomes indicates that Mdmd translocated to different genomic sites. Thus, an instructive signal in sex determination can arise by duplication and neofunctionalization of an essential splicing regulator (Sharma, 2017).

Evidence for evolutionary conservation of sex-determining genes

Evidence for the evolutionary conservation of a sex-determining mechanism is presented. The male sexual regulatory gene mab-3 from the nematode Caenorhabditis elegans is related to the Drosophila melanogaster sexual regulatory gene doublesex (dsx). Both genes encode proteins with a DNA-binding motif here named the 'DM domain'. mab-3 acts downsteam of tra-1. Both worm and fly genes control sex-specific neuroblast differentiation and yolk protein gene transcription; dsx controls other sexually dimorphic features as well. Despite the similarities dix has extra functions in each sex; thus, the two genes are not strictly equivalent. The form of DSX that is found in males can direct male-specific neuroblast differentiation in C. elegans. This structural and functional similarity between phyla suggests a common evolutionary origin of at least some aspects of sexual regulation. A human gene, DMT1, has been identifed that encodes a protein with a DM domain; DMT1 is expressed only in testis. DMT1 maps to the distal short arm of chromosome 9, a location implicated in human XY sex reversal. Proteins with DM domains may therefore also regulate sexual development in mammals (Raymond, 1998).

Similarity of DNA binding and transcriptional regulation by Caenorhabditis elegans MAB-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanisms

Doublesex in insects and MAB-3 in nematodes encode DM domain transcription factors that directly regulate expression of yolk protein genes by binding to similar DNA sites, suggesting that at least this aspect of sex determination has been conserved in evolution. Despite containing different numbers of DM domains, MAB-3 and DSX bind to similar DNA sequences. mab-3 mutations deregulate vitellogenin synthesis at the level of transcription, resulting in expression in both sexes, and the vitellogenin genes have potential MAB-3 binding sites upstream of their transcriptional start sites. MAB-3 binds to a site in the vit-2 promoter in vitro: this site is required in vivo to prevent transcription of a vit-2 reporter construct in males, suggesting that MAB-3 is a direct repressor of vitellogenin transcription. This is the first direct link between the sex determination regulatory pathway and sex-specific structural genes in C. elegans, and it suggests that nematodes and insects use at least some of the same mechanisms to control sexual development (Yi, 1999).

mab-3 is a direct tra-1 target gene regulating diverse aspects of C. elegans male sexual development and behavior

Sex determination is controlled by global regulatory genes, such as tra-1 in C. elegans, Sex lethal in Drosophila, or Sry in mammals. How these genes coordinate sexual differentiation throughout the body is a key unanswered question. tra-1 encodes a zinc finger transcription factor, TRA-1A, that regulates, directly or indirectly, all genes required for sexual development. mab-3 (male abnormal 3), acts downstream of tra-1 and is known to be required for sexual differentiation of at least two tissues. mab-3 directly regulates yolk protein transcription in the intestine and specifies male sense organ differentiation in the nervous system. It encodes a transcription factor related to the products of the Drosophila sexual regulator doublesex (dsx), which also regulates yolk protein transcription and male sense-organ differentiation. MAB-3 has two copies of a nonclassical 'zinc finger' DNA-binding motif called a DM domain. The DM domain was first identified in Dsx. As expected from its unusual sequence, the DM domain is structurally distinct from other zinc fingers, and it binds in the DNA minor groove. The similarities between mab-3 and dsx has led to a suggestion that some aspects of sex determination may be evolutionarily conserved. mab-3 is also required for expression of male-specific genes in sensory neurons of the head and tail and for male interaction with hermaphrodites. These roles in male development and behavior suggest further functional similarity to dsx. In male sensory ray differentiation it has been found that MAB-3 acts synergistically with LIN-32, a neurogenic bHLH transcription factor. Expression of LIN-32 is spatially restricted by the combined action of the Hox gene mab-5 and the hairy homolog lin-22, while MAB-3 is expressed throughout the lateral hypodermis. mab-3 transcription is directly regulated in the intestine by TRA-1A, providing a molecular link between the global regulatory pathway and terminal sexual differentiation (Yi, 2000).

mab-3, in concert with other genes including lin-32, is required for V ray differentiation in the male tail. Genetic epistasis analysis and comparison of mutant phenotypes indicate that mab-3 and lin-32 act later in ray neuroblast differentiation than mab-5 and lin-22, but whether their expression is regulated by these genes and how they interact functionally has been unclear. Using reporter genes, lin-32 expression was found to be regulated positively by mab-5 and negatively by lin-22, while mab-3 expression, in contrast, appeared to be independent of these genes. Thus, the crucial determinant of where V rays form appears to be lin-32 rather than mab-3. Several lines of evidence suggest that mab-3 acts to enhance the activity of lin-32 to promote ray formation. (1) mab-3 mutant males, while severely defective in V ray formation, do produce a small number of V rays, and thus mab-3 is not absolutely essential for ray formation. (2) lin-22 mutations cause the ectopic expression of lin-32 in the anterior lateral hypodermis, but this causes ectopic ray formation only if mab-3 is also present. (3) Ectopic expression of LIN-32 can restore V ray formation to mab-3 mutants. This result must be interpreted with caution as it involves overexpression, but it suggests that mab-3 is dispensable for V ray formation if sufficient LIN-32 is present. The reciprocal is not the case: MAB-3 overexpression does not suppress ray defects in lin-32 mutants. This result argues against models in which mab-3 and lin-32 perform the same function in ray formation. In such models the total activity of MAB-3+LIN-32, rather than the activity of one protein or the other, is crucial for ray formation. Ectopic HLH-2 expression also restores V ray formation to mab-3 mutants, but less efficiently, perhaps by increasing the concentration of a complex with LIN-32 (Yi, 2000).

