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 (Dsx^F) 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. Dsx^F 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, Dsx^F and Dsx^M compete with each other for the regulation of target genes. In addition to directing sex-specific differentiation, Dsx^F plays an important role in sexual behavior. Wild-type males ectopically expressing Dsx^F are actively courted by other males. This acquisition of feminine sex appeal is likely due to the induction of female pheromones by Dsx^F. More extreme behavioral abnormalities are observed when Dsx^F 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 Dsx^F and that it is not regulated by the sex-specific splicing cascade (Waterbury, 1999).

Using constitutively expressed dsx^F transgene, it was asked if Dsx^F 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. Dsx^M has been shown to have a positive role in sex-comb formation; intermediate sex combs form when Dsx^M is expressed in females carrying a dsx^Dom allele and ectopic sex combs form on all six legs in males or females when Dsx^M is expressed ubiquitously under hsp70 control. However, Dsx ^F does not appear to affect sex-comb formation in otherwise wild-type males when dsx^F is ectopically expressed using an actin-dsx^F or an hs70-dsx^F transgene. These results led to the idea that Dsx^F does not play a role in the formation of sex combs (Waterbury, 1999).

A single copy of the hsp83-dsx^F transgene has no readily apparent effects on sex-comb formation in otherwise wild-type males. To look more closely for a competitive balance between Dsx^F and Dsx^M on sex comb formation, endogenous copies of dsx were removed. By reducing the level of endogenous dsx, Dsx^F 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 Dsx^F acts to negatively regulate sex comb formation in females and indicates that a competition exists between Dsx^F and Dsx^M when both protein forms are present. This is the first example of a negative role for Dsx^F (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: Dsx^F activates and Dsx^M represses. Yp-1 is expressed in both the fat body and the ovaries of females; however, Dsx only regulates expression within the fat body. Dsx^F 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-dsx^F transgene in otherwise wild-type males is sufficient to activate yp-1 expression in the fat body. Thus Dsx^F 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 Dsx^M 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-dsx^F 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 Dsx^F 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-dsx^F 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 Dsx^M can repress yp-1 transcription in the absence of Ix protein. Given that Dsx^F can induce yp-1 expression in males, an examination was carried out to see if this induction is dependent upon ix. Males carrying the dsx^F transgene, but homozygous for ix^- and wild type for dsx are phenotypically wild type and fertile. However, without ix, Dsx^F 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 dsx^F transgene is also dependent upon the ix gene. These findings suggest that Dsx^F 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 Dsx^F protein (Waterbury, 1999).

Dsx^F 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 dsx^F transgene has any effects on male courtship behavior. As a (partial) control for these experiments, the effect of another transgene, hsp83-tra^F, 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-tra^F transgene is expected to more strongly feminize XY animals than hsp83-dsx^F; however, the feminization of XY animals by the hsp83-tra^F is not complete in all tissues, and male-specific structures, such as the Muscle of Lawrence, are still observed. When hsp83-tra^F 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).

dsx^F transgene males are fertile and can and will mate with females. The measured courtship index of dsx^F transgene males demonstrates that they court virgin females with as much interest as wild-type males. The duration of copulation of dsx^F 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 dsx^F transgene males would discriminate between females and males. Unlike dsf^-, fru^-, or hsp83-tra^F males, dsx^F 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-tra^F pseudofemales also elicited high levels of courtship from wild-type males. Given that dsx^F 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 dsx^F transgene males produce female attractants that are responsible for eliciting courtship behaviors by other males (Waterbury, 1999).

While the dsx^F 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 dsx^F males from XX females in this experiment, the XY animals were marked with the Y chromosome-linked eye marker B^s. The defect caused by B^s 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 B^s 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 dsx^F 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-tra^F transgene males (Waterbury, 1999).

Because the dsx^F 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 dsx^F pseudofemales actively reject courting wild-type males, they do allow themselves to be mated. Unlike wild-type females, however, the dsx^F 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 dsx^F pseudofemales. In addition dsx^F 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 dsx^F transgene males is that ectopic expression of Dsx^F 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 dsx^F 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, dsx^F 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 Dsx^F 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 dsx^F transgene males. One copy of the dsx^F 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 Dsx^F cannot be overcome by Dsx^M. Production of the antiaphrodisiac 7-T is also reduced by Dsx^F. The amount of 7-T decreases nearly 10-fold from 836.0 ng/fly in wild-type males to 93.0 ng/fly in dsx^F 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 dsx^F expression. (1) Similar results were obtained in all dsx^F 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-tra^F resembles that of wild-type females. However, tra^F 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 Dsx^F in the presence of Dsx^M, although quantitated amounts of cVA do decrease from 160.0 ng/fly in control males to 45.0 and 107.0 ng/fly in dsx^F;+/+ and dsx^F; 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 Dsx^F but only efficiently in the complete absence of Dsx^M (Waterbury, 1999).

Given that ix appears to have a role in yolk protein production in dsx^F 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 Dsx^F and Ix function together to promote the production of female-specific pheromones. This conclusion is further supported by the pheromone profile of dsx^F transgene males mutant for ix. Where one copy of the dsx^F 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 dsx^F 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 Dsx^F 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 dsx^F transgene males, it would appear that ix expression is not directly dependent upon the Sxl - tra splicing cascade. While the possibility that Dsx^F 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 Dsx^F, enabling Dsx^F 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 dsx^F 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 Dsx^F 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 Dsx^F. 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-2^ts 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-2^ts 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 FRU^M is not expressed within the internal genitalia. The DSX^M and DSX^F 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 DSX^F positively regulates its sex-specific expression. The microarray data for CG17012 are consistent with this conclusion and do not reveal any effect of DSX^M 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 DSX^F, and that DSX^M 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 DSX^M, 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 DSX^F 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 DSX^F 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 DSX^M, but rather of negative action by DSX^F 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 DSX^M (XY wild type, XX tra and XX dsx^D pseudomales), suggesting that DSX^M positively regulates their expression. Expression also appears higher in XX dsx null compared with XX wild-type animals, suggesting negative regulation by DSX^F 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 DSX^F 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).

Chinmo prevents transformer alternative splicing to maintain male sex identity

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform tra^F. In turn, Tra^F alternatively splices dsx to the female isoform dsx^F. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs; cyst stem cells) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. This feminization occurs by transcriptional and post-transcriptional regulation of tra^F. chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). tra^F splicing in chinmo-deficient CySCs leads to the production of Dsx^F at the expense of the male isoform DsxM, and both Tra^F and Dsx^F are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, this study shows that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. This work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of tra^F production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility (Brmai, 2018).

This study shows that that one single factor, Chinmo, preserves the male identity of adult CySCs in the Drosophila testis by regulating the levels of canonical sex determinants. CySCs lacking chinmo lose DsxM expression not by transcriptional loss but rather by alternative splicing of dsx pre-mRNA into dsx^F. These chinmo-mutant CySCs ectopically express Tra^F and Dsx^F, and both factors are required for their feminization. Furthermore, the results demonstrate that tra alternative splicing in cyst cells lacking chinmo is achieved independently of Sxl. Instead, this work strongly suggests that tra^F production in the absence of chinmo is mediated by splicing factors Vir and Fl(2)d. It is proposed that male sex identity in CySCs is maintained by a two-step mechanism whereby tra^F is negatively regulated at both transcriptional and post-transcriptional levels by Chinmo (see Model for adult somatic sex maintenance in the Drosophila somatic gonad). In this model, loss of chinmo from male somatic stem cells first leads to transcriptional upregulation of tra pre-mRNA as well as of vir and fl(2)d. Then the tra pre-mRNA in these cells is spliced into tra^F by the ectopic Vir and Fl(2)d proteins. The ectopic Tra^F in chinmo-deficient CySCs then splices the dsx pre-mRNA into dsx^F, resulting in loss of Dsx^M and gain of Dsx^F, and finally induction of target genes usually restricted to follicle cells in the ovary (Brmai, 2018).

Chinmo has motifs associated with transcriptional repression and its loss clonally is associated with ectopic transcription. One interpretation of the data is that Chinmo directly represses tra, vir, and fl(2)d in male somatic gonadal cells. As the binding site and potential co-factors of Chinmo are not known, future work will be needed to determine whether Chinmo directly regulates expression of these genes. It is also noted that ~50% of chinmo-mutant testes still feminize in the genetic absence of tra or dsx^F. These latter data indicate that Chinmo regulates male sex identity through another, presumably parallel, mechanism that does not involve canonical sex determinants. However, this tra/dsx-independent mode of sex maintenance downstream of Chinmo is not characterized and will require the identification of direct Chinmo target genes (Brmai, 2018).

Previous work has shown that JAK/STAT signaling promotes chinmo in several cell types, including CySCs (Flaherty, 2010). Since JAK/STAT signaling is itself sex-biased and restricted to the embryonic male gonad, it is presumed that activated Stat92E establishes chinmo in male somatic gonadal precursors, perhaps as early as they are specified in the embryo. Because loss of Stat92E from CySCs does not result in an apparent sex transformation phenotype, the interpretation is favored that Stat92E induces expression of chinmo in CySCs but that other sexually biased factors maintain it. One potential candidate is Dsx^M, which is expressed specifically in early somatic gonads and at the same time when Stat92E activation is occurring in these cells. In fact, multiple Dsx^M ChIP-seq peaks were identified in the chinmo locus, suggesting potential regulation of chinmo by Dsx^M. This suggests a potential autoregulatory feedback loop whereby Dsx^M preserves its own expression in adult CySCs by maintaining Chinmo expression, which in turn prevents tra^F and dsx^F production (Brmai, 2018).

