doublesex


DEVELOPMENTAL BIOLOGY

Embryonic

Sex-specific apoptosis, dependent on doublesex, regulates sexual dimorphism in the Drosophila embryonic gonad

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B: Loh, 2000), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).

To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).

During Drosophila embryogenesis, Sox100B is expressed in a number of cell types, including the gonad (Loh, 2000). Since Sox100B is closely related to Sox9 (an important sex determination factor in humans and mice), whether Sox100B expression is sexually dimorphic in Drosophila was tested. Interestingly, it was found that after gonad coalescence (stage 15), Sox100B expression in the gonad is male-specific. Sox100B immunoreactivity is not observed in the coalesced female gonad, whereas it is detected in a posterior cluster of SGPs in the male gonad. While this expression pattern is seen in most wild-type backgrounds (including Canton-S and faf-lacZ), in certain 'wild-type' lines, such as w1118, a few Sox100B-positive cells are observed in the posterior of the coalesced female gonad (however, this is still clearly distinguishable from the number of Sox100B-positive cells in the male). Unlike Eya and Wnt-2, Sox100B is not expressed in all SGPs, since it is usually absent from female gonads and from the anterior region of the male gonad and does not colocalize with the SGP marker 68-77. Sox100B expression appears restricted to the posterior cluster of SGPs that is observed only in the male gonad. Thus, like Sox9 expression in vertebrates, Sox100B exhibits a male-specific pattern of expression in the Drosophila embryonic gonad, suggesting that it may indeed be an ortholog of Sox9 (DeFalco, 2003).

After having identified sexually dimorphic markers of the embryonic gonad, these markers were used to investigate how sexual dimorphism is established. It was asked whether proper gonad formation is necessary for the establishment of sexual dimorphism by examining Sox100B expression in fear-of-intimacy (foi) mutant embryos. In foi mutants, germ cells migrate and associate normally with the SGPs, but these two cell types fail to coalesce into a round and compact gonad. Despite the failure of gonad coalescence, a cluster of Sox100B-expressing cells was still observed at the posterior of the male gonad, while no Sox100B-expressing cells are observed in the female at this stage. Whether the presence of germ cells is necessary for the establishment of sexual dimorphism in the embryonic gonad was examined. Embryos that lack germ cells due to a hypomorphic mutation in oskar, a gene required for germ cell formation, were examined. Other aspects of embryonic development occur normally in these embryos, including the formation and coalescence of the SGPs. Agametic gonads show identical sexual dimorphism to wild-type embryos. Sox100B is coexpressed with Eya in the cluster of somatic cells in the posterior of the male gonad, but Sox100B expression is not observed in the female gonad. Thus, sexual dimorphism of the embryonic somatic gonad does not require proper gonad morphogenesis or the presence of germ cells (DeFalco, 2003).

The posterior cluster of Eya and Sox100B coexpressing cells could result from sex-specific differences in gene expression within the cells of the gonad. Alternatively, it could reflect a difference in gonad morphology, in which these cells are only present in males and not in females. To distinguish between these possibilities, the morphology of the male and female coalesced (stage 15) gonad was examined, using approaches that do not depend on cell-type-specific SGP markers. First, a CD8-GFP fusion protein was expressed broadly in the mesoderm. The fusion of the extracellular and transmembrane regions of mouse CD8 with GFP allows for visualization of cell and tissue morphology. A cluster of mesodermal cells is consistantly observed attached to the posterior of the male gonad that is not observed in the female. In blind experiments, the sex of the embryo can be predicted based on the presence of this posterior cluster of cells. Male and female gonads were also examined by transmission electron microscopy (TEM). Male and female embryos were first sorted using an X chromosome-linked GFP expression construct and then processed separately for TEM. In this analysis, a cluster of cells that is not present in the female gonad was consistently at the posterior of the male gonad. Both the size and morphology of these cells indicate that they are somatic cells rather than germ cells. Thus, the observed sexual dimorphism reflects a change in gonad morphology, not just a change in gene expression. Since the additional cells at the posterior of the male gonad express at least some markers in common with SGPs (e.g., Eya), these cells are referred to as male-specific SGPs (msSGPs) (DeFalco, 2003).

Since no sex-specific differences were observed in SGP proliferation in the gonad, it seems unlikely that the SGPs are dividing to produce the msSGPs. Therefore, Sox100B was used as a marker for the msSGPs to determine where and when these cells are first specified. At stages prior to gonad coalescence (stages 12 and 13), a cluster of Eya/Sox100B double-immunopositive cells is observed posterior and ventral to the developing clusters of SGPs, which express Eya alone. Interestingly, this cluster of Eya/Sox100B double-positive cells is initially observed in both males and females and appears identical, although Eya expression may be somewhat lower in the female cluster. During stage 13, as the SGPs and germ cells associate closely along PS 10-12, the Eya/Sox100B double-positive cells move toward the gonad in both sexes. In males, these cells join the posterior of the coalescing gonad. In contrast, these cells do not join the gonad in females, and only Eya-positive, Sox100B-negative cells are found in the coalesced gonad. It is concluded that the Eya/Sox100B double-positive cells are the msSGPs and that they form separately from the SGPs. These cells are initially specified in both males and females and move anteriorly to join the gonad in males. In females, these cells do not form part of the gonad, as judged by the above morphological analysis, and are no longer detected using available markers (DeFalco, 2003).

Since the msSGPs develop separately from the SGPs, it was of interest to address where the msSGPs arise and what controls their specification. By marking the anterior of each parasegment using an antibody against Engrailed, it was determined that the msSGPs are specified in PS13. This observation is consistent with these cells arising posterior to the SGPs, which form in PS 10, 11, and 12. Other Sox100B expression is observed in nongonadal tissues. Whether, like the SGPs, the msSGPs are specified in the dorsolateral domain of the mesoderm was also addressed. Mesodermal cell types that form in this region, such as the SGPs and the fat body, require the homeodomain proteins Tinman and Zfh-1 for their specification. However, in embryos double-mutant for tinman and zfh-1, the msSGPs are still specified, even though the SGPs fail to develop. Thus, msSGPs do not arise from the dorsolateral domain, consistent with the fact that the msSGPs are first observed in a position ventral to the SGPs. The msSGPs also differ from the SGPs in terms of their requirements for the homeotic gene abd-A. SGP specification absolutely requires abd-A, while msSGPs are still present in these mutants. Thus, despite the fact that the msSGPs and the SGPs share expression of some molecular markers such as Eya and Wnt-2, their specification is under independent control (DeFalco, 2003).

Since the msSGPs express both Eya and Sox100B, the requirements for each of these genes in msSGP specification was investigated. In eya mutants, Sox100B-positive cells are still observed posterior to the germ cells at early stages, in a position where the msSGPs normally develop. Since the SGPs are not maintained in these mutants, the germ cells disperse and the gonad does not coalesce. Therefore, it is impossible to tell if the msSGPs would join the posterior of the male gonad in eya mutants. However, initial msSGP specification does not require eya. Similarly, in a deletion that removes the Sox100B locus, a large cluster of Eya-positive cells was still observed at the posterior of the male gonad that does not appear in females. Thus, the initial development of the msSGPs does not require Sox100B. Expression of Eya and Sox100B are mutually independent and are likely to be downstream of factors controlling initial msSGP specification (DeFalco, 2003).