The results are most consistent with a model in which mab-3 plays a permissive role in V ray formation in concert with lin-32. In wild-type males, mab-5 directly or indirectly activates lin-32 expression only in the V5- and V6-derived neuroblasts R1-R6. The combined expression of mab-3 and lin-32 in R1- R6 results in their differentiation into V rays. In the anterior body seam (V1-V4 lineages), mab-3 is expressed but lin-32 is not, because it is repressed by lin-22, and this prevents sensory ray formation. The repression of lin-32 by lin-22 could be direct, or it may be mediated by mab-5, since lin-22 mutants ectopically express mab-5 in the V1-V4 lineages. Co-expression of mab-3 is necessary for full lin-32 activity, but this requirement can be bypassed by elevating the level of LIN-32 expression. This model predicts that ectopic LIN-32 expression in the anterior body seam should result in ectopic rays, which has been shown to be the case. The regulation of lin-32, an achaete-scute homolog, by lin-22, a hairy homolog, suggests that the regulatory relationship of these genes may be conserved between flies and worms (Yi, 2000).

mab-3 might potentiate the activity of lin-32 by any of several mechanisms, which are not mutually exclusive. One possibility is that MAB-3 and LIN-32 physically interact to generate a more active form of LIN-32. A second possibility is that MAB-3 regulates a gene that affects the activity of LIN-32. It could repress an inhibitor of LIN-32 or activate an enhancer of LIN-32 activity. A third possibility is that MAB-3 and LIN-32 may regulate some of the same downstream targets, and that more LIN-32 is required to achieve proper regulation of these genes when MAB-3 is absent. Mechanistic studies and searches for regulatory targets of MAB-3 and LIN-32 should help address these possibilities (Yi, 2000).

An intriguing finding is that mab-3 reporters are expressed in a number of sensory neurons in the male head and the tail whose formation is not prevented by mab-3 mutations. All of these cells are good candidates for mediating male mating behavior, and indeed some have been shown to be required for specific aspects of male mating. This raises the possibility that mab-3 plays additional behavioral roles in the male nervous system. The possible role of mab-3 in male mating behavior was tested using two approaches. First, whether mab-3 is required for the expression of genes implicated in male mating behavior was investigated. Of three genes assayed, mab-3 is required for normal expression of two: lov-1 in the head and srd-1 in the tail. Next to be tested was whether mab-3 males exhibit defective interaction with hermaphrodites was tested. While wild-type males show a strong preference for hermaphrodites over males, mab-3 males are not attracted to either sex and rapidly leave. This defect cannot result entirely from lack of V rays, since mab-5 mutants still show a preference for hermaphrodites over males. Taken together, these results strongly suggest that mab-3 is required in the nervous system for expression of genes that mediate early, and perhaps also later, steps of male mating behavior. In this regard, mab-3 further resembles doublesex, which is required for male courtship behavior in Drosophila. Additional assays will be needed to distinguish whether mab-3 mutant males are defective in taxis to hermaphrodites, sustained interaction with hermaphrodites once located, or both. It also will be important to determine in which cells mab-3 is required in order to successfully carry out each of the different aspects of male behavior. The finding that mab-3, like dsx, is required for male mating behavior further suggests that these two genes may be conserved from an ancient sexual regulator (Yi, 2000).

It is now clear that tra-1 coordinates sexual development and behavior via a group of downstream regulatory genes, including egl-1 and mab-3. These genes provide an interface between the global sex-determination pathway, with tra-1 at its terminus, and the expression of the genes responsible for terminal differentiation and function of sexually dimorphic cells throughout the animal. mab-3 serves as a direct link between tra-1 and terminal differentiation in the intestine, and as an indirect link in the nervous system, playing key roles in both the formation and the function of male neurons. Even by regulating both mab-3 and egl-1 expression, tra-1 directs the sexually dimorphic development of only a small proportion of cells. An important goal for the future will be to identify the genes that link tra-1 to sexually dimorphic development elsewhere in the animal (Yi, 2000).

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. MAB-3 promotes expression of the proneural Atonal homolog 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. It is therefore suggested that regulation of Hes genes by DM domain proteins may be a general mechanism for specifying sex-specific neurons (Ross, 2005).

MAB-3 promotes development of male-specific sensory neurons by regulating two bHLH factors. In V ray precursors, MAB-3 indirectly promotes expression of the proneural protein LIN-32 by preventing expression of REF-1, a distant homolog of the Hes family of neurogenic proteins. REF-1 is a negative regulator of lin-32; lin-32::gfp expression is dramatically reduced in the mab-3 V ray lineage, but is restored by the introduction of a ref-1 mutation. This REF-1-mediated repression of lin-32 is necessary to prevent V ray formation in mab-3 mutants, as evidenced by the observation that ref-1 mutations restore V ray development in mab-3 mutants. Furthermore, ectopic ref-1 expression is sufficient to cause V ray defects in wild-type males. These results indicate that MAB-3 acts in parallel to Hox proteins to promote activation of lin-32 by preventing expression of ref-1, a gene with antineural activity (Ross, 2005).