Recent studies on tissue-specific sex maintenance demonstrate that while the Sxl/Tra/Dsx hierarchy is an obligate and linear circuit during embryonic development, at later stages it is more modular than previously appreciated. For example, Sxl can regulate female-biased genes in a tra-independent manner. Additionally, Sxl and Tra^F regulate body size and gut plasticity independently of the only known Tra^F targets, ^dsx and fru. Negative regulation of the Tra^F-Dsx^F arm of this cascade is required to preserve male sexual identity in CySCs but unexpectedly is independent of Sxl. Because depletion of Vir or Fl(2)d significantly blocks sex transformation and both are required for tra alternative splicing in the ovary, this work reveals they can alternatively splice tra pre-mRNA even in the absence of Sxl. This is the first demonstration of Sxl-independent, Tra-dependent feminization. These results raise the broader question of whether other male somatic cells have to safeguard against this novel mechanism. Because recent work has determined that sex maintenance is important in systemic functions regulated by adipose tissue and intestinal stem cells, it will be important to determine whether Chinmo represses tra^F in these settings. Finally, since the transcriptional output of the sex determination pathway is conserved from Drosophila (Dsx) to mammals (DMRT1), it is possible that transcriptional regulation of sex determinants plays a similar role in adult tissue homeostasis and fertility in higher organisms (Brmai, 2018).

Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons

For successful mating, a male animal must execute effective courtship behaviors toward a receptive target sex, which is female. Whether the courtship execution capability and upregulation of courtship toward females are specified through separable sex-determining genetic pathways remains uncharacterized. This study found that one of the two Drosophila sex-determining genes, doublesex (dsx), specifies a male-specific neuronal component that serves as an execution mechanism for courtship behavior, whereas fruitless (fru) is required for enhancement of courtship behavior toward females. The dsx-dependent courtship execution mechanism includes a specific subclass within a neuronal cluster that co-express dsx and fru. This cluster contains at least another subclass that is specified cooperatively by both dsx and fru. Although these neuronal populations can also promote aggressive behavior toward male flies, this capacity requires fru-dependent mechanisms. These results uncover how sex-determining genes specify execution capability and female-specific enhancement of courtship behavior through separable yet cooperative neurogenetic mechanisms (Ishii, 2020).

This study has uncovered distinct yet cooperative roles of dsx and fru on male-type social behaviors through a specific subset of P1/pC1 neurons. For courtship behaviors, this study found that NP2631 ∩ dsx^FLP neurons are specified in a fru-independent manner, and in males, their capacity to generate courtship behaviors does not require fruM. However, activation of NP2631 ∩ dsx^FLP neurons in fruF males failed to increase courtship selectively toward female targets. These results suggest that dsx plays a major role in establishing a neuronal circuit that enables the male flies to execute courtship behavior, whereas fru is critical for enhancing courtship behavior toward females, likely through proper recognition of target sex. The fact that the sex of the target flies influences the function of P1/pC1 subsets implies that information about target sex can modulate the neural circuit units downstream of these neurons, and encourages revision of the linear circuit model for sexually dimorphic social behaviors. In contrast, the complete specification and courtship-promoting functions of P1^a neurons require both dsx and fru, revealing genetic and functional heterogeneity within P1/pC1 neurons. Lastly, NP2631 ∩ dsx^FLP and P1^a neurons require a fruM-dependent mechanism to promote male-type aggressive behavior. This suggests that neither of these neurons are part of the execution mechanism for male-type aggressive behavior, and that the genetic mechanisms specifying execution components for courtship and aggressive behaviors are different (Ishii, 2020).

Electrical stimulation of various parts of the brain has been known to elicit complex behaviors, including social behaviors, for almost a century. Recent technological advances have allowed researchers to identify specific, genetically labeled populations of neurons that can induce mating and aggressive behaviors upon acute optogenetic stimulation in both mice and in flies, even toward suboptimal targets (such as inanimate objects). These findings seem consistent with the idea that neuronal activation can override most contexts and generate specific behaviors depending on the identity of stimulated cells. However, interactions with a target animal can transmit important information which a tester animal may use to choose appropriate behaviors. In fact, attacks triggered by optogenetic stimulation of ventrolateral hypothalamus (VMH) in male mice tend to last longer toward castrated males than toward female targets, and chemogenetic activation of progesterone receptor-expressing VMH neurons appears to induce more attacks toward male than toward female targets. While effects on target sex are not consistently documented, the current results and previous observations in mice show that the target sex has a significant impact on behavioral choice even for optogenetically induced social behaviors. These results suggest that sensory or behavioral feedback from target animals can impact the operation of what may appear to be an 'execution mechanism' for a given behavior (Ishii, 2020).

Identification of neural sites where the information about the target sex is integrated with the activity of both NP2631 ∩ dsx^FLP and P1^a neurons will be an important step in understanding how such context cues modulate ongoing neural activity and, ultimately, behavioral outcome. While a 'command'-like center that irreversibly executes courtship or aggressive behaviors, like recently characterized egg-laying controlling neurons, may exist, it is also possible that information about target sex (and its behavioral response) can be injected at multiple levels of a neural circuit, thereby ensuring the target sex-specific execution of sexually dimorphic social behaviors. This is conceptually analogous to the neural control of fine motions, which can be constantly adjusted by sensory feedback and efference copies all the way down to the motoneuron level (Ishii, 2020).

Recently, the importance of addressing sex as a biological variable has been widely recognized. In the context of social behaviors, this variable in the tester animals can be critical for uncovering the underlying neural mechanisms (Ishii, 2020).

The functional segregation of dsx and fru that was observed in this study can be considered analogous to the organizational and activation functions of sex hormones in mammals. Differential exposure to gonadal steroid hormones, mostly through estrogen receptors, specifies neural circuits that are necessary for sex-specific reproductive behaviors, whereas hormonal surges in the adult stage (such as testosterone or progesterone) orchestrate activation of sex-specific behaviors. It is postulated that dsx has an organizational function for the courtship execution circuit, whereas fru is important for the appropriate activation of the circuit (Ishii, 2020).

The results do not mean that fru is not necessary for the establishment of all neuronal components involved in courtship. Nonetheless, the result suggests that the wing extension execution circuit that connects NP2631 ∩ dsx^FLP neurons and relevant motoneurons is specified even in the absence of fruM, which is consistent with previous observations that fruF males are capable of expressing at least a part of courtship behavior. While a specification role for dsx on P1/pC1 neurons has been previously reported, the current study showed for the first time the behavioral role of a specific P1/pC1 subset (NP2631 ∩ dsx^FLP neurons) in fruF males. dsx is important for the specification of a few other behaviorally relevant sexual dimorphisms in the Drosophila nervous system. For instance, the sexually dimorphic axon development of leg gustatory receptor neurons, which includes aphrodisiac pheromone sensors, requires dsx function. The neural connectivity and function of TN1 neurons, which are pre-motor neurons important for the production of pulse song, are also specified by dsx. Several classes of abdominal ganglia neurons involved in male copulation also express dsx. Although relatively few in number, these examples display the importance of dsx in key neuronal populations for organizing circuit components that are essential for the execution of courtship behaviors. It is noteworthy that dsx is involved in sex-determination across a variety of animal phyla, whereas fru's role in sex-determination seems confined to insects. This suggests that dsx may be evolutionarily more ancient in the context of sex-determination than fru, which can account for its dominance over fru when specifying sexually dimorphic neurons that co-express dsx and fru (Ishii, 2020).

The proposal that fruM may be important for enhancing courtship behavior specifically towards females is consistent with the fact that many characterized fru-expressing neurons are involved in processing sex- and species-specific sensory cues. Namely, P1^a neurons, as well as more broadly defined P1/pC1 neurons accessed by different genetic reagents, are known to respond to sex-specific chemical cues, underscoring their critical role in sensory integration for courtship. fruM can play the 'activation' role for courtship by establishing sensory circuits that transmit sex-specific sensory information to P1/pC1 neurons, or by enabling P1/pC1 neurons to properly integrate and transform such neural inputs. Neuroanatomical defects of P1^a neurons in fruF males could disrupt either process (Ishii, 2020).

Gain control of sex-specific sensory cues can be one neuronal mechanism for the 'activation' function, but courtship behavior can be enhanced in other ways as well. For instance, behavioral persistence or context-dependent intensity adjustment can result in an increase of the overall courtship vigor. Recently, a new class of fru-expressing neurons downstream of P1^a neurons has been found to mediate the persistence of courtship behavior triggered by P1^a neuronal activation (Jung, 2020). Even if fruM is not absolutely necessary for the formation of the minimal wing extension execution circuit, it can have a significant impact on the generation of effective wing extension toward female target flies (Ishii, 2020).

While it is concluded that the role of fru is not necessarily to specify the execution mechanism for courtship behavior, fruM females can still perform wing extensions or dsx^- expressing neurons in females can elicit wing extensions, suggesting that the residual execution mechanism for at least a part of courtship behavior may be specified in a sex-invariant manner. The presence of a latent mating execution circuit in female brains is also suggested in mice. Because the courtship songs produced by females or fruM females are defective, male-type splicing of dsx nonetheless seems to be instrumental in organizing the proper execution mechanism for Drosophila courtship behavior (Ishii, 2020).

In striking contrast to wing extensions, this study found that activation of neither NP2631 ∩ dsx^FLP nor P1^a neurons in fruF males induced lunges. This result points to the existence of a fruM-dependent execution mechanism for male-type aggressive behaviors, likely downstream of these neurons. In Wohl (2020), it was found that at least one group of fruM-dependent neurons can promote male-type aggressive behaviors independent of dsx. Therefore, a separation of the courtship execution mechanism and the aggression execution mechanism by two sex-determining genes is likely accomplished by a partial separation of underlying neural circuits. The aggression-promoting function of NP2631 ∩ dsx^FLP and P1^a neurons likely reflects their roles to coordinate aggression and courtship depending on internal and external conditions, instead of a simple decision switch that triggers fixed types of behavior (Ishii, 2020).