Since the msSGPs are initially specified in both males and females, a determination was made of how these cells receive information about their sexual identity that allows them to behave differently in the two sexes. tra plays a key role in the sex determination pathway in Drosophila and is required to promote female differentiation in somatic tissues. tra mutant gonads were examined to test if tra function is required for gonad sexual dimorphism (XX embryos are masculinized by mutations in tra). Sox100B-immunopositive cells are observed in the posterior somatic gonad of both XX and XY tra mutant embryos in a manner comparable to wild-type males. Analysis of the Sox100B expression pattern in the gonad reveals that there are no differences between XX and XY tra mutants, or between either of these genotypes and wild-type males. Conversely, when Transformer is expressed in XY embryos (UAS-traF, tubulin-GAL4), Sox100B-immunopositive cells are no longer observe in these gonads, and they now appear similar to wild-type females (DeFalco, 2003).

In most somatic tissues, the principle sex determination factor downstream of tra is dsx. Unlike tra, dsx is required for both the male and female differentiation pathway, since both XX and XY dsx mutant adults show an intersexual phenotype. However, in the somatic gonad, dsx mutant XY embryos are indistinguishable from wild-type males and show no change in Sox100B expression. Thus, unlike in most somatic tissues, this early characteristic of male development does not require dsx. In XX embryos that are mutant for dsx, a completely masculinized phenotype is observed, in which Sox100B expression in the gonad is similar to a wild-type male. When a dominant allele of dsx, dsxD, is used to express DsxM (dsxD/dsx) in XX embryos, these gonads are no more masculinized than dsx null XX gonads. Therefore, while DsxF is required for the proper female phenotype in XX gonads, it appears that the male Sox100B expression pattern is the 'default' state in the absence of dsx function (DeFalco, 2003).

Since the msSGPs join the posterior of the male gonad but are no longer detected in the female, the basis for the sexually dimorphic behavior of these cells was investigated. In the female, these cells could turn off Sox100B and Eya and contribute to some other tissue, or they might be eliminated altogether. To test this latter hypothesis, whether msSGPs are eliminated by sex-specific programmed cell death in the female was addressed. Since programmed cell death occurs in a caspase-dependent manner, the gonad phenotype was examined in embryos in which caspase activity was inhibited by expressing the baculovirus p35 protein in the mesoderm. In these embryos, XX gonads now appear masculinized; Sox100B-positive cells (msSGPs) persist and join the posterior of female gonads, and coexpress Eya, as in wild-type male embryos. There are not as many Sox100B-positive cells in females as in males, suggesting that p35 may not be completely suppressing cell death. The presence of such cells in the female gonad does not appear to drastically affect ovary formation or oogenesis, since embryos develop into fertile adult females (DeFalco, 2003).

To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).

It is concluded that proper information from the sex determination pathway is required to control the sexually dimorphic behavior of the msSGPs. The female phenotype in the embryonic gonad is dependent on both tra and dsx. Interestingly, it seems that the male phenotype is the default state; in the absence of any tra or dsx function, msSGPs in both XX and XY embryos behave as in wild-type males. This is a different situation than in most other tissues, in which dsx is required in both sexes to promote proper sexual differentiation. In particular, while no role is found for DsxM in this process, DsxF is positively required either to establish the female fate in the posterior somatic gonad or to repress the male fate. This role for DsxF in msSGP development is analogous to its role in the genital disc, in which DsxF is required to block recruitment of btl-expressing cells into the disc; in both cases, dsx female function serves to repress incorporation of a male-specific cell type. Since the msSGPs are initially specified in a sex-independent manner, this may account for the fact that the persistence of these cells (the male phenotype) is the default state. It will be of interest in the future to address the role of the msSGPs in testis development, and how genes such as dsx, eya, and Sox100B act in this process (DeFalco, 2003).

Although sex determination schemes vary widely in the animal kingdom, there is evidence that the molecular and cellular pathways used to control sexual dimorphism may be conserved, even between vertebrates and invertebrates. One example is Sox9, which has been implicated as an ancestral sex-determining gene in vertebrates given its male-specific gonad expression in diverse species such as human, mouse, turtle, and chicken. A potential Drosophila ortholog of Sox9, Sox100B, is expressed in a male-specific manner in the embryonic somatic gonad. The manner of Sox100B expression is reminiscent of that in the mouse; Sox9 is initially expressed in both sexes, but is maintained and upregulated in the male gonad. It will be very interesting to compare the role that Sox100B plays in the development of the Drosophila testis to the one played by Sox9 in vertebrates (DeFalco, 2003).

Molecular conservation is also observed amongst the members of the Dsx/Mab-3 Related Transcription Factor (DMRT) family. DMRT family members have been shown to be essential for sex-specific development in Drosophila (Dsx), C. elegans (mab-3), medaka fish (DMY), and mice (DMRT1) and have been implicated in human sex reversal. This study demonstrates that dsx is essential for proper sex-specific development of the msSGPs. Thus, increasing evidence indicates that DMRT family members are also conserved regulators of sexual dimorphism (DeFalco, 2003).

Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or 'niches', which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. This study investigates the formation of the testis GSC niche, or 'hub', during the late stages of embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).

The evidence indicates that an embryonic hub, which appears to give rise to the adult hub and create the male GSC niche, forms during the late stages of embryogenesis. A subset of anterior SGPs initiates expression of several molecular markers that are also expressed in the adult hub. These SGPs segregate into a tight cluster in a distinct region of the gonad, and a subset of germ cells organizes around these SGPs in a manner similar to the organization of GSCs around the adult hub. Since spermatogenesis begins by early larval stages, it is possible that the embryonic hub already forms a functional GSC niche. The formation of the hub, or indeed any stem cell niche, can be divided into the distinct issues of niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).

The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression during stage 17 also express every other marker of adult hub identity tested, including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that these cells are specified as hub cells at this time. The fate of the anterior SGPs that lose esg expression and do not form part of the hub is unknown. An intriguing possibility is that these cells could form another important somatic cell type: the cyst progenitor cells (somatic stem cells) that associate with the hub along with the GSCs (Le Bras, 2006).

Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to be regulated by esg in other tissues. It has been reported, however, that esg is required for hub maintenance, and that the hub is severely defective at later stages in esg mutants that survive embryogenesis. Thus, esg is critical for the male GSC niche, but is either not important for the initial formation of this structure, or acts redundantly with another factor (Le Bras, 2006).

It has been possible to follow the morphogenesis of the hub from the time of gonad formation until the embryonic hub is fully formed. At the time of gonad coalescence, anterior SGPs interact with other SGPs, and with the germ cells, in a manner that is indistinguishable from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in their relationship to other SGPs and germ cells. Hub cells segregate away from other SGPs to one pole of the gonad, and coalesce tightly with one another. In addition, hub cells do not ensheath the germ cells at this stage. Instead, a defined interface between hub cells and germ cells forms which is labeled by DE- and DN-cadherin, but not Fasciclin 3. Thus, hub cells appear to maximize their interactions with one another, and minimize their interactions with other cells in the gonad, although they clearly still contact a subset of germ cells (Le Bras, 2006).