Two conserved ref-1 regulatory elements (A and B) have been identified and putative MAB-3 binding sites have been identified within these elements. Wild-type MAB-3, is required to prevent ref-1::gfp expression during V ray development. Based on these observations, the following model is proposed. In wild-type V ray precursors, MAB-3 promotes LIN-32-mediated V ray development by binding within one or both of the conserved elements to prevent activation of ref-1 by an unknown factor, X. In the mab-3 mutant V ray lineage, ref-1 is inappropriately activated by X and disrupts V ray formation by preventing Hox-mediated activation of lin-32. ref-1-inactivating mutations relieve this repression of lin-32, restoring normal V ray formation in mab-3 ref-1 double mutants (Ross, 2005).

ref-1 is initially expressed in the posterior hypodermal seam cells in young males and is downregulated when mab-3 is first expressed. Although the identities of ref-1 activators are unknown, a binding site for one such factor may overlap the MAB-3 binding site in element B; disruption of this MAB-3 site eliminates ref-1 expression in the seam. Thus, MAB-3 may repress ref-1 by physically interfering with binding or function of activators bound to nearby sites. Similarly, overlapping binding sites for DSX and a bZIP transcription factor coordinate regulation of yolk expression in Drosophila. The structure of DM domain proteins may be particularly suited for interaction with transcription factors that bind overlapping DNA sites. DSX binds in the minor groove of DNA, which might allow close apposition with major groove binding transcription factors (Ross, 2005).

It is possible that the weak mab-3-suppressing mutation ref-1(ez6) reduces ref-1 expression by disrupting a second positive regulatory site in element A. However, ref-1 transgenes containing the ez6 lesion are expressed in the seam and rescue the ref-1(ez11) V ray phenotype. The rescuing activity and expression of ref-1 transgenes driven by ez6 mutant regulatory sequences may be a consequence of high copy number of the reporter or may indicate a minor role for this element (Ross, 2005).

All sex-specific development in the C. elegans soma occurs downstream of the zinc finger transcription factor TRA-1, the terminal global regulator in the sex determination cascade. However, the connection between TRA-1 and male-specific effectors that drive V ray development remains obscure. MAB-3 represses ref-1 expression in males to allow specification of V rays by LIN-32. While it might follow that REF-1 normally prevents V ray formation in hermaphrodites, this does not appear to be the case. Although ref-1 is expressed in hermaphrodite seam cells, ref-1 mutant hermaphrodites do not produce ectopic V rays or express ectopic lin-32::gfp. TRA-1 must somehow prevent V ray formation in hermaphrodites. TRA-1 might regulate lin-32 expression directly or might prevent lin-32 activation indirectly by regulating Hox gene activity. EGL-5 expression in the V6 lineage is sex specific and could be regulated by TRA-1. MAB-5 is expressed in the V6 lineage in both sexes, but TRA-1 might modulate MAB-5 activity, for example by controlling factors that modify MAB-5 posttranslationally (Ross, 2005).

Male-specific regulation of ref-1 by MAB-3 also must require additional regulators. Although mab-3 is expressed in hermaphrodites, it only represses ref-1 in males. It is possible that mab-3 requires a male-specific coregulator. Alternatively, MAB-3 may be posttranslationally modified such that it is active only in males (Ross, 2005).

Proteins of the Hes family of neurogenic regulators typically share a characteristic bHLH domain, an Orange domain that may confer functional specificity, and a C-terminal WRPW sequence required for interaction with the corepressor Groucho (Gro). Although the bHLH domains of REF-1 are most similar to those of the Hes family, the overall resemblance of REF-1 to Hes proteins is weak. The six C. elegans REF-1-like proteins are unusual in that they each possess two bHLH domains. Furthermore, the REF-1 bHLH domains are only 28% and 22% identical to the bHLH domain of Hairy and lack a basic domain proline that is conserved in other Hes proteins. By contrast, the bHLH domain of LIN-22, a second C. elegans Hairy homolog, is 51% identical to that of Hairy. Additionally, REF-1 lacks an Orange domain and contains a C-terminal FRPWE, rather than WRPW, sequence (Ross, 2005).

Despite sequence and structural differences, REF-1 bears striking functional homology to other Hes proteins. In flies, Hairy and E(spl) proteins progressively limit domains of neurogenesis in the peripheral nervous system by interfering with the activity of proneural factors like Achaete (Ac), Scute (Sc), and Atonal. During sensory bristle formation, Hairy binds directly to an ac-sc enhancer to restrict spatial expression of ac. E(spl) proteins act later, in response to Notch signaling, to downregulate proneural gene expression in presumptive epidermal cells by interfering with an autostimulatory feedback loop. E(spl) proteins also antagonize proneural proteins by interfering with activation of proneural target genes. In both cases, E(spl)-mediated repression can occur by direct DNA binding or by protein-protein interactions with proneural activators that are bound to their own sites. Repression by either mechanism requires recruitment of Gro. Vertebrate Hes proteins can act as transcriptional repressors and are also thought to prevent activation by sequestering the MASH or MATH proneural proteins in inactive heterodimer complexes (Ross, 2005).

Like Hes proteins, REF-1 prevents neurogenesis by negatively regulating a proneural protein, LIN-32. ref-1-dependent reduction of lin-32::gfp expression in mab-3 mutant males suggests that REF-1 is likely to repress lin-32 transcription. Consistent with this, REF-1 proteins with substitutions in the first basic domain (mu220 and ez11) fail to repress neurogenesis and lin-32::gfp expression, suggesting that DNA binding is required. In addition, the lin-32 promoter contains many potential REF-1 binding sites (E boxes and N boxes). It is unclear whether REF-1 interacts with the Gro homolog UNC-37 to negatively regulate lin-32. ref-1 transgenes lacking the FRPWE domain weakly rescue the ref-1 phenotype, suggesting that this sequence is partially dispensable for ref-1 function. It is possible that another sequence mediates REF-1/UNC-37 interactions or that REF-1 interacts with a different corepressor. The possibility cannot be excluded that REF-1 regulates lin-32 posttranscriptionally, perhaps by forming an unstable heterodimer with LIN-32 or by interfering with a positive feedback mechanism that would normally increase lin-32 expression (Ross, 2005).