Both dsx and fru encode transcription factors. The sexually dimorphic morphology and wiring specificity of many fru-expressing neurons are determined in a cell-autonomous manner, suggesting that dsx and fru define Drosophila sex at a cellular level through regulation of a specific set of target genes. Mammalian sex hormones ultimately exert their effects through nuclear steroid receptors, which serve as transcription factors. Thus, both in flies and in mammals, organismal sex can be regarded as a collective phenotype of genetic 'sexes' that can be reduced down to the cellular leve. To understand how sex at the neuronal level influences sexually dimorphic behaviors, cell-type specific manipulation of sex-determining genes is required. The current study focused on neural functions in a whole animal mutant, which prevents addressing the role of either dsx or fru specifically within NP2631 ∩ dsx^FLP or P1^a neurons. For example, it is ot known whether NP2631 ∩ dsx^FLP neurons in fruF males failed to enhance courtship behavior toward female targets because of the absence of fruM within this population, or because of the lack of fruM in other neuronal populations, or both. In addition, the current approach does not address if it is the presence of dsxM or the absence of dsxF that is important for the specification of male-type NP2631 ∩ dsx^FLP neurons or P1^a neurons (Ishii, 2020).

It is important to note that sex specification is a developmental process of transformation. Both at genetic and organismal levels, one sex is not a loss-of-function mutant of the other. Loss-of-function manipulations at the cellular level, by cell type-specific RNA interference or CRISPR interference -based approaches, may show that either dsx or fru is necessary for the proper development or function of the given neurons, but may be insufficient to illuminate the genetic origin of the sex-specific transformation at the cellular level. In addition, temporally and spatially precise manipulation of genes during development remains difficult. This can create a difficulty interpreting the effects of either knock-down or over-expression of sex determining genes, which are dynamically regulated from early developmental stages. Creation of neuronal mutant clones may circumvent this problem, but the tra mutation, which has been previously used to convert a 'neuronal sex', cannot dissociate the roles of dsx and fru (Ishii, 2020).

Faced with these often overlooked limitations of cell-type specific gene manipulations, it would be informative to characterize what types of transformations are observed in mutants of sex-specific splicing at an organismal level, as in this and other studies. Although a constitutive mutants have above-mentioned limitations, they nonetheless establish fundamental functional differences among sex-determining genes, as well as benchmarks for the efficacy for cell-specific manipulations techniques. Although clearly out of the scope of the current study, electron microscopy-based connectome reconstructions of fruF male and fruM female brains could provide useful information for understanding the transformative nature of sex specification in the brain (Ishii, 2020).

Lastly, the serendipitous finding that NP2631 ∩ dsx^FLP and P1^a neurons contain genetically and functionally distinct populations underscores the importance of characterizing neuronal cell types in greater detail. How to determine cell types remains a challenge in neuroscience, but genetic access to a finely defined population of neurons even within what is considered as a single class of neurons can be the key to understand how a neural circuit generates complex behaviors such as social behaviors (Ishii, 2020).

In the posterior part of male brains, 'P1' neurons, as defined by fru-expressing cluster, and pC1 neurons, as defined by dsx-expressing cluster, extensively overlap. This raises a question about the distinction between 'P1' and 'pC1' neurons. Furthermore, recent single cell level analyses of the neurons that belong to the male 'P1' cluster or 'pC1' cluster revealed surprising neuroanatomical and functional diversity, raising a possibility that P1/pC1 neurons may be functionally heterogeneous as well (Ishii, 2020).

Surprisingly, this study found that behaviorally relevant NP2631 ∩ dsx^FLP and P1^a neurons, as well as NP2631 ∩ fruFLP and P1^a neurons, seldom overlap. Optogenetic stimulation of NP2631 ∩ dsx^FLP and P1^a neurons triggers social behaviors in temporally distinct manners. Moreover, fru has a different impact on the specification and function of these two neuron groups, suggesting that little overlap of NP2631 ∩ dsx^FLP and P1^a neurons does not necessarily reflect arbitrary labeling bias within a single homogeneous neuronal population by different genetic reagents. Instead, these observations support the idea that of P1/pC1 neurons consist of functionally diverse subtypes (Ishii, 2020).

It is acknowledged that the genetic reagents used in this study are likely insufficient to resolve the possible heterogeneity within either NP2631 ∩ dsx^FLP or P1^a neurons. Differential expression patterns of FruM proteins within both clusters alone suggest that such heterogeneity almost certainly exists. Recent advances in whole-brain neural reconstruction using electron microscopy images will provide a foundation for precise characterization of Drosophila neurons, as has been recently used for the female-type 'pC1' cluster. A large number of 'split-GAL4' collections will allow universal access to the specific subpopulations. These types of tools will facilitate cross-study comparisons of neuroanatomical and behavioral data, and will serve as a catalyst to understand the logic of neural control of behavior in general. With the advance of single cell-level genetic and epigenetic profiling techniques, the importance of precisely characterizing the targeted neuronal types will only grow not only in Drosophila, but in every model organism. Reproducible access to each neuronal type can uncover functional units for a given behavior at even finer detail, which will be fundamental for deconstructing the dynamics of neural circuits that are responsible for generating social behaviors in a context-dependent manner. Such knowledge will be also critical for establishing theoretical models that account for brain operations and population-level dynamics of animals engaging in social interactions (Ishii, 2020).

Nucleoporin107 mediates female sexual differentiation via Dsx

A missense mutation in Nucleoporin107 (Nup107; D447N) underlies XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modeling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of bone morphogenetic protein (BMP signaling hyperactivation). Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex- and tissue-specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells (Shore, 2022).

This study has shown that Nup107 activity in the somatic component of the gonad is necessary for the proper development and function of the ovaries. In which somatic cell type is the activity of Nup107 necessary? The fact that KD of Nup107 using the tj-Gal4 driver resulted in larval and adult aberrant phenotypes indistinguishable from those induced by the Nup107 loss of function mutation, indicates that the ovarian function of Nup107 is primarily required in the Tj-expressing cells. Notably, the tj-Gal4 driver is not expressed in terminal filament (TF) cells either at larval or adult stages. TF cells together with the cap cells (CCs) constitute the ovarian stem cell niche. During larval ovarian development, Tj is expressed in the ICs, CCs, and follicle stem cell (FSC) progenitors. CCs, which are derived from the intermingled cells (ICs), are formed at the base of fully formed TFs at the transition from the third larval instar to prepupal stage. Therefore, the aberrant PGCs and ICs observed in the larval gonads are not due to impairment of Nup107 or Dsx activities in CCs. Likewise, FSC progenitors are also not the candidate cells for the site of action of Nup107 activity as they reside posterior to ICs with a minimal physical contact with only a few posteriorly located PGCs. Together these data imply that Nup107 acts specifically in ICs enabling them to effectively interact with the PGCs. Consistent with this notion, loss of Nup107 affected the behavior of ICs such that these cells showed varying degrees of failure to mingle with the PGCs. A severe failure of ICs and PGCs to interact in the larval gonad is expected to cause an ovarian-dysgenesis-like phenotype as it is essential for the germarium development and ovariole formation. Likewise, a milder failure of intermingling in the larval gonad would allow for the formation of adult ovaries. However, in the adult ovary Nup107 activity is further required in ECs for the formation of their cellular extensions and regulation of differentiation of the GSCs. Thus, it will be important to determine in future studies the functional relationship between Nup107 and the signaling pathways which previously were shown to regulate the formation of these cellular extensions (Shore, 2022).

These studies have revealed that Nup107, a ubiquitously expressed nuclear envelope protein, is a crucial player during female gonad formation. How does an essential housekeeping protein critical for nuclear transport, perform such a sex- and tissue-specific function(s)? Two possible scenarios, not necessarily mutually exclusive, are envisioned to explain how the specific mutation in Nup107 results in ovary-specific aberrant phenotypes. In the first scenario, Nup107 would specifically mediate nucleocytoplasmic translocation of factor(s) or downstream effector(s) required for ovarian development. Indeed, recent studies have demonstrated that Nup107 is involved in translocation of specific factors. For instance, in the event of DNA damage, Nup107 directly interacts with the apoptotic protease activating factor 1 (Apaf-1 also known as Drosophila ARK) and mediates its transport into the nucleus to elicit cell-cycle arrest. Furthermore, it has been shown in tissue culture that specific Nucleoporins, including Nup107, are required for nuclear translocation of SMAD1, an important downstream effector of the Dpp/BMP pathway (Shore, 2022).

Alternatively, accumulating evidence has documented that in addition to their primary function in regulating the exchange of molecules between the nucleus and cytoplasm, NPC components may contribute to genome organization and tissue-specific regulation of gene expression in a nuclear transport-independent manner. Consequently, such moonlighting activities may not be confined to the nuclear envelope which is the primary native location of these proteins. For instance, mammalian Nup107-160 complex (a subcomplex of the NPC of which Nup107 is a key component) has recently been shown to shuttle in and out of GLFG nuclear bodies containing Nup98, a nucleoporin that regulates multiple aspects of gene regulation. Consistently, Nup107 was shown to regulate levels of specific RNAs through gene imprinting (Sachani, 2018). Furthermore, using an RNAi-based assay, Nup107 was identified as a positive regulator of OCT4 and NANOG expression in human ESCs. In this regard, it is noteworthy that the recently published chromatin-binding profile of Nup107 suggested that Nup107 specifically targets active genes (Gozalo, 2020). Altogether these data support the possibility that Nup107 affects transcription of specific target genes in a tissue- and sex-specific manner either directly or indirectly (Shore, 2022).