It is apparent that the changes in cell–cell contact and morphology that occur during hub formation require changes in cell adhesion. Indeed, characteristic changes have been found in expression of the homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur during hub formation; all three are significantly upregulated in the embryonic and adult hub. Increased homophilic adhesion among hub cells could account for their ability to maximize their contacts with one another, and sort away from other SGPs. However, no changes were observed in embryonic hub formation in mutants for these cell adhesion molecules. Thus, these proteins, and possibly others, may act redundantly in this process (Le Bras, 2006).

It is clear that a subset of germ cells organizes specifically with the developing hub as it forms. During the last stage of hub formation, germ cells become oriented in a rosette distribution around the developing hub in a manner characteristic of GSCs in the adult. These may represent the subset of germ cells that will become GSCs. The presence of DE- and DN-cadherin at sites of hub–germ cell contact suggests that cadherin-mediated adhesion may be important for niche–GSC interaction in the testis, as has been observed in the ovary. Interestingly, germ cells are not required for hub formation. Analysis of a number of hub identity markers indicates that these cell form normally from a subset of anterior SGPs in embryos that lack germ cells. The hub does not appear as well compacted in these embryos, consistent with observations of the adult hub, indicating that hub–germ cell contact (or hub–germ cell signaling) affects the final shape of the hub. Nevertheless, the GSC niche can form in the absence of one of its stem cell populations (somatic stem cells may still be present). It will be of great interest in the future to determine if the subset of germ cells organized around the male embryonic hub are, indeed, developing GSCs, and to study how their transition to stem cell identity might be regulated by the niche (Le Bras, 2006).

The formation of the male GSC niche is a sex-specific characteristic of anterior SGPs. Male-specific expression of esg and hub formation both require the sex determination genes tra and dsx. In some tissues, DSXM is required to promote male development and repress female development, while the opposite is true for DSXF. Interestingly, it was found that embryonic hub development is entirely masculinized in dsx null mutants; XX and XY individuals appear identical when mutant for dsx and both resemble wild type males. Thus, no role is seen for DSXM in promoting embryonic hub formation, while DSXF is required in females to repress hub formation. Since esg is expressed male-specifically, it is one candidate for being directly regulated by DSX (Le Bras, 2006).

We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the mechanism for how sexual dimorphism is created differs between the two cell types. msSGPs are present only in males because they have undergone sex-specific apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs. These cells appear to remain present in both sexes, but only form a hub in males. Thus, although the sex determination genes tra and dsx regulate sex-specific development of both cell types, the cellular mechanisms employed are different. Finally, as was observed for the hub, development of the msSGPs is completely masculinized in dsx mutant embryos. Thus, for both of these cell types, the male pattern of development in the embryonic gonad is the default state in the absence of dsx function, and it is the role of DSXF to repress male development in females. However, DSXM may well play a role in development of one or both of these gonad cell types at later stages, since proper testis development in males clearly requires dsx (Le Bras, 2006).

The sex determination pathway must also ensure that GSC niches form in females and are different from those in males. Recently, it has been shown that germ cells populating the anterior of the gonad in female embryos are predisposed to become GSCs in the adult ovary, while germ cells populating the posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad may promote GSC identity, similar to what is proposed to happen in the male during hub formation. One possibility is that anterior SGPs give rise to GSC niches in both sexes, while genes such as tra and dsx control whether these niches will be male or female (Le Bras, 2006).

In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).

Larval

In the ventral nervous system, a sex to abdominal neuroblasts undergo sex-specific mitotic divisions during larval and early pupal stages. Animals mutant for several sex-determining genes were analyzed to determine the genetic regulation of neuroblast commitment to the male or female pattern of division and the time during development when these decisions are made. The choice of the sexual pathway taken by sex-specific neuroblasts depends on the expression of doublesex. In the absence of any functional dxs+ products, the sex-specific neuroblasts fail to undergo any postembryonic divisions in male or female larval nervous systems. From the analysis of intersexes generated by dominant alleles of dsx, it has been concluded that the same neuroblasts provide the sex-specific neuroblasts in both male and female central nervous systems. The time when neuroblasts become committed to generate their sex-specific divisions has been identified by shifting transformer-2 temperature sensitive flies between the male- and female-specifying temperatures at various times during larval development. Neuroblasts become determined to adopt a male or female state at the end of the first larval instar, a time when abdominal neuroblasts enter their first postembryonic S-phase (Taylor, 1992b).

The gene doublesex controls which genital primordium of the genital disc will grow and which will be repressed. The female genital primordium develops from A8 and the male genital primordium develops from A9. Therefore, the gene doublesex must act in concert with another regulatory gene(s) to determine the genital primordium that develops in each sex. A possible candidate for this additional regulatory element is the homeotic gene Abd-B, since this gene is reponsible for specification of posterior segments. Under normal conditions, the female genital primordium can develop either into genitalia or remain in the repressed state, producing no adult structures. In mutant conditions for Abdominal-B m transcript, it can develop into an abdominal tergite plus sternite. Similarly, under normal conditions, the male genital primordium can develop into either normal genitalia or remain in the repressed state, forming no adult strucures. In Abd-B r transcript mutants it develops into rudimentary genitalia (Sa‡nchez, 1997 and references).

The development of the genital primordia is based on two processes: cell proliferation and sexual differentiation. Cell proliferation refers to the capacity of each genital primordium to grow or to be kept in the repressed state. Sexual differentiation refers to the type of adult structure formed by each genital primordium. It is proposed that the control of cell proliferation in the male and female genitalia requires the concerted action of Abd-B and doublesex, either directly or indirectly, through the expression of the genes dpp and wingless. Thus, in female genital discs, the repressed male primordium does not express dpp whereas the repressed female primordium of the male genital discs expresses a reduced level of dpp. This reduced level seems to be insufficient to stimulate cell growth. In contrast, when strong dpp levels are obtained in the repressed female primordium of male discs, repressed female primordia overproliferate in mutants for patched or costal-2, as well as in the discs where uniform ectopic expreession of hedgehog is produced. The genes dpp and wg, however, do not participate in the sexual differentiation process, which depends on sexual cytodifferentiation genes. Thus the growth of repressed female primordia of the patched mutant male discs would give rise to no adult female genital structures since the genetic sex is male (S‡anchez, 1997 and references).

Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain

The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. This study developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. The power of CLIn was demonstrated in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. 18 dopaminergic neurons from three distinct clusters were mapped to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. Type II lineages that produce doublesex- and fruitless-expressing neurons were identified, and whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain was examined. Blocking apoptosis in these lineages results in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism (Ren, 2016).

The relationship between neuron classes and lineages is complex in the Drosophila brain, where analogous neurons of a given class may arise from distinct lineages and a single lineage can yield multiple neuron classes. Therefore, a method was developed that would enable mapping and and analysis of neuron classes with respect to lineage identity using intersectional transgenic strategies. Specifically, the neuron class of interest expresses the GAL4 transcriptional activator from a class-specific transgene, while the lineage(s) of interest expresses the KD recombinase from a lineage-specific transgene. The KD recombinase activity triggers production of another recombinase, Cre, under the control of the deadpan (dpn) promoter, which is active in all NBs. Cre recombinase activity then triggers the simultaneous production of the LexA::p65 transcriptional activator and loss of the GAL4 inhibitor, GAL80, in all subsequently born progeny within the lineage(s). The LexA::p65 activates reporter-A expression within lineages of interest via lexAop. Because all other neurons outside lineage(s) of interest express GAL80, GAL4 is only active in neurons of the LexA::p65-expressing lineage(s) and thus can positively mark these neurons by activating expression of a reporter-B under UAS control. One can therefore subdivide any complex set of neurons that express a class-specific GAL4 transgene based on their developmental lineage(s). Consequently, CLIn enables the unambiguous determination of the lineage origins of particular neuron classes, which is essential for understanding the development and organization of the Drosophila brain (Ren, 2016).