Both ref-1 and lin-32 are required for normal development of two neuronal structures derived from seam cells. REF-1 negatively regulates development of V5- and V6-derived sensory rays and production of the postdeirid, a neuroblast normally derived from V5. In contrast, LIN-32 promotes sensory ray and postdeirid formation (Ross, 2005)

The Hes protein LIN-22 prevents neurogenesis in anterior seam cells V1-V4 . In lin-22 mutants, V1-V4 undergo a V5-like lineage to produce a postdeirid and two sensory rays. The ectopic postdeirid depends on lin-32, suggesting that LIN-22 negatively regulates lin-32 in V1-V4. REF-1 and LIN-22 appear to affect postdeirid production regionally, with LIN-22 acting in the anterior and REF-1 in the posterior seam (Ross, 2005).

Ectopic sensory ray production in lin-22 mutants requires Hox, lin-32, and mab-3 activity, suggesting that LIN-22 acts upstream of the network of V ray regulators. ref-1 and lin-22 interact to inhibit V ray formation in V1-V4. ref-1 mutations cause ectopic ray formation in mab-3; lin-22 double mutants, which normally do not produce V1-V4-derived rays. This suggests that, at least in mab-3 mutants, LIN-22 acts upstream of REF-1 in a hierarchy of bHLH proteins controlling V ray neurogenesis (Ross, 2005).

During Drosophila peripheral neurogenesis, Hairy acts early to establish a prepattern of cells competent to become neurons. E(spl) proteins subsequently define the subgroup of these cells that will form sensory organs. The inappropriate neurogenesis in V1-V4 in lin-22 mutants suggests that LIN-22, like Hairy, acts globally to define which seam cells are competent to produce neuronal lineages. The experiments suggest that REF-1, like E(spl), may then act downstream within these lineages to refine which cells will become neurons (Ross, 2005).

One mechanism by which DM domain proteins regulate sexual dimorphism is the sex-specific modulation of developmental programs. For example, DSXF inhibits Wingless and FGF pathway activity and DSXM sex specifically inhibits Dpp signaling. Although direct targets are not known, this inhibition is likely to occur by transcriptional regulation of key pathway components. The DM domain protein MAB-3 represses the Hes family bHLH protein REF-1 in males to modulate sex-specific nervous system development (Ross, 2005).

Hes proteins regulate both the extent of neurogenesis and the specification of neuronal subtypes. In Drosophila, E(spl) mutations lead to ectopic neurogenesis, while overexpression prevents neurogenesis. In the mouse brain, Hes proteins control timing of cell differentiation to regulate brain size, shape, and cell arrangement, possibly via interactions with cell-cycle regulators. Thus, it is clear that sex-specific regulation of Hes activity in the developing nervous system could achieve sexual dimorphism in organ shape, size, cell fate, or timing of differentiation. MAB-3/REF-1 interactions provide an example of such regulation (Ross, 2005).

mab-3 mutant males produce epidermal cells at the expense of neuronal cells, a phenotype like that caused by Hes overexpression in other organisms. In addition, V ray precursor cell divisions of mab-3 mutants are often delayed relative to wild-type. This delay may reflect an interaction between ref-1 and cell-cycle regulators, similar to that seen for mouse Hes proteins. While no interactions between bHLH proteins and DSX have been described, these seem likely based on functional homology between MAB-3 and DSX. Male sex combs are a likely candidate for this mode of regulation, since bristle formation in flies is regulated by Hes proteins. DSX also regulates sexual dimorphism in abdominal neuroblasts, which undergo more cell divisions in males than in females. It is possible that this sex-specific proliferation is controlled by DSX/bHLH interactions (Ross, 2005).

This work establishes that sex-specific regulation of REF-1 and LIN-32 by MAB-3 can regulate development of male-specific neurons in C. elegans. Future studies will reveal whether sex-specific regulation of bHLH proteins by DM domain transcription factors is a conserved mechanism for generating sexual dimorphism in the nervous system (Ross, 2005).

dmd-3, a doublesex-related gene regulated by tra-1, governs sex-specific morphogenesis in C. elegans

Although sexual dimorphism is ubiquitous in animals, the means by which sex determination mechanisms trigger specific modifications to shared structures is not well understood. In C. elegans, tail tip morphology is highly dimorphic: whereas hermaphrodites have a whip-like, tapered tail tip, the male tail is blunt-ended and round. The male-specific cell fusion and retraction that generate the adult tail are controlled by the previously undescribed doublesex-related DM gene dmd-3, with a secondary contribution from the paralogous gene mab-3. In dmd-3 mutants, cell fusion and retraction in the male tail tip are severely defective, while in mab-3; dmd-3 double mutants, these processes are completely absent. Conversely, expression of dmd-3 in the hermaphrodite tail tip is sufficient to trigger fusion and retraction. The master sexual regulator tra-1 normally represses dmd-3 expression in the hermaphrodite tail tip, accounting for the sexual specificity of tail tip morphogenesis. Temporal cues control the timing of tail remodeling in males by regulating dmd-3 expression, and Wnt signaling promotes this process by maintaining and enhancing dmd-3 expression in the tail tip. Downstream, dmd-3 and mab-3 regulate effectors of morphogenesis including the cell fusion gene eff-1. Together, these results reveal a regulatory network for male tail morphogenesis in which dmd-3 and mab-3 together occupy the central node. These findings indicate that an important conserved function of DM genes is to link the general sex determination hierarchy to specific effectors of differentiation and morphogenesis (Mason, 2008).