In Drosophila melanogaster, Sex-lethal (Sxl) is the master determinant of somatic sexual identity, regulating a splicing dependent regulatory cascade resulting in the presence of alternatively spliced sex-specific isoforms of Dsx protein, Dsx-F and Dsx-M, in females and males, respectively. Subsequent dimorphic sexual development including sex-specific gonad morphogenesis is under the control of these Dsx isoforms. Consistently, Dsx proteins deploy components of the housekeeping machinery to achieve sex-specific development of the gonads. Thus, such 'maintenance' factors are unlikely to be involved in any regulatory capacity. The data challenge this notion and demonstrate the presence of sexually dimorphic circuitry downstream of a 'housekeeping' nuclear envelope protein, Nup107, which regulates the expression the female form of dsx (Shore, 2022).

The similar sex-specific and ovary-restricted phenotype associated with compromised Nup107 activity in both humans and flies implies common underlying molecular mechanisms. This study has identified Dsx as the primary target acting downstream of Nup107 in Drosophila ovarian development. The mammalian homologues of Dsx, the Dmrt family of transcription factors, also function during sex-specific gonad development. However, in mammals the main function of Dmrt genes in the gonad is to promote male-specific differentiation. While detailed functional analysis is not available, it is plausible that in mammals, another key female-specific transcription factor, like Foxl2 (female-specific forkhead box L2) may act downstream of Nup107 to substitute for DsxF in flies (Shore, 2022).

A previous study showed that the key components of the stem cell niche, that is the hub in males and the TFs in the case of females, are still formed in the absence of dsx, but this happens in a stochastic manner in both XX and XY dsx null mutant individuals. These results indicate that in the context of the developing stem cell niche, Dsx may not act in an instructive manner, but is instead required to ensure that the proper program (male or female) is selected, which does not require Dsx activity for the execution of subsequent sex-specific development. Nevertheless, their findings clearly demonstrated that the resulting adult ovaries and testes are improperly formed, consist of aberrant structures, arguing that Dsx activity, is critical for proper gonad development outside of the stem cell niche. Observations made in this study are consistent with this suggestion. Moreover, the experimental strategies and results differ in two important ways. First, the current experiments have relied on reduction of only the female form of dsx that is dsx-F which allowed for sex determination and thus no 'male' structures or cellular identity transformations were observed. Second, in the current experiments tj-Gal4 driver was used, which is not expressed in the stem cell niche (TF cells) but in other somatic gonadal cells. This experimental design enabled uncovering a novel developmental function of Dsx-F in ICs and their adult descendants, ECs. Supporting this notion it has ben shown that RNAi KD of dsx also resulted in small ovaries (Shore, 2022).

This study found that the secreted metalloprotease AdamTS-A is an important downstream component in the Nup107-Dsx axis, as KD of AdamTS-A results in phenotypes similar to those elicited by loss of either Nup107 or dsx. In the adult ovary, these aberrant phenotypes include loss of EC membrane protrusions and expanded BMP signaling. This raised the question of how AdamTS-A regulates the range of BMP signaling. The ECM, which is produced and secreted by cells, has the structure of a complex fibrillar meshwork and provides structural support and tissue integrity, playing an active role in regulating cell behavior. ECM proteoglycans sequester and modulate chemical signals, including growth factors and guidance molecules. Furthermore, type IV collagens, major components of the ECM, were shown to restrict Dpp signaling in the ovary. This is particularly intriguing, since in C. elegans Gon-1, the homolog of AdamTS-A, was shown to genetically interact with a type IV collagen (EMB-9) in the regulation of gonadogenesis. This raised the possibility that AdamTS-A, secreted by ECs, restricts Dpp movement in the germarium through cleavage of ECM components. However, by knocking down coracle in ECs, this study has shown that disruption of their cellular protrusions, which encapsulate the germ cells, leads to expansion of BMP signaling. This implies that the activity provided by these cellular extensions is necessary and sufficient for restricting the BMP signal (Shore, 2022).

Thus, Nup107, Dsx, and AdamTS-A all function in ECs and are necessary for the formation and maintenance of the cellular protrusions which are required for restricting the BMP signal emanating from the GSC's niche. Further, it appears that Adam-TS-A activity in the ECM is required for the formation and/or maintenance of these cellular protrusions. The results indicate that in this context AdamTS-A regulates BMP signal distribution indirectly via regulation of the cellular protrusion maintenance. It is also possible that AdamTS-A utilizes these cellular extensions in order to reach the ECM away from the ECs in the GSC region, where it acts to restrict Dpp trafficking (Shore, 2022).

Overall, these results support a model where Nup107 regulates the expression of dsx, either directly or indirectly, while Dsx directly regulates the transcription of multiple target genes including AdamTS-A. These observations have also uncovered that DsxF controls somatic niche function by calibrating the range and/or strength of Dpp/BMP signaling, possibly via modulation of the level and/or activity of the ECM components. Thus, it will be critical to elucidate how activities of nonsex-specific components such as Nup107 are coordinated with sex-specific regulation to achieve the precise specification and patterning underlying gonad development. This is of particular significance since modulation of BMP signaling circuitry is inextricably linked with the establishment and maintenance of stem cell fate. Importantly, as in the case of Nup107, BMP signaling is also required in a nonsex-specific manner in a variety of developmental contexts. These observations therefore open new avenues toward the critical examination of how a productive molecular dialog is established between nonsex-specific housekeeping machinery and versatile intersecting developmental pathways, in order to ultimately achieve proper sex-specific gonadogenesis crucial for fertility, and transmission of genetic information (Shore, 2022).

Female copulation song is modulated by seminal fluid

In most animal species, males and females communicate during sexual behavior to negotiate reproductive investments. Pre-copulatory courtship may settle if copulation takes place, but often information exchange and decision-making continue beyond that point. This study shows that female Drosophila sing by wing vibration in copula. This copulation song is distinct from male courtship song and requires neurons expressing the female sex determination factor DoublesexF. Copulation song depends on transfer of seminal fluid components of the male accessory gland. Hearing female copulation song increases the reproductive success of a male when he is challenged by competition, suggesting that auditory cues from the female modulate male ejaculate allocation. These findings reveal an unexpected fine-tuning of reproductive decisions during a multimodal copulatory dialog. The discovery of a female-specific acoustic behavior sheds new light on Drosophila mating, sexual dimorphisms of neuronal circuits and the impact of seminal fluid molecules on nervous system and behavior (Kerwin, 2020).

This study reports a novel acoustic behavior during Drosophila reproduction, female specific copulation song. It occurs in D. melanogaster as well as in its sibling species D. simulans, D. mauritiana and D. sechellia. While the acoustic parameters of male courtship song display marked inter-species differences, female song structure in D. melanogaster, D. simulans and D. mauritiana species is very similar. This is in line with the proposed function of male song as a prezygotic isolating barrier. In contrast, such a function seems unlikely for female copulation song, which occurs after a mating partner has been chosen. The results revise the notion that only male Drosophila melanogaster sings. This study has identified neuronal components of a female song circuit with shared and distinct elements compared to its male counterpart. Silencing of a specific motor output neuronal class required for flight, dlm mns, abolishes female song completely, whereas it only decreases the amplitude of male song. Male song depends critically on dsx+ fru+ neurons, many of which are male specific or sexually dimorphic. For female song, dsx+ fru- neurons of the ventral nerve cord, but not dsx+ fru+ neurons necessary for song. Future work dissecting thoracic circuits will reveal how sex specific wing motor patterning is generated and how neuronal dimorphisms explain the different acoustic parameters of male and female song (Kerwin, 2020).

The composition of male ejaculate, critically affects female singing behavior, whereas the quality of male pre-copulatory courtship (visual, olfactory and gustatory input) is likely to have little impact. Absence of sperm in the presence of seminal fluid increases singing. Males generally depleted of ejaculate or specifically lacking secondary cell products (SCPs) in their seminal fluid barely elicit any female song. More song in the absence of sperm could be due to a potential increase in seminal fluid or a greater accessibility of SCPs, some of which are normally bound to sperm. Alternatively, sperm might suppress female singing. Sperm is transferred in a discrete, ~1 min long bout around 7-8 min after start of copulation. In contrast, seminal fluid transfer starts immediately after the initiation of copulation and is thought to continue until disengagement. This transfer pattern might explain the higher probability of female song at the beginning and end of copulation (Kerwin, 2020).

Since females mutant for two mechanosensory channels expressed in the female reproductive tract (Ppk and Piezo) still sing in copula, it is unlikely that transfer of ejaculate elicits female singing via mechanical stimulation. It is hypothesized that SCPs provide a chemical cue for the female, analogous to female pheromones triggering male courtship. It will be interesting to unravel if a single SCP or a more complex mixture is needed for female song initiation, by which receptor(s) and sensory neurons the transfer is detected and how the signal is relayed to motor patterning circuits (Kerwin, 2020).

These experiments demonstrate that female copulation song influences female remating and reproductive success when females have the possibility to remate. How could hearing female song exert such an effect? Female remating probability is impacted by the composition of male ejaculate. The latter is not fixed, but can be modulated by the male to adjust the amount of seminal fluid and sperm transferred to the presence of rivals and female condition (so-called strategic ejaculate allocation). Since seminal fluid is depleted after several matings, strategic allocation has been predicted to be adaptive for males. Based on these previous findings, it is proposed that female copulation song directly affects male seminal fluid allocation and by this decreases female remating. Since an effect of song playback is seen, it is assumeed that males detect female song with their auditory system. However, this does not rule out that males can also detect female song by mechanosensation. Female wing vibrations during singing might also dissipate pheromones and thereby influence olfaction (Kerwin, 2020).