The CLIn system unambiguously establishes the correspondence between cell classes and their lineage origins and enables the subdivision of a given neuronal class among certain NB lineages. It also allows interrogation of serially derived neuronal diversity. One can therefore map individual neurons of a given class with respect to their lineage and temporal origins in an effort to unravel the intricate neuron class-lineage relationships in the brain (Ren, 2016).

Revealing diverse cell classes of a lineage, by carefully choosing different GAL4 drivers that each distinguish a particular cell class, will allow better characterization of progeny heterogeneity within a lineage. It is therefore possible to explore how cellular diversity is generated during development. For example, it will be interesting to determine whether a specific cell class develops from one fixed temporal window. Moreover, comparing the cell-class diversity of different lineages will provide insight into the developmental heterogeneity of stem cells (Ren, 2016).

Conversely, for cell classes that originate from multiple lineages, CLIn analysis reveals the distribution of a cell class among different lineages. Vertebrate studies found that neurons of the same lineage origin, compared to neurons of the same class but different lineage origins, are more likely structurally connected via gap junction and have similar network functions. In Drosophila, lineage has been shown to be a developmental and a functional unit. Thus, assigning a cell class to different lineages may reveal the particular function of a neuronal subset within a cell class (Ren, 2016).

Moreover, the CLIn system permits incorporations of additional effectors driven by the GAL4-UAS system or the LexA system to manipulate cell class or lineage, respectively. The toolkit of effectors for different purposes is growing rapidly over recent years. Multiple reporter constructs are available to label specific sub-domains of the cell (dendrite, axon, or synapse). Effectors that affect cell viability could eliminate or immortalize specific neurons or glia. Effectors that alter membrane activity can be used to modulate neural activity. In addition, CLIn enables distinguishing gene’s functions in whole lineage including stem and progenitor cells versus only in a subset of lineage progeny by independent gene manipulations via lexAop versus UAS systems (Ren, 2016).

However, the CLIn system requires further improvement to reach its full potential. In particular, the drivers for targeting various NB subsets remain to be fully characterized. Moreover, their targeting efficiency and specificity could vary individually. Engineering drivers based on genes known to be expressed in defined subsets of embryonic NBs may provide an initial complete set of more reliable NB drivers. An additional challenge for the study of type II lineages is how to selectively target INP sublineages. Via the current dpn enhancer, the frequencies of INP1 sublineages are very low compared with that of NB lineages (Ren, 2016).

Type II NBs yield supernumerary neurons plus glia, which are expected to be highly diverse in cell classes. CLIn unambiguously assigned various neuronal classes to common type II lineages. In this study, the majority of progeny remained negative for the drivers employed. Revealing the full spectrum of neuronal heterogeneity within type II lineages requires characterization of additional cell-class drivers (Ren, 2016).

Diverse cell classes could arise from a single INP. Single-cell lineage analysis has shown that one INP can produce multiple morphological classes of neurons most likely pertaining to different functional classes. Temporal mapping by CLIn revealed the birth of both TH-GAL4 and dsxGAL4 neurons in early windows of larval type II lineages. This lends further support to the production of diverse neuronal classes by common INPs. Examining INP clones labeled by CLIn did validate that the first larval-born INP of the DM6 lineage makes one fruGAL4 neuron in addition to two TH-GAL4 neurons (Ren, 2016).

Per the limited cell-lineage analysis along the NB axis of type II lineages, sibling INPs produce morphologically similar series of neurons that differ in subsets of terminal neurite elaborations. These phenomena indicate expansion of related neurons across sibling INP sublineages. Assuming production of about 50 sibling INPs and in the absence of apoptosis, one would expect composition of 50 cell units for each neuronal class made by one type II NB. Notably, rescuing apoptotic dsx- or fru-expressing neurons throughout lineage development did restore complements of 50 or so cells in several, but not all, type II lineages. However, most type II lineages yield very few, if any, TH-GAL4 neurons. For instance, the DL1 lineage produces two TH-GAL4 neurons that innervate the upper FB layers. Temporal mapping of the DL1 lineage reveals the existence of multiple (n > 3) morphologically distinct INP clones that contain neurons projecting to the FB upper layers, similar to the DL1 TH-GAL4 neurons. Thus, morphologically similar neurons may belong to different functional classes, highlighting the challenges in sorting out neuronal classes and their interrelationships in the brain (Ren, 2016).

Pioneering studies in C. elegans showed that the acquisition of neurotransmitter identity could be achieved through distinct mechanisms. A shared regulatory signature consisting of three terminal-selector types of transcription factors regulates the terminal identity of all dopaminergic neurons. By contrast, different combinations of terminal selectors act in distinct subsets of glutamatergic neurons to initiate and maintain their glutamatergic identity. In the present study, it was observed that six type II lineages produce 18 dopaminergic neurons but all during early larval neurogenesis. The derivation of TH neurons from multiple neuronal lineages at similar temporal windows argues for their specification by combinations of different lineage-identity genes with common temporal factors (Ren, 2016).

Previous analysis of fruGAL4 neurons has uncovered 62 discrete MARCM clones in the fly central brain that might arise from an equal number of lineages. Ten clones show dimorphic cell numbers, and 22 clones exhibit dimorphic trajectories. Contrasting the stochastic clonal labeling of only fruGAL4 neurons, CLIn allows determination among a collection of lineages of whether a given lineage yields any fruGAL4 neurons. Based on the additional lineage information, two clones (pIP-j and pIP-h) were attributed as being partial clones of another two full-sized clones (pIP-g and pMP-f). Moreover, a more focused approach reveals sexual dimorphism of fru-expressing neurons in all type II NB lineages (Ren, 2016).

The presence of many more dsx- or fru-expressing neurons in male than female brains is proposed to result from selective loss of specific neurons in females through apoptosis. However, blocking apoptosis increased dsx- or fru-expressing neurons in both male and female lineages. This is consistent with a previous report showing that sex-independent apoptosis occur widely in type II lineages. Although the number of apoptosis-blocked female neurons was similar, if not identical, to that of the control male neurons, blocking apoptosis unexpectedly increased the number of male dsx- or fru-expressing neurons such that there were more neurons in the apoptosis-blocked male than female lineages. This unmasks the original potential of the male and female NBs to produce different numbers of dsx- or fru-expressing neurons in type II lineages (Ren, 2016).

Distinct fates have been reported for male and female NBs in the abdominal ganglion of Drosophila CNS. In this study, the male isoform of Dsx, DsxM, promotes additional NB divisions in males relative to females. Similarly, it has been reported that DsxM specifies additional cell divisions in the male, relative to female, central brain NBs that give rise to the pC1 and pC2 clusters. The proliferation of Drosophila intestinal stem cells is also determined by their sexual identity, although this is controlled by genes other than dsx and fru. Consistent with the notion that male and female NBs may possess distinct fates, this study found that male type II lineages contain more neurons committed to express dsx or fru, which possibly results from the greater number of NB divisions in males, as shown in the previous study. Elucidating the underlying molecular mechanisms of sex-specific neuron numbers in the central brain will require additional studies of the sex-dependent production and specification of different dsx- or fru-expressing neurons in the apoptosis-blocked type II NB lineages (Ren, 2016).