This study found that dmd-3, a previously undescribed member of the DM family, is both necessary and sufficient for male tail tip morphogenesis, a process that generates one of the most prominent sexual dimorphisms in the C. elegans soma. A secondary partially redundant role was found of the related gene mab-3 in this process. Together, these findings lead to a model in which dmd-3 instructively specifies tail tip morphogenesis by integrating multiple developmental signals and regulating at least two downstream events. The temporal control of dmd-3 is specified by the heterochronic pathway through the regulator lin-41. (Mutations in the lin-41 regulator let-7 also cause Lep phenotypes, indicating that let-7 also acts in this pathway, though this possibility has not been tested directly). Positional cues regulate dmd-3 through a Wnt pathway that includes the ligand LIN-44 and its downstream target tlp-1. Interestingly, this cue seems to be most important for the maintenance and amplification of dmd-3 expression; the initial positional or cell-type activator of dmd-3 remains unknown. Finally, the male-specificity of dmd-3 expression arises through regulation by the master sexual regulator TRA-1A (Mason, 2008).

doublesex/mab3 related-1 (dmrt1) is essential for development of anterior neural plate derivatives in Ciona

Ascidian larvae have a hollow, dorsal central nervous system that shares many morphological features with vertebrate nervous systems yet is composed of very few cells. This study shows that a null mutation in the gene dmrt1 in the ascidian Ciona savignyi results in profound abnormalities in the development of the sensory vesicle (brain), as well as other anterior ectodermal derivatives, including the palps and oral siphon primordium (OSP). Although the phenotype of the mutant embryos is variable, the majority have a complete loss of the most anterior structures (palps and OSP) and extensive disruption of sensory structures, such as the light-sensitive ocellus, in the sensory vesicle. dmrt1 is expressed early in the blastula embryo in a small group of presumptive ectodermal cells as they become restricted to anterior neural, OSP and palp fates. Despite the early and restricted expression of dmrt1, no defect was observed in the mutant embryos until the early tailbud stage. It is speculated that the variability and late onset in the phenotype may be due to partially overlapping activities of other gene products (Tresser, 2010).

This paper describes a mutant line in the ascidian Ciona savignyi that disrupts the development of anterior neural plate derivatives. The genetic lesion responsible for this phenotype maps to a member of the doublesex/mab-3-related (dmrt) family of transcription factors. The Drosophila doublesex and the Caenorhabditis elegans mab-3 genes share a common DNA-binding motif known as the DM domain. These genes were originally characterized for their roles in sex determination in flies and worms. Subsequently, DM domain-containing genes have also been found in most vertebrate species, many of which are expressed in the gonads and play a role in sex determination. Several members of the vertebrate dmrt family are expressed outside the gonads and are involved in a wider range of developmental processes. The DM-containing gene terra is involved in somite formation and left-right asymmetry in both zebrafish and chickens. In Xenopus dmrt4 is expressed in the olfactory placodes and plays a role in the neurogenesis of the olfactory epithelium. Dmrt3 in mouse may play a similar role (Tresser, 2010 and references therein).

In the ascidian, dmrt1 is expressed in the anterior nervous system, which is derived from the anterior animal (a-Line) cells of the eight-cell-stage embryo. An FGF signal (presumably FGF 9/16/20) from vegetal blastomeres induces neural fate in the a-Line cells. FGF, along with foxA-α, activates several neural genes including otx, nodal and dmrt1. Otx plays a crucial role in maintaining anterior neural identity and suppressing epidermal fate. Recently, knockdown experiments in ascidians have shown that dmrt1, along with otx, plays a role in promoting the expression of six 1/2, six 3/6 and meis in the developing brain as well as promoting the expression of foxC, which promotes expression of palp-specific genes (Tresser, 2010 and references therein).

A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates

A novel zebrafish zinc-finger gene, terra, contains a DNA binding domain similar to that of the Drosophila dsx gene. However, unlike dsx, terra is transiently expressed in the presomitic mesoderm and newly formed somites. Expression of terra in presomitic mesoderm is restricted to cells that lack expression of MyoD. In vivo, terra expression is reduced by hedgehog but enhanced by BMP signals. Overexpression of terra induces rapid apoptosis both in vitro and in vivo, suggesting that a tight regulation of terra expression is required during embryogenesis. Terra has both human and mouse homologs and is specifically expressed in mouse somites. Taken together, these findings suggest that terra is a highly conserved protein that plays specific roles in the early somitogenesis of vertebrates (Meng, 1999).

Zebrafish terra transcripts are most abundant in the presomitic mesoderm and the first 2-3 newly formed somites; they rapidly disappear after the segmented somites are formed. The expression pattern of terra suggests that this putative transcription factor plays a role in zebrafish somitogenesis. Indeed, some genes that have a similar expression pattern in mouse embryos, such as paraxis, Notch1 and delta-like gene 1. Gene knock out experiments have shown that play critical roles in the formation of somites. Preliminary data, using a transient dominant interference approach, indicate that inhibition of terra activity in zebrafish embryos results in the following: the absence of epithelial somites on one or both sides of the neural tube; the formation of irregularly shaped somites, or a truncated tail. These results indicate the importance of terra expression during early zebrafish somitogenesis. Mouse terra expression also occurs early and is restricted to the dermamyotome in developing somites, suggesting a role in the initial compartmentalization of somites. Currently, transgenic zebrafish are being generated that will express stable dominant interference alleles of terra (Meng, 1999).