It is further proposed that a feedback loop might coordinate female singing and fine-tune ejaculate transfer. At the beginning of copulation, male SCPs trigger female song, cueing the male that his partner is responsive to seminal fluid components. During the subsequent course of copulation, female song influences further ejaculate transfer. Here, the function of female song could be to entice allocation of costly components from the male. Alternatively, female song might help to proportion overall ejaculate composition to match individual physiological needs. Females might not be able to predict male ejaculate composition by assessing male pre-copulatory courtship. There is no evidence that females can prematurely terminate copulations. Copulation song might thus be a way for females to give feedback to and influence males with whom they have chosen to copulate, modulating allocation behavior to their benefit (Kerwin, 2020).

This first investigation of female copulation song has not yet unraveled its evolutionary significance, and it is only possible to speculate about potential roles in sexual conflict and sexual selection. This study found evidence that female song can delay remating. So far, it is not known if this is the only or most important function of female singing or might be only a secondary effect of changed ejaculate composition. Delayed remating could be adaptive for females under certain conditions, when it leads to efficient use of the sperm from the first male before it is replaced by the ejaculate of a subsequent mate. This might be in the interest of females, since it allows for mixed paternity and genetic diversity of their offspring (Kerwin, 2020).

In a working model, the SCPs stimulating female singing are not necessarily identical with the seminal fluid components that are differentially transferred. In the future, comprehensive screening of the numerous SCPs which are altered in expression levels in iab-6cocu mutant males, which have defective secondary cells, as well as analysis of seminal fluid composition in song playback vs. silence copulations by ELISA or quantitative proteomics are needed to test these hypotheses. Identifying the factors which are differentially transferred in response to female song is crucial for building hypotheses about the adaptive value of female song (Kerwin, 2020).

In general, it can be in the interest of females to influence ejaculate allocation, receipt of seminal fluid components, and, ultimately, paternity of their offspring by active signaling. This study proposes copulation song as a new mechanism by which male reproductive competition, most likely via male allocation behavior, can be influenced by females (Kerwin, 2020).

A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior

Although males and females largely share the same genome and nervous system, they differ profoundly in reproductive investments and require distinct behavioral, morphological, and physiological adaptations. How can the nervous system, while bound by both developmental and biophysical constraints, produce these sex differences in behavior? This study uncovered a novel dimorphism in Drosophila melanogaster that allows deployment of completely different behavioral repertoires in males and females with minimum changes to circuit architecture. Sexual differentiation of only a small number of higher order neurons in the brain leads to a change in connectivity related to the primary reproductive needs of both sexes-courtship pursuit in males and communal oviposition in females. This study explains how an apparently similar brain generates distinct behavioral repertoires in the two sexes and presents a fundamental principle of neural circuit organization that may be extended to other species (Nojima, 2021).

Sexually reproducing species exhibit sex differences in social interactions to boost reproductive success and survival of progeny. Comparing and contrasting the anatomy, activity, and function of sexually dimorphic neurons in the brain of males and females across taxa are starting to reveal the fundamental principles of neural circuit organization underlying these sex differences in behavior. A variety of alternative neuronal circuit configurations have been proposed to generate sexually dimorphic behaviors. Many studies have identified sex differences in sensory inputs in various species; however, such differences in higher order brain circuits that organize species- and sex-specific instinctive behaviors in response to sensory cues are still poorly characterized (Nojima, 2021).

Sex is determined early in an animal's development and initiates many irreversible sexual differentiation events that influence how the genome and the environment interact to give rise to sex-specific morphology and behavior. In Drosophila, selective expression of two sex determination transcription factors (TFs), Doublesex (Dsx) and Fruitless (Fru), define cell-type-specific developmental programs that govern functional connectivity and lay the foundations through which innate sexual behaviors are genetically predetermined. Because both fru- and dsx-expressing neurons are essential for male and female reproductive behaviors, studies in the adult have focused on neurons that express these TFs to identify anatomical or molecular sex differences in neuronal populations. This allows entry to the neural circuits underlying sex-typical behaviors and identification of the neuronal nodes that control component behaviors and behavioral sequencing (Nojima, 2021).

Dsx proteins, which are part of the structurally and functionally conserved Doublesex and Male-abnormal-3 Related Transcription factors (DMRT) protein family, are critical for sex-specific differentiation throughout the animal kingdom. In the insect phylum, Dsx proteins act at the interface between sex determination and sexual differentiation, regulating a myriad of somatic sexual differences both inside and outside the nervous system. The dsx gene has functions in both sexes: its transcripts undergo sex-specific alternative splicing to encode either a male- or female-specific isoform. dsx expression is highly regulated in both male and female flies, as shown by its temporally and spatially restricted expression patterns through development, with only a select group of neurons expressing dsx. The dsx gene is expressed in some 150 and 30-40 neurons per hemisphere in the male and female brains, which reside in 10 and 7 to 8 discrete anatomical clusters, respectively. This restricted expression of dsx in higher order neurons in the brain suggests these neurons may act as key sex-specific processing nodes of sensory information (Nojima, 2021).

To study the fundamental principles of neural circuit organization underlying sex differences in behavior, this study identified and mapped dsx⁺ sexual dimorphisms in the CNS. This analyses revealed that all dsx⁺ clusters are either sexually dimorphic or sex specific; none are sexually monomorphic. To examine higher order processing differences between the sexes, this study focused on the dsx⁺ anterior dorsal neuron (aDN) cluster, as it is present in both sexes yet has sexually dimorphic dendritic arborizations associated with sensory perception. These anatomical differences lead to sex-specific connectivity, with male aDN inputs being exclusively visual, while female inputs are primarily olfactory. Finally, this study shows that this unique sexually dimorphic neuronal hub that reroutes distinct sensory pathways gives rise to functionally distinct social behaviors between the sexes: visual tracking during courtship in males and communal egg-laying site selection in females (Nojima, 2021).

This study identified a small cluster of two neurons per hemisphere in the central brain, which reconfigures circuit logic in a sex-specific manner. Perhaps most surprising is the seemingly unrelated behaviors these equivalent neurons control in each sex-visual tracking during courtship in males and communal egg laying in females. Ultimately, these circuit reconfigurations lead to the same end result-an increase in reproductive success. These findings highlight a flexible strategy used to structure the nervous system, where relatively minor modifications in neuronal networks allow each sex to respond to their social environment in a sex-appropriate manner (Nojima, 2021).

The behavioral function of the male aDN cluster appears to be related to visual aspects of courtship behavior. A set of visual projection neurons, LC10a, was previously identified as involved in tracking and following behaviors in the male during courtship; however, no apparent sex differences in their anatomy or their physiological responses to visual stimuli were detected. It would seem these sex differences in behavior arise from the sex-specific downstream connectivity of LC10a neurons in the central brain. This study identified aDNs connecting downstream to LC10a in males only. aDN inactivation mirrors visual tracking defects displayed upon LC10a inactivation; therefore, the male aDN cluster confers sex specificity to visually guided tracking of females during courtship (Nojima, 2021).

This study also identified AL5a neurons to be downstream of LC10a in both sexes. Interestingly, it has been reported that AL5a is likely upstream of the fru⁺ cluster Lv2/pIP-b/pIP8 thought to exchange and integrate visual information from the right and left hemispheres of the brain. This male-specific connectivity is compatible with a potential role for AL5a in mediating visual information necessary for wing choice during courtship, a behavior these neurons have been shown to elicit when activated (Nojima, 2021).

The two LC10a downstream clusters that this study identified, aDN and AL5a, also show differences in their anatomical connectivity and physiological responses. Whereas AL5a is downstream of LC10a in both sexes, aDN is only connected to LC10a in the male. Despite direct anatomical connectivity between LC10a and aDN in males, functional connectivity was only uncovered under conditions of pharmacological disinhibition. This observation might hint at inhibitory modulation of aDN that depends on the male's internal state, e.g., his mating drive, or additional cues that influence his courtship arousal. A previous study found that, in sexually satiated males, calcium responses in courtship 'decision-making' P1 neurons were absent when stimulating upstream neurons but could be restored to the levels observed in naive males by application of PTX. It is tempting to speculate that inhibition in the LC10a -> aDN pathway is similarly linked to sexual arousal. In contrast, AL5a responses to LC10a stimulation occurred in the absence of PTX and were markedly larger in AL5a than in aDN. The variation in calcium signals could be due to the considerable difference in cell numbers comprising each cluster (2 aDN versus 24 AL5a) or due to inputs from different AOTu regions. aDNs sample from the whole glomerulus region, whereas the AL5a cluster is restricted to the dorsal part of the AOTu, suggesting they extract information from broad versus specific parts of the visual field, respectively. Future investigation will be aimed at linking the clusters' anatomical differences with their differential processing of visual information to facilitate distinct behavioral roles (Nojima, 2021).

In females, the aDN cluster does not receive visual information but appears to sample from a range of sensory modalities, with information received via the antennal lobe dominating its inputs, suggesting its involvement in a complex behavior requiring multisensory integration. One such behavior is female egg-laying site selection, which is critical to the success of offspring. For Drosophila, offspring survival rates depend on the selection of oviposition sites that are shared with conspecifics, a process known to rely on olfaction (Nojima, 2021).

This study has shown that aDNs are highly integrated into circuitry known to regulate oviposition. The excitatory oviEN, which is anatomically similar to the aDNs, responds to information about substrate suitability via gustatory and mechanosensory cues in the legs and directly influences aDN output. Silencing oviEN function suppresses egg laying itself, whereas silencing aDN does not affect the overall number of eggs laid. Instead, aDN-silenced females are no longer able to show a preference to lay eggs communally, losing a female-specific social behavior essential for offspring survival. While both oviEN and aDN output directly onto the oviposition motor program (through oviDNs), oviENs are the largest contributors to oviDN dendritic budgets, with aDN being relatively minor contributors. Thus, the aDN cluster acts as a modulator of egg laying choice, whereas the oviEN more generally affects the mechanics of egg laying (Nojima, 2021).