Lineage mapping based on morphology provides limited information about neuronal classes. Given the intricate relationship between neuronal classes and cell lineages, CLIn is needed to resolve the detail even in fly brains where invariant neuronal lineages exist. This is critical for fully understanding how cell lineages guide the formation of variant neural circuits with distinct combinations of neuronal classes and types (Ren, 2016).

In mammalian brains, extensive neuronal migration obscures the roles of cell lineages in the global organization of neural networks. However, clonally related neurons preferentially make local connections. Moreover, ample evidence exists for the heterogeneity of mammalian neural stem cells and the control of neuronal identity by spatiotemporal patterning of neural progenitors. Untangling of a further sophisticated brain and its development may absolutely require examination of cell lineages and neuronal classes at the same time. Systems like CLIn with its emphasis on the relationship between cell class and lineage potentially aid greatly in the study of mammalian brain development, anatomy, and function (Ren, 2016).

Effects of Mutation or Deletion

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. The roles of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway have been examined. It was asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. TraF, the female form of transformer, is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by TraF can be blocked by the male form of the Dsx protein (DsxM). Expression of the female form of dsx, DsxF, in XY animals also induces germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, these findings indicate that the somatic signaling pathway is redundant at the level of tra and dsx. Finally, these studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination (Waterbury, 2000).

Transplantation experiments and clonal analysis have suggested that germline sexual identity in XX animals depends upon a combination of cell-autonomous factors that somehow assess the X/A ratio and nonautonomous factors that signal sexual identity from the soma to the germline. A plausible pathway for linking somatic sexual identity to the mechanism that generates the nonautonomous signal is the well-characterized Sxl -> tra/tra-2 -> dsx cascade. In previous studies, the effects were tested of mutations in tra, tra-2, and dsx on the sexual identity of germ cells in XX animals. Unexpectedly, only in the case of the sex-nonspecific gene, tra-2, does loss-of-function mutation lead to a switch in sexual identity of the XX germ cells from female to male. To account for these findings, it has been proposed that the somatic signal must be generated by a novel tra-2-dependent regulatory cascade. Since dsx is dispensable for this process in XX animals, it has been postulated that an unidentified tra-2 regulatory target, z, directly or indirectly generates the signal. To explain the fact that XX germ cells retain partial female identity in tra mutants, it has been suggested that there must be another gene q whose activity overlaps or is redundant with tra. In this view, both tra and q would be able to function with the cofactor tra-2 to promote the female-specific expression of z (Waterbury, 2000).

This model was tested by introducing transgenes that ectopically express the female forms of tra and dsx into XY animals and by assaying their effects on germline sexual identity. The findings are generally consistent with predictions of the original model: there were some unexpected results that altered an understanding of the nature of the germline sex determination process and the role of dsx. Experiments with the tra transgene are considered below (Waterbury, 2000).

According to the model, ectopically expressed tra is predicted to activate the regulatory cascade that signals female identity from the soma to the germline. Activation of this signaling pathway should require tra-2 and the target gene z, while dsx would be dispensable. The results are generally consistent with these predictions. Ectopically expressed Tra switches the sexual identity of germ cells in XY animals from male to female, turning off male-specific germline markers and inducing female-specific markers. This switch in sexual identity is blocked by mutations in tra-2, but is not prevented by loss-of-function mutations in dsx (Waterbury, 2000).

In XX animals, the available evidence indicates that the tra/q -> tra-2 -> z feminization pathway functions in the soma. Hence, an expectation of the model is that ectopically expressed Tra would also feminize XY germ cells through its action in the soma, not in the germline. However, since the hsp83 promoter is known to be active in both soma and germline, it is possible that Tra protein ectopically expressed in XY germ cells feminizes these cells by a novel mechanism that is independent of the somatic signaling pathway that normally operates in XX animals. Two lines of evidence argue against this: (1) since several of the hsp83-traF lines were recovered from males, it would appear that expression of Tra in XY germ cells is not in itself sufficient to feminize these cells; (2) the available evidence suggests that ectopically expressed Tra feminizes the germline in XY animals by a pathway resembling that used in XX animals; it requires tra-2 but is independent of a functional dsx (Waterbury, 2000).

Although the hsp83-traF transgene does not require dsx to feminize the germline of XY animals, feminization can be prevented if the only source of Dsx protein is provided by an allele that constitutively expresses DsxM. This result was unexpected since DsxM has no effect on the sexual identity of the germline in XX animals. There are several possible explanations for this discrepancy. It has been proposed that there is another gene, q, which occupies the same position in the regulatory cascade as tra. If this gene is downstream of Sxl, as expected, it would be expressed in the male mode in XY; P[hsp83-traF] animals and hence would not contribute to the production of the feminizing signal. Because DsxM alters the development of the soma surrounding the germline and consequently the cell-cell contacts between soma and germline, the signal produced by tra alone might not be sufficient to feminize. A second possibility is that XY germ cells are intrinsically less responsive to the feminizing signal than XX germ cells. For example, given the lack of strong evidence for germline dosage compensation, the signal could be sensitive to a twofold difference in X-linked genes. A third possibility is that DsxM produces a masculinizing signal that is able to counteract the effects of the feminizing signal produced by TraF. At the present, these explanations cannot be distinguished (Waterbury, 2000).

Since the dsx gene can be removed or expressed exclusively in the male mode without affecting germline sexual identity in XX animals, it has been suggested that dsx has no role in germline sex determination. However, contrary to this suggestion, ectopic expression of DsxF in XY; dsx- animals can feminize the germline and this feminizing activity can be blocked if DsxM is also present in the soma (Waterbury, 2000).

Why is DsxF capable of feminizing XY germ cells, yet dispensable in XX animals? One way to reconcile these two observations is to postulate that z regulates the synthesis of the feminizing signal ('fes') instead of encoding the signal itself. If this were the case, both Z and DsxF could independently promote the production of fes. In females, since q and tra would be active, Z would be able to induce sufficient levels of fes to feminize the germline in the absence of DsxF or in the presence of DsxM. Furthermore, since the female and male Dsx proteins recognize the same target sequences, ectopically expressed DsxF would be able to activate fes synthesis in XY animals only when DsxM is absent (Waterbury, 2000).

In the revised model for the somatic signaling pathway, Sxl has been placed at the top of the regulatory cascade where it is responsible for activating the female-specific expression of both tra and q. While Sxl is known to be required for sex-specific regulation of tra, it should be noted that there is no evidence that it is responsible for controlling the activity of q. However, unless q is itself a target for the X/A counting system, there are no other known mechanisms that could promote female expression. If q is downstream of Sxl, results with SxlM1,fm3 and SxlM1,fm7 (revertant alleles of SxlM1) suggest that q is regulated by a different mechanism than tra. q and/or tra, together with tra-2, would then activate the female-specific expression of z and dsx. The female products of z and dsx would in turn direct the synthesis of the feminizing signal. By this model, the germline would assume male identity whenever Sxl is off in the soma. However, it is not clear whether the male pathway requires production of a male somatic signal by the male form of Dsx (or Z) or occurs in XY germ cells by default in the absence of a female signal. In favor of the former possibility is the finding that constitutively expressed DsxM prevents Tra from feminizing XY germ cells. However, functional dsx is not required in XY animals to select male identity (Waterbury, 2000).