A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes

The genes that determine the development of the male or female sex are known in Caenorhabditis elegans, Drosophila, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. In the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal DMRT1 gene, named DMRT1Y. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make DMRT1Y a candidate for the medaka male sex-determining gene (Nanda, 2002).

DMY is a Y-specific DM-domain gene required for male development in the medaka fish

Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. Recombinant breakpoint analysis has been used to restrict the sex-determining region in medaka fish (Oryzias latipes) to a 530-kilobase (kb) stretch of the Y chromosome. Deletion analysis of the Y chromosome of a congenic XY female further shortened the region to 250 kb. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only the DM-related PG17 was Y specific; it was thus named DMY. Two naturally occurring mutations establish DMY's critical role in male development. The first heritable mutant -- a single insertion in exon 3 and the subsequent truncation of DMY -- results in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. 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 testicular development and is a prime candidate for the medaka sex-determining gene (Matsuda, 2002).

A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators

Deletion of the distal short arm of chromosome 9 (9p) has been reported in a number of cases to be associated with gonadal dysgenesis and XY sex reversal, suggesting that this region contains one or more genes required in two copies for normal testis development. Recent studies have greatly narrowed the interval containing this putative autosomal testis-determining gene(s) to the distal portion of 9p24.3. DMRT1 has been identified as a human gene with sequence similarity to genes that regulate the sexual development of nematodes and insects. These genes contain a novel DNA-binding domain, which has been named the DM domain. DMRT1 maps to 9p24. 3 and in adults is expressed specifically in the testis. The possible role of DM domain genes in 9p sex reversal has been investigated. A second DM domain gene, DMRT2, has been identified that also maps to 9p24.3. Point mutations in the coding region of DMRT1 and the DM domain of DMRT2 are not frequent in XY females. Fluorescence in situ hybridization analysis shows that both genes are deleted in the smallest reported sex-reversing 9p deletion, suggesting that gonadal dysgenesis in 9p-deleted individuals might be due to combined hemizygosity of DMRT1 and DMRT2 (Raymond, 1999).

Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation

The only molecular similarity in sex determination found so far among phyla is between the Drosophila doublesex (dsx) and Caenorhabditis elegans mab-3 genes. dsx and mab-3 contain a zinc finger-like DNA-binding motif called the DM domain, perform several related regulatory functions, and are at least partially interchangeable in vivo. A DM domain gene called Dmrt1 has been implicated in male gonad development in a variety of vertebrates, on the basis of embryonic expression and chromosomal location. Such evidence is highly suggestive of a conserved role(s) for Dmrt1 in vertebrate sexual development, but there has been no functional analysis of this gene in any species. Murine Dmrt1 is shown in this study to be essential for postnatal testis differentiation, with mutant phenotypes similar to those caused by human chromosome 9p deletions that remove the gene. As in the case of 9p deletions, Dmrt1 is dispensable for ovary development in the mouse. Thus, as in invertebrates, a DM domain gene regulates vertebrate male development (Raymond, 2000).

Dmrt1 mRNA is expressed both in germ cells and in Sertoli cells. Thus, the germ cell death caused by mutation of Dmrt1 could reflect either a defect in the germ cells themselves or a defect in Sertoli cells, which promote germ cell survival and differentiation. To compare Dmrt1 protein expression with Dmrt1 mRNA expression, an antibody was generated against the C-terminal portion of the protein. Immunohistochemistry reveals that in the embryonic testis Dmrt1 protein accumulates primarily in Sertoli cell nuclei, with little or no expression detectable in germ cells. Starting at P1, Dmrt1 levels rise in germ cells and reach high levels by P7, just before meiosis begins. The similar timing of increased Dmrt1 expression in germ cells and the onset of germ cell death in the Dmrt1 -/- mutant testis suggests a possible cell-autonomous function for Dmrt1 in maintaining the germ line. From P7 through adult stage, Dmrt1 protein is present in Sertoli cells and undifferentiated germ cells, but not in differentiating germ cells. In adult testis, Dmrt1 is expressed in Sertoli cells in all regions of the seminiferous tubules, but is expressed dynamically in premeiotic germ cells (spermatogonia), with high expression only in regions of the seminiferous tubule that are early in the spermatogenic cycle. This further suggests that Dmrt1 may play a role in pre-meiotic germ cells, for example, regulating entry to meiosis or controlling the mitotic cell cycle. The antibody also confirms that no Dmrt1 protein is detectable in Dmrt1 -/- testes, demonstrating that this is a null allele (Raymond, 2000).

Dmrt1 -/- Sertoli cells overproliferate, fail to adopt a differentiated morphology, and then die postnatally. To characterize these phenotypes further, expression of several Sertoli cell markers was examined. Prior to P7, no defects are apparent. For example, the early marker Gata-4 is expressed normally in Dmrt1 -/- Sertoli cells during embryonic and early postnatal development, as are other early testis markers, including Dhh, Ptch2, and Mis. The failure of germ cell migration in Dmrt1 -/- at P7 is clearly visible in sections stained for Gata-4, with most Dmrt1 -/- germ cells failing to move from the center of the tubule to the margin. This may result from defects in the germ cells, the Sertoli cells, or both, and may reflect a failure of Sertoli/germ cell interaction. At P14, Gata-4 levels have decreased in Dmrt1 +/-, but remain high in Dmrt1 -/- Sertoli cells. The persistence of Gata-4 expression in Dmrt1 -/- Sertoli cells may reflect a cell-autonomous defect. Alternatively, it may result from absence of germ cell-dependent regulation, since Gata-4 expression also persists in the Sertoli cells of c-kit mutant testes, which also lack germ cells (Raymond, 2000).