As the oviposition of fertilized eggs is a female behavior that can only be displayed after mating, the behavioral programs required are likely inhibited in virgin females. The activity of the inhibitory neuron oviIN depends on female mating status and thus appears to act as a general inhibitor of egg-laying circuitry in virgin females. oviINs form axo-axonic synapses with both the aDN and oviEN, suggesting they gate their outputs by presynaptic inhibition in a state-dependent manner. Intriguingly, as both oviEN and oviIN form axo-axonic synapses with aDN, this suggests a potential gating mechanism by which their relative strengths inhibit or facilitate output from aDN onto downstream targets (Nojima, 2021).

Consistent with aDNs' behavioral function in egg-laying site selection, a female post-mating behavior, this study found differences in the aDN physiological responses in mated versus virgin females. Stimulation of OSNs resulted in significantly stronger aDN calcium responses in mated females compared to virgins. This finding might hint at a state-dependent inhibition of olfactory inputs into aDN in females, potentially analogous to the inhibition of visual inputs to aDN observed in males. The difference in physiological responses between mated and virgin females was not observed when stimulating PNs, which are downstream of OSNs but upstream of aDN. There are different possible explanations for this discrepancy, including differences in the populations of neurons targeted by the driver lines used to target PNs versus OSNs or inhibition in virgin females occurring at the level of OSN to PN connectivity; therefore, activating PNs directly bypasses the state-dependent inhibition. In addition to state-dependent effects, there also seemed to be differences in the calcium responses in different neuronal compartments. This finding could be explained by the position of the input synapses of different upstream neurons into the aDN (e.g., dendritic versus axonic). The exact mechanism of how aDN integrates these different inputs and transforms them into an output that guides egg-laying site selection remains to be examined (Nojima, 2021).

The principal output of the female aDN is the previously undescribed SMP156 neuron, which itself outputs primarily in the IB, where its axons show cross-hemisphere connectivity, suggesting it acts as integrators of sensory information from different directions. The major SMP156 output neuron type (IB011) projects to the lobula in the opposite hemisphere, potentially integrating olfactory and visual information as observed in other flying insects during pheromone orientation. Olfactory navigation requires comparisons of left and right inputs, e.g., when male moths orient themselves toward conspecific females in response to sex pheromones. Determination of position and direction applies to males pursuing females and females following pheromonal cues to locate a communal egg-laying site. It is proposed that the aDN cluster in females selectively integrates sensory information, relaying it to SMP156, which confers directionality and processes information relevant to locating an appropriate egg-laying site. In the absence of a male connectome for comparison, it can only be speculated about potential shared downstream connectivity. As the male aDN output sites are mainly overlapping with female sites in the SMP, it is possible that the male visual pathway also inputs into SMP156, or a similar neuron associated with the IB, potentially feeding back onto visual pathways, supporting appropriate tracking of the female. A male connectome and more genetic tools will help reveal the full extent of downstream functional connectivity and convergence between the sexes (Nojima, 2021).

As fundamental features of most animal species, sexual dimorphisms and sex differences have particular importance for the function of the nervous system. These innate sex-specific adaptations are built during development and orchestrate interactions between sensory information and specific brain regions to shape the phenotype, including the emergent properties of the sex-specific neural circuitry. Evolutionary forces acting on these neural systems have generated adaptive sex differences in behavior. In Drosophila, males compete for a mate through courtship displays, while a female's investment is focused on the success of their offspring. These sex-specific behaviors are guided by the perception and processing of sensory cues, ensuring responses lead to reproductive success. This study has shown how a sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior and provides insight on how similar mechanisms may be implemented in the brains of other sexually dimorphic species (Nojima, 2021).

Doublesex regulates fruitless expression to promote sexual dimorphism of the gonad stem cell niche

Doublesex (Dsx) and Fruitless (Fru) are the two downstream transcription factors that actuate Drosophila sex determination. While Dsx assists Fru to regulate sex-specific behavior, whether Fru collaborates with Dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs. This study reports that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously-studied aspects of sex-specific Fru expression, which is regulated by Transformer (Tra)-mediated alternative splicing, this study shows that male-specific expression of fru in the gonad is regulated downstream of dsx, and is independent of tra. fru genetically interacts with dsx to support maintenance of the niche throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, this study identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the niche, indicating that fru is likely a direct target for transcriptional regulation by Dsx. These findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra (Zhou, 2021).

In sexually reproducing animals, the proper production of gametes and successful copulation are equally critical for reproductive success. It is therefore important that both the gonad and the brain know their sexual identity. The Doublesex/Mab-3 Related Transcription Factors act downstream of sex determination and play an evolutionarily conserved role to establish and maintain sexual dimorphism in the gonad. Meanwhile, sexual dimorphism in other tissues such as the brain is controlled, to varying degrees in different animals, through autonomous control by the sex determination and non-autonomous signaling from the gonads. In many invertebrate species, another sex-determination gene fruitless (fru), which encodes multiple BTB-Zinc finger transcription factors, plays a central role in controlling mate choice, courtship behavior and aggression. How sex determination in the gonad and the nervous system are related and coordinated in these species remains unclear (Zhou, 2021).

The founding member of the DMRT family is Drosophila doublesex (dsx). dsx and fru undergo sex-specific alternative mRNA splicing by the sex determination factor Transformer (Tra), together with its co-factor Transformer-2 (Tra-2), to produce transcripts encoding sex-specific protein isoforms. It was once thought that dsx controls sexual dimorphism outside the nervous system while fru regulates sex-specific nervous system development and behavior. But more recent evidence shows that dsx cooperates with fru to specify sex-specific neural circuitry and regulate courtship behaviors. However, whether fru acts along with dsx to control sexual dimorphism outside the nervous system remains unknown (Zhou, 2021).

The fru gene locus contains a complex transcription unit with multiple promoters and alternative splice forms (see Fruitless is expressed male-specifically in the germline stem cell niche and is independent of Fru^M). Sex-specific regulation of fru was only known to occur through alternative splicing of transcripts produced from the P1 promoter, which produces the FruM isoforms. The downstream promoters (P2-P4) produce Fru isoforms (collectively named FruCom) encoded by transcripts that are common to both sexes and are required for viability in both males and females. fru P1 transcripts have only been detected in the nervous system, suggesting that sex-specific functions of fru are limited to neural tissue. However, FruCom is expressed in several non-neural tissues, including sex-specific cell types of the reproductive system. Further, from a recent genome-wide search for putative Dsx targets, fru was identified as a candidate for transcriptional regulation by Dsx. These data raise the possibility that fru functions cooperatively with dsx to regulate gonad development (Zhou, 2021).

Over the past decades, much effort has been focused on understanding the functions of fru in regulating sex-specific behaviors, yet it remained unclear whether fru plays a role in regulating sexual dimorphism outside the nervous system. The work presented in this study demonstrates that Fru is expressed male-specifically in the gonad stem cell niche, and is required for CySC maintenance, cyst cell survival, and for the maintenance of the hub during larval development. Further, male-specific expression of Fru in the gonad is independent of the previously described mechanism of sex-specific alternative splicing by Tra, and is instead dependent on dsx. fru appears to be a direct target for transcriptional regulation by Dsx. This work provides evidence that fru regulates sex-specific development outside the nervous system and alters traditional thinking about the structure of the Drosophila sex determination pathway (Zhou, 2021).

While it was previously reported that fru is expressed in tissues other than the nervous system, including in the gonad, a function for fru outside the nervous system was previously unknown. This study found that Fru is expressed in the developing and adult testis in the hub, the CySC, and the early developing cyst cells. Importantly, it was found that fru is important for the proper function of these cells (Zhou, 2021).

Fru is not expressed at the time of hub formation during embryogenesis, but expression is initiated during the L2/L3 larval stage. This correlates with a time period when the hub must be maintained and resist transforming into female niche structures: in dsx mutants, all gonads in XX and XY animals develop hubs, but in half of each, hubs transform into terminal filament cells and cap cells. fru is not required for initial hub formation, consistent with it not being expressed at that time. fru is also not, by itself, required for hub maintenance under the conditions that were possible to assay (prior to the pupal lethality of fru null mutant animals). However, under conditions where hub maintenance is compromised by loss of dsx function, fru clearly plays a role in influencing whether a gonad will retain a hub, or transform into TF. Fru expression in dsx mutant gonads correlates with whether they formed male or female niche structures, and removing even a single allele of fru is sufficient to induce more hubs to transform into TFs. Finally, ectopic expression of Fru in females is sufficient to inhibit TF formation and partially masculinize the gonad, but does not induce hub formation. Thus, it is proposed that fru is one factor acting downstream of dsx in the maintenance of the male gonad stem cell niche, but that it acts in combination with other factors that also regulate this process (Zhou, 2021).

This study also demonstrated that fru is required for CySC maintenance and for the survival of differentiating cyst cells. Loss of fru from the CySC lineage led to rapid loss of these CySCs from the testis niche. Since precocious differentiation of CySCs or an increase in their apoptosis was not observed, these mechanisms do not appear to contribute to CySC loss. One possibility is that fru is needed for CySCs to have normal expression of adhesion proteins and compete with other stem cells for niche occupancy. It has been shown that fru regulates the Slit-robo pathway and that robo1 is a direct target of fru in the CNS. Interestingly, the Slit-Robo pathway also functions in the CySCs to modulate E-cadherin levels and control the ability of CySCs to compete for occupancy in the niche. Therefore, fru may use similar mechanisms to maintain CySC attachment to the hub. fru also influences survival in the differentiating cyst cells, as an increase in cell death was observed in these cells in fru mutants. Several reports have demonstrated that fru represses programmed cell death in the nervous system. It was further indicated that the cell death gene reaper is a putative target of Fru. Thus, fru may play a role in repressing the apoptosis of cyst cells (Zhou, 2021).