One question raised by these studies is the role of the postulated autonomous X chromosome counting system in germline sex determination. In particular, it has been argued from pole cell transplantation experiments that this autonomous system overrides input from the soma in XY germ cells, forcing them to assume male identity. However, data has been presented indicating that the sexual identity of XY germ cells can be switched from male to female by ectopic expression of TraF and DsxF. If TraF and DsxF activate the signaling pathway(s) that normally functions in XX animals, this result would imply that there may be no cell-autonomous system that selects sexual identity by measuring the germ cell X/A ratio. In this view, the autonomous components of the germline sex determination system would play an entirely different role. They would be subordinate to the somatic signaling pathway, being responsible only for responding correctly to the somatic signal and having no role in making the actual choice. Of course, if the default pathway within the germline is male, then this pathway will be followed 'autonomously' in the absence of a feminizing signal from the soma (Waterbury, 2000).

From a phylogenetic perspective, the simplest solution for germline sex determination is that germ cells strictly follow the same sexual fate as that of the soma in securing the development of a fully functional organism. In fact, this appears to be the mechanism for germline sex determination in other dipteran species such as Musca domestica and Chrysomya rufifacies. In these organisms, somatic sex alone is necessary and sufficient to dictate sexual fate to the germline. Irrespective of their sexual karyotype, when germ cells are surrounded by ovarian tissue, eggs are produced, and when surrounded by testicular tissue, sperm are produced. Studies in the nematode C. elegans and in the mouse further support the idea that somatic sex is widely used to dictate the sexual fate of gametes. In Drosophila, it is believed that somatic sex is the primary determinant. Why then is this soma-to-germline signaling mechanism insufficient to direct complete female or male germline differentiation independent of the chromosome composition of the germ cells in Drosophila? A likely explanation is that XY and XX germ cells in Drosophila have lost the ability to respond equally well to somatic cues. For example, in XY; P[hsp83-traF] pseudofemales, most of the ovarioles have a tumorous ovary phenotype and ovarioles that have normal-looking egg chambers are observed very infrequently. Given that there is no strong evidence for germline dosage compensation, one plausible explanation for the abnormal development of these sex-transformed XY germ cells is that the dose of X-linked gene products is insufficient to properly execute an oogenic developmental program. The Sxl gene would be a good example of a gene that is required for oogenesis and, because of its autoregulatory activity, is highly sensitive not only to its own dose but also to the dose of other X-linked genes such as the splicing factor snf. It is reasonable to suppose that there may be a variety of steps in oogenesis (or spermatogenesis) that are sensitive to the dose of X-linked genes (Waterbury, 2000).

Within the germline, otu, ovo, and Sxl have been identified as candidate genes that respond to the feminizing signal from the soma and determine the sex of the germ cells. Mutations in all three genes have sex-specific effects on germline development. Perhaps the most striking result is the fact that loss-of-function ovo and otu mutations markedly reduce the viability of XX but not XY germ cells. Thus one important question is whether these mutations have similar effects on the viability of XY germ cells feminized by the hsp83-traF transgene. Somewhat surprisingly, it was found that otu and ovo mutations behave differently. Strong loss-of-function otu mutations reduce the viability of XY germ cells feminized by the traF transgene. This finding suggests that the lethal effects of strong otu mutations arise because the germ cells assume a female identity, and not because of their number of X chromosomes. In contrast, ovo mutations have no apparent effect on the viability of feminized XY germ cells. One explanation for this difference is that lethal effects are not observed in ovo mutants because the feminizing signal produced by the traF transgene in XY animals is weaker than the feminizing signal found in wild-type XX animals. Alternatively, it is possible that XX germ cell death in ovo mutants does not depend upon the choice of sexual identity, but rather is a function of the X chromosome dose (Waterbury, 2000).

If otu, ovo, or Sxl functions as a master sex determination switch within the germline, one would expect to find that mutations in any of these genes would completely block the feminization of germ cells much like mutations in Sxl prevent feminization in the soma. While the results indicate that none of these genes fits this criterion for a master regulatory switch, effects are observed in the expression of sex-specific markers. Mutations in all three genes prevent the traF transgene from inducing the expression of female bruno (and Sxl) gene products. However, in all three cases the transgene still induces the expression of female orb gene products. One interpretation of these findings is that the sex determination pathway in the germline is split into at least two branches -- one branch that contains bruno and Sxl and another branch that contains orb. For both bruno and orb, sex-specific expression depends upon the activation of distinct sex-specific promoters. If these two genes are in independent branches of the germline sex determination pathway, this would imply that there must be distinct 'male' and 'female' transcription factors for the four promoters. Moreover, it seems likely that one important function of the somatic signaling system would be to control the expression of these transcription factors. Clearly, it will be important to identify these transcription factors and to learn how they are regulated (Waterbury, 2000).

A pair of muscles span the fifth abdominal segment of male but not female adults. To establish whether genes involved in the development of other sexually dimorphic tissues control the differentiation of sex-specific muscles, flies mutant for five known sex-determining genes were examined for the occurrence of male-specific abdominal muscles. Female flies mutant for alleles of Sex-lethal, defective in sex determination, or null alleles of transformer or transformer-2 are converted into phenotypic males that form male-specific abdominal muscles. Both male and female flies, when mutant for null alleles of doublesex, develop as nearly identical intersexes in other somatic characteristics. Male doublesex flies produce the male-specific muscles, whereas female doublesex flies lack them. Female flies, even when they inappropriately express the male-specific form of doublesex mRNA, fail to produce the male-specific muscles. Therefore, the wild-type products of the genes Sex-lethal, transformer and transformer-2 act to prevent the differentiation of male-specific muscles in female flies. However, there is no role for the genes doublesex or intersex in either the generation of the male-specific muscles in males or their suppression in females (Taylor, 1992a).

The intersex (ix) gene is a terminally positioned gene in the sex determination heirarchy. The null phenotype of ix is to transform diplo-X animals into intersexes while leaving haplo-X animals unaffected. The ix+ product functions in a cell-autonomous manner, and it is required at least until the termination of cell division in the abdomen. In addition, the ix+ product is required to function with the female-specific product of doublesex to implement appropriate female sexual differentiation in diplo-X animals (Chase, 1995).