Expression of the later Sertoli cell marker Gata-1 also is abnormal in Dmrt1 -/- testes. Gata-1 normally is expressed in Sertoli cells from about P10, but expression is delayed and reduced, although not absent, in Dmrt1 -/- mutants, which further confirms the failure of Sertoli cells to complete differentiation in the Dmrt1 -/- testis. Gata-1 expression in adult Sertoli cells is dependent on the stage of the spermatogenic cycle. It will be of interest to determine whether the cyclical expression of Dmrt1 in spermatogonia and of Gata-1 in the adjacent Sertoli cells are functionally related (Raymond, 2000).

The role of Dmrt1 in mammalian sexual development has been genetically tested. Murine Dmrt1 is necessary in the male gonad for survival and differentiation of both somatic and germ-line cells. Sertoli cell morphology and gene expression are abnormal in Dmrt1 -/- and the phenotype of the mutant testis differs from that of other mutants simply lacking germ cells. Thus, the defects observed must be caused at least in part by a failure of Sertoli cell differentiation. The loss of germ cells in Dmrt1 -/- may be an indirect effect of Sertoli cell inadequacy or a cell-autonomous defect, possibilities that are currently being tested by cell-specific targeting (Raymond, 2000).

The defects observed in Dmrt1 -/- mutant mice resemble those in humans with distal 9p deletions, with two important differences. First, in mouse, Dmrt1 is recessive, whereas human 9p deletions in some cases are haploinsufficient for testis differentiation. There are several possible explanations for this difference, which are not mutually exclusive. (1) There may be an inherent difference in dose sensitivity in the pathway(s) responsible, because other genes required for testis development are more dose sensitive in human than in mouse. (2) Genetic background may be important. Human 9p hemizygosity causes testis defects of variable severity and is incompletely penetrant, even when the critical region for testis development is removed by large deletions, suggesting that background effects may be significant. (In addition, the degree of haploinsufficiency of 9p24.3 in humans may be overestimated, as small deletions with no testis phenotype would go undetected.) Experiments are under way to test whether heterozygous murine Dmrt1 mutants have a phenotype on different genetic backgrounds. (3) It is possible that 9p deletions remove an additional gene(s) involved in testis development and thus, although DMRT1 is recessive, combined hemizygosity with another gene(s) can cause defective testis development in XY individuals retaining a copy of DMRT1 (Raymond, 2000 and references therein).

Another important difference between mouse and human is that some 9p-deleted XY patients have Mullerian duct remnants and feminized external genitalia, whereas, perhaps surprisingly, no defects outside of the gonads have been observed in Dmrt1 -/- mice. This finding indicates earlier defects in Sertoli cell function in human than those observed in the Dmrt1 -/- mouse. The possibility that genetic background effects are obscuring earlier functions in the mouse cannot be eliminated. However, male-specific DMRT1 mRNA expression occurs at an earlier developmental stage in the human gonad than in mouse, and thus DMRT1 might play an earlier role in human testis differentiation than Dmrt1 does in mouse. Birds and reptiles also have male-specific Dmrt1 expression prior to gonad differentiation, and so Dmrt1 may be required earlier in gonad development in these species as well (Raymond, 2000).

Of what significance is the similarity of Dmrt1 to the invertebrate sexual regulators dsx and mab-3? The data presented here demonstrate that in mammals, as in nematodes and insects, a DM domain transcription factor controls male sexual development. Does this functional similarity imply a close evolutionary relationship between Dmrt1 and the invertebrate sexual regulators? Currently, this question cannot be satisfactorily answered for several reasons. First, comparison of protein sequence alone is not very helpful in this case. Even dsx and mab-3, which perform a number of related biological functions and can be functionally interchangeable, show quite limited sequence similarity, restricted primarily to the DM domain. Of the 12 nematode DM domain genes, the one most similar in sequence to dsx is not mab-3. Several vertebrate DM domain genes have been identified, but it is not yet clear whether Dmrt1 is the one most closely related to dsx and mab-3. Not all vertebrate DM domain genes are involved in testis development. However, other DM domain genes are expressed in the embryonic mouse gonad, so it is possible that multiple members of this gene family are involved in vertebrate gonad development. Thus, on the basis of sequence comparison, one cannot yet conclude that Dmrt1 is the mammalian ortholog of dsx and mab-3 (Raymond, 2000).

A second issue confounding the evolutionary question is the fundamentally different biology of mammalian sex determination as compared with that of invertebrates. In worms and flies, sex determination occurs throughout the body, so if dsx and mab-3 are orthologs, they can reasonably be expected to control a number of similar aspects of sexual dimorphism. However, in mammals, sex determination occurs in the embryonic gonad and secreted sex hormones induce sexual dimorphism elsewhere. Thus, there is no a priori expectation that Dmrt1 should regulate yolk protein transcription or control nervous system sexual dimorphism, as dsx and mab-3 do, or that Dmrt1 should function anywhere outside of the gonad (Raymond, 2000).

Confounding the situation further, sex determination evolves rapidly and the role of sex-determining genes can change rapidly. An extreme example is Sry, which plays a pivotal role in mammalian sex determination but does not exist in birds and reptiles. Similarly, Sox9 appears to play an early sex-determining role in mammals but a later role in testis differentiation in other vertebrates. Nevertheless, given the evolutionary lability of sex-determining genes, it is particularly striking that Dmrt1 has been found in all vertebrates in which it has been sought. Furthermore, Dmrt1 is unique in being expressed very early and sex specifically in the gonad of all classes of vertebrates so far examined, regardless of the sex-determining mechanism used, whether chromosomal or environmental. Determining the evolutionary relationships of dsx, mab-3, and Dmrt1 will require the examination of a greater range of species. What is now clear, however, is that DM domain genes play an essential role in sexual development in at least three phyla and probably in others (Raymond, 2000).