In summary, fru function is clearly important for male niche maintenance and the function of the CySCs and their differentiating progeny. This provides clear evidence that fru regulates sex-specific development in tissues other than the nervous system. Whether additional tissues are also regulated by fru remains to be determined (Zhou, 2021).

Previously, it was thought that the only mechanism by which sex-specific functions of fru were regulated was through Tra-dependent alternative splicing of the P1 transcripts. fru null alleles are lethal in both sexes and Fru proteins derived from non-P1 promoters were thought to be sex-nonspecific and not to contribute to sex determination. Thus, fru and dsx were considered as parallel branches of the sex determination pathway, each independently regulated by Tra. This study demonstrates that fru can also be regulated in a manner independent of tra and dependent on dsx, and provides evidence that fru is a direct target for transcriptional regulation by Dsx (see Proposed model of the Drosophila sex determination pathway. First, Fru expression in the testis is independent of the P1 transcript that is regulated by Tra. A P1 Gal4 reporter is not expressed in the testis and a mutation that prevents FruM expression from P1 does not affect Fru immunoreactivity in the testis. Second, in animals that simultaneously express the female form of tra (Tra on) and the male form of Dsx [XX; dsxD/Df(3R)dsx3], Fru is expressed in the male mode in the testis, demonstrating that it is regulated by dsx and not tra. Finally, an evolutionarily conserved Dsx consensus binding site upstream of the P4 promoter is required for proper expression levels of a fru P4 reporter in the testis. Together, these data demonstrate a novel mode for fru regulation by the sex determination pathway, where sex-specific expression of fru is regulated by dsx. It also means that the large number of fru transcripts that do not arise from the P1 promoter can be expressed in a sex-specific manner to contribute to sexual dimorphism (Zhou, 2021).

The male and female forms of Dsx contain the same DNA binding domain and can regulate the same target genes, but often have opposite effects on gene expression. Prior to this study, the documented Dsx targets (Yolk proteins 1 and 2, bric-a-brac and desatF), along with other proposed targets, were all expressed at higher levels in females than males. Thus, for these targets, DsxF acts as an activator and DsxM acts as a repressor (or DsxM has no role). Interestingly, fru is the first identified Dsx target that is expressed in a male-biased manner. Thus, for direct regulation of fru, DsxM would activate expression while DsxF represses. Mechanistically for Dsx, this implies that the male and female isoforms are not dedicated repressors and activators, respectively, but may be able to switch their mode of regulation in a tissue-specific or target-specific manner. Mouse DMRT1 has also been shown to regulate gene expression both as transcriptional activator and repressor. Thus, it is quite possible that bifunctional transcriptional regulation is a conserved characteristic of DMRTs (Zhou, 2021).

It is possible that dsx regulation of fru occurs in the nervous system as well, where it co-exists with direct regulation of fru alternative splicing by Tra. It was originally thought that alternative splicing of the fru P1 transcript by tra was essential for male courtship behavior. However, more recently it was found that these animals could exhibit male courtship behavior if they were simply stimulated by other flies prior to testing. Interestingly, the courtship behavior exhibited by these males was dependent on dsx. It is proposed that fru might still be essential for male courtship in these fru P1-mutants, but that sex-specific fru expression is dependent on transcriptional regulation of other fru promoters by Dsx (Zhou, 2021).

If sex-specific fru function can be regulated both through alternative splicing by Tra and through transcriptional regulation by Dsx, it raises the question of what is the relationship between these two modes of regulation? It is proposed that regulation of fru by Dsx is the more ancient version of the sex determination pathway and that additional regulation of fru by Tra evolved subsequently, through the acquisition of regulatory RNA elements in the fru P1 transcript. This model is supported by studies of fru gene structures in distantly related Dipteran species, and species of other insect orders, that illustrate the considerable variability in the organization of sequences controlling fru splicing. Further, in some insects, no evidence for alternative splicing of fru has been found, yet fru still plays an important role in males to control courtship behaviors. Finally, in the Hawaiian picture-winged group of subgenus Drosophila, the fru orthologues lack the P1 promoter, and non-P1 fru transcripts exhibit male-specific expression, similar to what is proposed for non-P1 fru transcripts in D. melanogaster. Thus, it appears that regulation of fru by dsx may be the evolutionarily more ancient mechanism for sex-specific control of fru, while Tra-dependent splicing of P1 transcripts is a more recent adaptation. More broadly, tra is not conserved in the sex determination pathway in the majority of animal groups, while homologs of Dsx, the DMRTs, are virtually universal in animal sex determination. Thus, if Fru orthologs are involved in the creation of sexual dimorphism in the body or the brain in other animals, they cannot be regulated by Tra but may be regulated by DMRTs (Zhou, 2021).

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 Dsx^F and Dsx^M 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 Dsx^F-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).

A double-negative gene regulatory circuit underlies the virgin behavioral state

Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, it was recently shown that loss of miR-iab-4/miR-iab-8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. This study used genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 -| Hth -| Dsx) specifies the virgin behavioral state (Garaulet, 2021).

Females of diverse invertebrate and vertebrate species coordinate multiple behavioral programs with their reproductive state. Mature female virgins are receptive to male courtship and copulation, but following mating and/or pregnancy, they decrease sexual activity and modulate behaviors to generate and foster their children. Behavioral remodeling associated with the female reproductive state includes increased aggression and nest building in avians and mammals and decreased male acceptance, increased egg-laying, and appetitive/metabolic changes in insects. The genetic and neurological control of this process has been intensively studied in fruit flies, where sexual activity induces the post-mating switch, a host of behavioral changes collectively known as post-mating responses (PMRs) (Garaulet, 2021).

In Drosophila, as in other species, 'virgin' is typically considered the default behavioral state, because factors that induce PMRs are transferred in seminal fluids during copulation. Among these, Sex Peptide (SP) is necessary and sufficient to drive most female post-mated behaviors. SP signals via uterine SP sensory neurons (SPSNs). Some SPSN+ neurons contact abdominal interneurons in the ventral nerve cord (VNC) that express myoinhibitory peptide, which input into a restricted population of ascending neurons (SP abdominal ganglion [SAG] neurons) that project to the posterior brain, including pC1 neurons. This outlines an ascending flow of information for how a seminal fluid peptide can alter female brain activity. The brain integrates this with auditory and visual cues to coordinate diverse behaviors mediated by distinct lineages of descending neurons and VNC populations that modulate specific behaviors according to internal state and external stimuli (Garaulet, 2021).

Recently, it was found that post-transcriptional suppression of the homeobox gene homothorax (hth) within the VNC is critical to implement the virgin behavioral state (Garaulet, 2020). Of note, deletion of the Bithorax Complex (BX-C) locus mir-iab-4/8, point mutations of their binding sites in hth, or deletion of the hth neural-specific 3' UTR extension bearing many of these microRNA (miRNA) sites all cause mutant female virgins to perform mated behaviors. Thus, the failure to integrate two post-transcriptional regulatory inputs at a single target gene prevents females from appropriately integrating their sexual internal state with external behaviors (Garaulet, 2021).

Recognition of the transcription factor Hth as a target of regulatory circuits for virgin behavior implies that downstream loci may serve as a functional output for this process. This study used molecular genetic profiling to identify a critical requirement for Doublesex (Dsx) to implement the female virgin behavioral state. Dsx has been well studied with respect to differentiation of sexually dimorphic traits, but its roles in post-mitotic neurons are little known. This study found that expression of Dsx in the VNC mediates virgin behavior, and that modulation of Dsx in only a few abdominal VNC neurons is sufficient to convert the suite of female virgin behaviors into mated conducts (Garaulet, 2021).

Recent work established how miRNA mediated suppression of the transcription factor Hth to safeguard the virgin female behavioral state (Garaulet, 2020). Using engineered alleles and spatio-temporal hth manipulations, this study demonstrated a developmental requirement for post-transcriptional regulation of Hth within the abdominal ganglion of the CNS for female behavior. However, Hth was not required in otherwise wild-type VT-switch neurons for execution of virgin behaviors, implying that expression of Hth in the abdominal VNC must normally be prevented. This involves integration of two mechanisms: a high density of BX-C miRNA binding sites (miR-iab-4/8) within the hth-HD 3' UTR, as well as a neural-specific 3' UTR elongation, which unveils many of these sites only on neural hth isoforms (Garaulet, 2021).

This study has extended this regulatory axis by showing that loss of BX-C miRNAs, acting through derepressed Hth, leads to downregulation of the Dsx in the abdominal VNC. Dsx is well-known as a master sex determination transcription factor, and it shows localized expression in specific CNS domains. However, although the activity of Dsx-expressing neurons per se has been implicated in the switch in females, the functions of Dsx in post-mitotic neurons are less well defined. This work reveals that Dsx itself is a central component in specifying virgin behavior, because its restricted suppression in as few as four (SAG-1+) neurons is sufficient to induce post-mated behaviors. It remains to be better defined how SAG-1 neurons are affected by depletion of Dsx. No overt differentiation defects were observed, but an effect of masculinization cannot be ruled out. Otherwise, the recent work suggests an activity defect in a general population of switch neurons in the miRNA mutant (Garaulet, 2020), but more direct analysis of dsx-depleted SAG-1 neurons awaits (Garaulet, 2021).