Sexual differentiation in Drosophila is controlled by a short cascade of regulatory genes, the expression pattern of which determines all aspects of maleness and femaleness, including complex behaviors displayed by males and females. One sex-determining gene is transformer (tra): tra activity is needed for female development. Flies with a female karyotype (XX) but which are mutant for tra develop and behave as males (and are termed X2 flies). In such flies, a female phenotype can be restored by a transgene that carries the female-specific cDNA of tra under the control of a heat-shock promoter. This transgene, called hs[trafem], also transforms XY animals into sterile females. When these XX and XY 'females' are raised at 25 degrees C, however, they display vigorous male courtship while at the same time, as a result of their female pheromone pattern, they are attractive to males. Intriguingly, their male courtship behavior is indiscriminately directed toward both females and males. When expression of tra is forced by heat shock, applied during a limited period around puparium formation, male behavior is abolished and replaced by female behavior. The temperature-sensitive period extends from shortly before puparium formation into early metamorphosis. Cuticular hydrocarbons function as pheromonal cues between the sexual partners. Consistent with their attractiveness to males, the normal female pattern of pheromones is found in transformed flies, irrespective of the heat shock. Could the indiscriminate male behavior of these flies result from continuous self-stimulation by their own female pheromones? The female pheromone pattern was replaced with the male pattern without affecting the CNS. This was achieved by introducing the male-determining mutation dsxD, which acts downstream of tra. This mutation causes XX flies to produce a male pheromone pattern. They are no longer attracted to males. Similarly, X2 and XY 'females', now with male body and pheromonal status due to dsxD, are completely unattrative to males; but they still court both sexes. Such flies even manage to copulate with females, showing that X2 and XY 'females' are capable of performing the full repertoire of male courtship. These results rule out self-stimulation and leave a disturbed nervous system as the most probable cause for the indiscriminate mating behavior. It is concluded that sexual behavior is irreversibly programmed during a critical period as a result of the activity or inactivity of a single control gene. Such programming takes place during a period of accelerated growth in the brain and especially in the mushroom bodies, suggesting that sexual behavior is 'hard wired' in the CNS (Arthur, 1998).

Epistasis experiments show that dissatisfaction acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade. Analysis of sex-specific neural and behavioral phenotypes suggests that genes regulating these phenotypes act downstream of tra. If so, XX;dsf- animals masculinized by mutations in tra will have the male-specific ventral neuronal phenotype shown by XY;dsf- males. To test this, ventral abdominal innervation of XX;tra- and XX;dsf-; tra- individuals were examined. XX;dsf-;tra- animals show the dsf phenotype while their dsf+; tra- siblings do not. Thus, it is inferred that dsf acts downstream of tra for development of ventral abdominal neuromuscular junctions. At the same time, these data rule out models in which some alternative pathway involving upstream elements such as the X chromosome to autosome ratio or Sxl, which are identical in both tested XX genotypes, independently regulates this process. To test dsf dependence on dsx function, advantage was taken of a gain-of-function dsx mutant, dsxD, which expresses the male Dsx protein regardless of tra and tra2 activity. In the absence of a wild-type dsx allele, both sexes develop external male morphology. Even so, XX animals express tra and XY flies do not. If dsf acts independently of dsx, it is expected that XX; dsf-; dsxD/Df (tra ON) animals will have normal ventral innervation and XY; dsf-; dsxD/Df (tra OFF) animals will have abnormal ventral innervation: the dsf mutant phenotype. If dsf is dependent on the activity of dsx, then XX and XY; dsf-; dsxD/Df animals should have equivalent and mutant phenotypes. The neurons in ventral A5 were examined for different genotypes of dsxD/Df mutant animals. XX; dsf/Df; dsxD/Df are wild type in appearance while XY;dsf/Df; dsxD/Df have a dsf phenotype. This result is consistent with the idea that dsf is part of a dsx-independent pathway (Finley, 1997).

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. The gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). The sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males (Keisman, 2001a).

In the male primordium of both male and female discs, the regulation of dac varies according to the genetic sex of the cell. Genetically female clones in the male (A9 derived) primordium of the male genital disc are unable to express dac in the lateral male (dpp-dependent) domain, but are able to express dac when they extended medially, towards the source of Wg. Conversely, in the female genital disc, genetically male clones in the repressed male primordium (A9) lose their ability to express dac in the medial, wg-dependent domain, and begin to express dac laterally, presumably in response to Dpp. Finally, dac expression is abnormal in intersexual genital discs from dsx mutant larvae: the male primordium of dsx genital discs expresses dac in both the endogenous, lateral male domains, and in a slightly weaker medial domain that corresponds roughly to the region where tra + clones are able to activate dac. Thus, it is concluded that in the male primordium, the sex determination pathway determines how a cell will regulate dac (Keisman, 2001a).

In the female primordium the results fail to show a role for the sex determination pathway in dac regulation. If such a role exists, it would be expected that genetically male clones in the female primordia of a female genital disc would activate dac laterally, like their counterparts in the male primordia. They do not, even when they take up much of the presumptive dpp-expressing domain. It would also be expected that such clones would repress dac medially. Only a few clones were observed to extend into the medial wg-expressing domain, and as expected these appear to repress dac. Interpretation of these results is complicated by the fact that changing the genetic sex of a cell in the genital disc can cause it to enter the 'repressed' state. Thus, for example, if a genetically male clone represses dac when it intersects the medial dac domain in the female primordia, it can be concluded either that the sex determination pathway regulates dac expression or that the cells, which are now male, have adopted a repressed state and are generally unresponsive. A similar caveat prevents interpreting the failure of tra2IR clones to activate dac ectopically in the female primordium. That tra + clones in the male primordium of male genital discs enter such a generally non-responsive state was not of concern, because these clones both repress and activate dac expression. The expression pattern of dac in the female primordium of a dsx mutant genital disc is also difficult to interpret. dac is not activated ectopically in the lateral domains of the dsx female primordium, which is consistent with the failure of tra2IR clones to cause such activation. However, even the medial, wg-dependent dac domain is frequently absent or severely reduced in the dsx female primordium, and thus the authors are reluctant to draw any conclusions from the absence of ectopic dac laterally (Keisman, 2001a).

A model is proposed for dac regulation in the male primordium, in which the different isoforms of Dsx protein modulate dac regulation by wg and dpp. In the absence of dsx, both wg and dpp can activate dac, producing the two domains of dac expression observed in the male primordium of a dsx disc. In the female, Dsxf modulates dpp activity so that dpp becomes a repressor of dac; Dsxf may also potentiate the activation of dac by wg. In the male, Dsxm modulates wg activity so that it becomes a repressor of dac, leaving dpp alone to activate dac. In support of this model, it is noted that the Dsx proteins act in a similar manner to positively or negatively modulate the effect of tissue-specific regulators on the yp genes (Keisman, 2001a and references therein).

Reproduction in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. A putative pheromone receptor, GR68a, is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila (Bray, 2003).

If Gr68a encodes a male-specific pheromone receptor, it would be predicted that the sex determination genes, which control all aspects of sexual differentiation, would regulate its expression. Thus, Gr68a expression was investigated in chromosomally female (XX) flies that were sexually transformed into Ψ males by mutations in tra2 or dsx. Both types of Ψ males show the normal male expression pattern of the p[Gr68a]-Gal4 driver. Since sex-specific fru expression is directly controlled by Tra and Tra2, and hence, independent of dsx (i.e., XX; dsx Ψ males express no Frum), male-specific expression of Gr68a is fru independent. Thus, Gr68a is a dsx-dependent effector gene expressed in chemosensory neurons of taste bristles in the foreleg, which is consistent with a function for this gene in pheromone recognition (Bray, 2003).