The doublesex-related gene, XDmrt4, is required for neurogenesis in the olfactory system

The Dmrt genes encode a large family of transcription factors whose function in sexual development has been well studied. However, their expression pattern is not restricted to the gonad, suggesting that Dmrt genes might regulate other developmental processes. This paper reports the expression and functional analysis of one member of this family: Xenopus Dmrt4 (XDmrt4). XDmrt4 is initially expressed in the anterior neural ridge and then becomes progressively restricted to part of the telencephalon and the olfactory placode/epithelium. XDmrt4 is induced at the anterior neural plate by a balance of neural inducers and caudalizing factors. Interference with XDmrt4 function by injection of a morpholino oligonucleotide or an inhibitory mutant resulted in a similar phenotype, the specific disruption of the olfactory placode expression of Xebf2 without affecting the expression of other placodal markers. Xebf2 belongs to a family of helix-loop-helix transcription factors implicated in neuronal differentiation, and later in embryogenesis XDmrt4-deficient embryos show impaired neurogenesis in the olfactory epithelium. Consistent with this finding, XDmrt4 is sufficient to activate neurogenin, Xebf2, and neural cell adhesion molecule expression in animal explants and is required for Noggin-mediated neuralization. Altogether, these results indicate that XDmrt4 is an important regulator of neurogenesis in the olfactory system upstream of neurogenin and Xebf2 (Huang, 2005).

Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA

The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences. A random oligonucleotide in vitro selection method was used to determine DNA binding sites for six of the seven proteins. These proteins selected sites resembling that of DSX despite differences in the sequence of the DM domain recognition helix, but they varied in binding efficiency and in preferences for particular nucleotides, and some behaved anomalously in gel mobility shift assays. DMRT1 protein from mouse testis extracts binds the sequence that was determined, and the DMRT proteins can bind their in vitro-defined sites in transfected cells. Some DMRT proteins can bind DNA as heterodimers. These results suggest that target gene specificity of the DMRT proteins does not derive exclusively from major differences in DNA binding specificity. Instead target specificity may come from more subtle differences in DNA binding preference between different homodimers, together with differences in binding specificity between homodimers versus heterodimers (Murphy, 2007).

Genome-wide analysis of DNA binding and transcriptional regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis

The DM domain proteins Doublesex- and MAB-3-related transcription factors (DMRTs) are widely conserved in metazoan sex determination and sexual differentiation. One of these proteins, DMRT1, plays diverse and essential roles in development of the vertebrate testis. In mammals DMRT1 is expressed and required in both germ cells and their supporting Sertoli cells. Despite its critical role in testicular development, little is known about how DMRT1 functions as a transcription factor or what genes it binds and regulates. This study combined ChIP methods with conditional gene targeting and mRNA expression analysis and identified almost 1,400 promoter-proximal regions bound by DMRT1 in the juvenile mouse testis and determined how expression of the associated mRNAs is affected when Dmrt1 is selectively mutated in germ cells or Sertoli cells. These analyses revealed that DMRT1 is a bifunctional transcriptional regulator, activating some genes and repressing others. ChIP analysis using conditional mutant testes showed that DNA binding and transcriptional regulation of individual target genes can differ between germ cells and Sertoli cells. Genes bound by DMRT1 in vivo were enriched for a motif closely resembling the sequence DMRT1 prefers in vitro. Differential response of genes to loss of DMRT1 corresponded to differences in the enriched motif, suggesting that other transacting factors may modulate DMRT1 activity. DMRT1 bound its own promoter and those of six other Dmrt genes, indicating auto- and cross-regulation of these genes. Many of the DMRT1 target genes identified in this study are known to be important for a variety of functions in testicular development; the others are candidates for further investigation (Murphy, 2010).

The mammalian Doublesex homolog DMRT6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis

In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. This study examined the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) in mice and find that Dmrt6 plays a crucial role in directing germ cells through the mitotic-to-meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain, a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing inappropriate expression of spermatogonial differentiation factors, including SOHLH1, SOHLH2 and DMRT1 as well as the meiotic initiation factor STRA8, and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant germ cells can complete spermatogonial differentiation and enter meiosis, but they show defects in meiotic chromosome pairing, establishment of the XY body and processing of recombination foci, and they mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 chromatin immunoprecipitation sequencing suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. These results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis (Zhang, 2014).

The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. This study reports the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). It was found to be sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. It is proposed that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects, and the potential use of fru and dsx for developing new genetic strategies in vector control is discussed (Salvemini, 2013).

DMRT1 is required for mouse spermatogonial stem cell maintenance and replenishment

Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system (see Drosophila spermatogenesis), with precise balance between self-renewal and differentiation. Prior work has established doublesex- and mab-3-related transcription factor 1 (Dmrt1) (see Drosophila dsx) as a conserved transcriptional regulator of male sexual differentiation. This study investigated the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. It was found that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf (see Drosophila CG4424), another transcription factor required for SSC maintenance. Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 (see Drosophila tap) normally do not contribute to SSCs marked by the Id4-Gfp (see Drosophila emc) transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. These data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion (Zhang, 2016).


doublesex: Biological Overview | Regulation | Protein Interactions and Regulation of Splicing | Developmental Biology | Effects of Mutation | References

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