Altogether, in contrast with highly branched regulatory networks that are bioinformatically inferred to lie downstream of individual miRNAs, this study revealed a linear, double-negative regulatory cascade comprising miRNAs and two transcription factors (see SAG-1 neurons specifically require Dsx for a suite of female virgin behaviors). These findings provide impetus to assess possible direct regulation of Dsx by Hth, as well as to elucidate Dsx targets that are relevant to female behavioral control. Overall, this study expands a genetic hierarchy that is essential for females to couple the virgin internal state with appropriate behaviors (Garaulet, 2021).

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).

Co-option of the bZIP transcription factor Vrille as the activator of Doublesex1 in environmental sex determination of the crustacean Daphnia magna

Divergence of upstream regulatory pathways of the transcription factor Doublesex (Dsx) serves as a basis for evolution of sex-determining mechanisms in animals. However, little is known about the regulation of Dsx in environmental sex determination. In the crustacean Daphnia magna, environmental sex determination is implemented by male-specific expression of the Dsx ortholog, Dsx1. Transcriptional regulation of Dsx1 comprises at least three phases during embryogenesis: non-sex-specific initiation, male-specific up-regulation, and its maintenance. This study demonstrates that the male-specific up-regulation is controlled by the bZIP transcription factor, Vrille (Vri), an ortholog of the circadian clock genes-Drosophila Vri and mammalian E4BP4/NFIL3. Sequence analysis of the Dsx1 promoter/enhancer revealed a conserved element among two Daphnia species (D. magna and D. pulex), which contains a potential enhancer harboring a consensus Vri binding site overlapped with a consensus Dsx binding site. Besides non-sex-specific expression of Vri in late embryos, male-specific expression was found in early gastrula before the Dsx1 up-regulation phase begins. Knockdown of Vri in male embryos showed reduction of Dsx1 expression. In addition, transient overexpression of Vri in early female embryos up-regulated the expression of Dsx1 and induced male-specific trait. Targeted mutagenesis using CRISPR/Cas9 disrupted the enhancer in males, which led to the reduction of Dsx1 expression. These results indicate that Vri was co-opted as a transcriptional activator of Dsx1 in environmental sex determination of D. magna. The data suggests the remarkably plastic nature of gene regulatory network in sex determination (Mohamad Ishak, 2017).

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, DSX^F inhibits Wingless and FGF pathway activity and DSX^M 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).

Ubiquitin-dependent regulation of a conserved DMRT protein controls sexually dimorphic synaptic connectivity and behavior

Sex-specific synaptic connectivity is beginning to emerge as a remarkable, but little explored feature of animal brains. This study describes a novel mechanism that promotes sexually dimorphic neuronal function and synaptic connectivity in the nervous system of the nematode Caenorhabditis elegans. A phylogenetically conserved, but previously uncharacterized Doublesex/Mab-3 related transcription factor (DMRT), dmd-4, is expressed in two classes of sex-shared phasmid neurons specifically in hermaphrodites but not in males. dmd-4 promotes hermaphrodite-specific synaptic connectivity and neuronal function of phasmid sensory neurons. Sex-specificity of DMD-4 function is conferred by a novel mode of posttranslational regulation that involves sex-specific protein stabilization through ubiquitin binding to a phylogenetically conserved but previously unstudied protein domain, the DMA domain. A human DMRT homolog of DMD-4 is controlled in a similar manner, indicating that these findings may have implications for the control of sexual differentiation in other animals as well (Bayer, 2020).

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).

Phylogenetic analysis and embryonic expression of panarthropod Dmrt genes

One set of the developmentally important Doublesex and Male-abnormal-3 Related Transcription factors (Dmrt) is subject of intense research, because of their role in sex-determination and sexual differentiation. This likely non-monophyletic group of Dmrt genes is represented by the Drosophila melanogaster gene Doublesex (Dsx), the Caenorhabditis elegans Male-abnormal-3 (Mab-3) gene, and vertebrate Dmrt1 genes. However, other members of the Dmrt family are much less well studied, and in arthropods, including the model organism Drosophila melanogaster, data on these genes are virtually absent with respect to their embryonic expression and function. This study investigate the complete set of Dmrt genes in members of all main groups of Arthropoda and a member of Onychophora, extending the data to Panarthropoda as a whole. The presence of at least four families of Dmrt genes (including Dsx-like genes) in Panarthropoda is confirmed, and their expression profiles during embryogenesis were examined. This work shows that the expression patterns of Dmrt11E, Dmrt93B, and Dmrt99B orthologs are highly conserved among panarthropods. Embryonic expression of Dsx-like genes, however, is more derived, likely as a result of neo-functionalization after duplication. These data suggest deep homology of most of the panarthropod Dmrt genes with respect to their function that likely dates back to their last common ancestor. The function of Dsx and Dsx-like genes which are critical for sexual differentiation in animals, however, appears to be much less conserved (Panara, 2019).

Transgenic and knockout analyses of Masculinizer and doublesex illuminated the unique functions of doublesex in germ cell sexual development of the silkworm

Masculinizer (Masc) plays a pivotal role in male sex determination in the silkworm, Bombyx mori. Masc is required for male-specific splicing of B. mori doublesex (Bmdsx; see Drosophila Dsx) transcripts. The male isoform of Bmdsx (BmdsxM) induces male differentiation in somatic cells, while females express the female isoform of Bmdsx (BmdsxF), which promotes female differentiation in somatic cells. Previous findings suggest that Masc could direct the differentiation of genetically female (ZW) germ cells into sperms. However, it remains unclear whether Masc directly induces spermatogenesis or if it promotes male differentiation in germ cells indirectly by inducing the expression of BmdsxM. In this study, genetic analyses were performed using the transgenic line that expressed Masc, as well as various Bmdsx knockout lines. Masc-expressing females with a homozygous mutation in BmdsxM showed normal development in ovaries. The formation of testis-like tissues was abolished in these females. On the other hand, Masc-expressing females carrying a homozygous mutation in BmdsxF exhibited almost complete male-specific development in gonads and germ cells. These results suggest that BmdsxM has an ability to induce male development in germ cells as well as internal genital organs, while BmdsxF inhibits BmdsxM activity and represses male differentiation. To investigate whether MASC directly controls male-specific splicing of Bmdsx and identify RNAs that form complexes with MASC in testes, RNA immunoprecipitation (RIP) was performed using an anti-MASC antibody. MASC was found to form a complex with AS1 lncRNA, which is a testis-specific factor involved in the male-specific splicing of Bmdsx pre-mRNA. Taken together, these findings suggest that Masc induces male differentiation in germ cells by enhancing the production of BmdsxM. Physical interaction between MASC and AS1 lncRNA may be important for the BmdsxM expression in the testis. Unlike in the Drosophila dsx, BmdsxM was able to induce spermatogenesis in genetically female (ZW) germ cells. This is the first report that the role of dsx in germ cell sexual development is different between insect species (Yuzawa, 2020).

Dmrt genes participate in the development of Cajal-Retzius cells derived from the cortical hem in the telencephalon

During development, Cajal-Retzius (CR) cells are the first generated and essential pioneering neurons that control neuronal migration and arealization in the mammalian cortex. CR cells are derived from specific regions within the telencephalon, that is, the pallial septum in the rostromedial cortex, the pallial-subpallial boundary, and the cortical hem (CH) in the caudomedial cortex. However, the molecular mechanism underlying the generation of CR cell subtypes in distinct regions of origin is poorly understood. This study found that double-sex and mab-3 related transcription factor (Dmrt) genes, that is, Dmrta1 and Dmrt3, were expressed in the progenitor domains that produce CR cells. The number of CH-derived CR cells was severely decreased in Dmrt3 mutants, especially in Dmrta1 and Dmrt3 double mutants. The reduced production of the CR cells was consistent with the developmental impairment of the CH structures in the medial telencephalon from which the CR cells are produced. It is concluded that Dmrta1 and Dmrt3 cooperatively regulate patterning of the CH structure and production of the CR cells from the CH during cortical development (Kikkawa, 2020).

Loss of Dmrt5 affects the formation of the subplate and early corticogenesis

Dmrt5 (Dmrta2) and Dmrt3 are key regulators of cortical patterning and progenitor proliferation and differentiation. This study shows an altered apical to intermediate progenitor transition, with a delay in SP neurogenesis and premature birth of Ctip2+ cortical neurons in Dmrt5-/- mice. In addition to the cortical progenitors, DMRT5 protein appears present in postmitotic subplate (SP) and marginal zone neurons together with some migrating cortical neurons. The altered split of preplate and the reduced SP and disturbed radial migration of cortical neurons into cortical plate in Dmrt5-/- brains and demonstrated an increase in the proportion of multipolar cells in primary neuronal cultures from Dmrt5-/- embryonic brains. Dmrt5 affects cortical development with specific time sensitivity that is described in two conditional mice with slightly different deletion time. A transient SP phenotype is observed at E15.5, but not by E18.5 after early (Dmrt5lox/lox;Emx1Cre), but not late (Dmrt5lox/lox;NestinCre) deletion of Dmrt5-/-. SP was less disturbed in Dmrt5lox/lox;Emx1Cre and Dmrt3-/- brains than in Dmrt5-/- and affects dorsomedial cortex more than lateral and caudal cortex. This study demonstrates a novel function of Dmrt5 in the regulation of early SP formation and radial cortical neuron migration. This study demonstrates a novel function of Dmrt5 in regulating marginal zone and subplate formation and migration of cortical neurons to cortical plate (Ratie, 2020).

Dmrt factors determine the positional information of cerebral cortical progenitors via differential suppression of homeobox genes

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. This study reports that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, Dmrt3 and Dmrta2 show a medial(high)/lateral(low) expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. It was also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. These findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex (Konno, 2019).

date revised: 1 March 2024

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