Isoform-specific control of male neuronal differentiation and behavior in Drosophila by the fruitless gene: Male serotonergic neurons exhibit abnormal differentiation in dsx-null animals

How the central nervous system (CNS) develops to implement innate behaviors remains largely unknown. Drosophila male sexual behavior has long been used as a model to address this question. The male-specific products of fruitless (fru) are pivotal to the emergence of this behavior. These putative transcription factors, containing one of three alternative DNA binding domains, determine the neuronal substrates for sexual behavior in male CNS. This study reports on the isolation of he first fru coding mutation, resulting in complete loss of one isoform. At the neuronal level, this isoform alone controls differentiation of a male-specific muscle and its associated motorneuron. Conversely, a combination of isoforms is required for development of serotonergic neurons implicated in male copulatory behavior. Full development of these neurons requires the male-specific product of doublesex, a gene previously thought to act independently of fru. At the behavioral level, missing one isoform leads to diminished courtship behavior and infertility. This study achieved the first rescue of a distinct fru behavioral phenotype, expressing a wild-type isoform in a defined subset of its normal expression pattern. This study exemplifies how complex behaviors can be controlled by a single locus through multiple isoforms regulating both developmental and physiological pathways in different neuronal substrates (Billeter, 2006).

Previously, fru and dsx have been described as acting independently in the sex-determination pathway, with FruM expressed specifically in the male CNS determining male sexual behavior and with dsx expressed in the soma determining the dimorphic morphology of the sexes. However, this study found that male serotonergic neurons exhibit abnormal differentiation in dsx-null animals and fail to differentiate in fru mutant males. These experiments evoke a mechanism for this apparent overlap in function. DsxM controls the formation of 20 neurons in the abdominal ganglion by prolonging neuroblast proliferation at the end of the larval stage. In dsx-null animals these neuroblasts completely fail to develop, and, notably, the number of male serotonergic neurons is reduced in mutant males. This DsxM-dependent proliferation of neurons appears to offer a substrate for FruM to induce serotonergic differentiation. A number of ectopic serotonergic neurons form in dsx-null females. Given that these females do not express FruM, the development of these neurons would appear normally to be inhibited by dsx (Billeter, 2006).

These experiments reveal a new feature of FruM function. Whereas dsx-null males exhibit less male serotonergic neurons, these retain their typical dorso-ventral patterning. Conversely, the ectopic serotonergic neurons in dsx-null females develop randomly either ventrally or dorsally. This lack of dorso-ventral patterning must be linked to the absence of FruM, given that females missing Tra develop a complete organized set of male serotonergic neurons. Constitutive expression of FruM in females also induces two clusters of serotonergic neurons, though with fewer neurons than wild-type males. This reinforces the contention that FruM is fundamental for controlling not only the serotonergic differentiation of these neurons but their structural organization into a functional circuit. These experiments also show that the male serotonergic neurons stem from at least two different populations of neurons, one requiring both DsxM and FruM, the other just FruM. Two lines of evidence support the idea that FruM acts directly on the neurogenesis of these cells: Their projections are greatly reduced in FruM-null males, suggesting that some of these neurons are absent, and the number of neurons expressing FruM in FruMC-null males is reduced. fru has been shown to prevent cell death in neurons in the male brain. FruM could use this mechanism to control the number of male serotonergic neurons during development (Billeter, 2006).

Although FruM is sufficient to determine most aspects of male sexual behavior, part of its function requires a male-specific neuronal substrate determined by dsx. It is predicted that DsxM/FruM cooperation extends beyond the formation of the male serotonergic neurons because FruM and Dsx colocalize in more than 100 neurons in the abdominal ganglion. Moreover, given that dsx is required for males to generate normal courtship song, it is anticipated their interaction to have a broader importance in the determination of male sexual behavior (Billeter, 2006).

Fruitless and Doublesex coordinate to generate male-specific neurons that can initiate courtship

Biologists postulate that sexual dimorphism in the brain underlies gender differences in behavior, yet direct evidence for this has been sparse. A male-specific, fruitless (fru)/doublesex (dsx)-coexpressing neuronal cluster, P1, was identified in Drosophila. The artificial induction of a P1 clone in females effectively provokes male-typical behavior in such females even when the other parts of the brain are not masculinized. P1, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Thus, the coordinated actions of two sex determination genes, dsx and fru, confer the unique ability to initiate male-typical sexual behavior on P1 neurons (Kimura, 2008).

An effort at identifying the neural centers for specific acts in courtship behavior was initiated by Hotta and Benzer (1976), who inferred the foci for a variety of behavioral acts in the brain by correlating the body surface markers or neural markers with the incidence of each act in sexually mosaic flies. Hall (1979) generated female-male (XX-XO) mosaic flies that were subjected to mating assays and then sacrificed, after which their brain sections were stained with the Acph-1 enzymatic marker to determine which parts of the brains of individual flies were female and which parts male. In this classic work with gynandromorphic flies, the conclusion was reached that the flies perform the early part of courtship behavior when a specific site located in the dorsal posterior brain is composed of male tissues, at least on one side of the brain. This conclusion was supported by a study in which part of a male brain was feminized with tra+. In addition, a cluster of cells in the posterior lateral protocerebrum has been suggested to mediate the initiation and early steps of male courtship by experiments in which transgenes that either activated or suppressed neural activities were focally expressed (Kimura, 2008 and references therein).

The present study succeeded in pinpointing a single neural cluster that plays an important role in initiating courtship by large-scale screens of tra1 mosaic females for the ability to perform male-typical behavior. The cluster is called P1, and is located in close proximity to the mushroom body. It is intriguing that P1 is located roughly in the area Hall (1979) assigned as the focus of male-courtship. He showed that 'SP3' ('supraoesophageal ganglion cortex site 3') is the 'domineering' focus for the early steps of male-type courtship behavior, which include tapping, following, and wing extension. To perform the late steps of male-type courtship behavior, flies need to have additional male tissues in other parts of the brain. Further study with gynandromorphs led to the identification of 'SP2' as a second focus for wing extension in male-type courtship behavior. Note that 'SP2' and 'SP3' represent the posterior brain sites distinct from current aSP2 and aSP3, which lie in the anterior brain (Kimura, 2008).

Female mosaic flies with the masculinized P1 cluster showed the early parts but not the late parts of male-type courtship, reminiscent of the behavior displayed by gynandromorphic flies with male 'SP3'. Thus, these two groups of flies share masculinized brain cells in a similar location and exhibit similar behavioral characteristics. P1 neurons have an input site and an output site in the lateral and medial protocerebra, to which sensory information on different modalities is conveyed by higher-order interneurons and from which descending interneurons extend their axons to motor centers in the brain and the ventral nerve cord. Although these structural features imply a role for P1 in the integration of multiple sensory inputs for the control of outputs for sexual behavior, this remains speculation, as the physiological properties of these neurons have not been characterized (Kimura, 2008).

Although the presence of P1 in the brain makes a fly more likely to engage in courtship, flies without P1 are able to perform male sexual behavior and other flies with P1 are not. This is not surprising in light of recent findings that the female flies with DsxF and devoid of Fru and DsxM perform male-type courtship behavior, provided that they are mutant for retained (Ditch, 2005; Shirangi, 2006; Shirangi, 2007). This may mean that entire motor patterns of male-type courtship behavior can be generated in the neural circuitry that is common to both sexes. A recent study using light-activated ion channels to stimulate fru-expressing neurons in the thoracic-abdominal ganglia reveals the presence of pattern-generating circuit for courtship song in both sexes (Clyne, 2008). P1 might function as a cluster of neurons that powerfully drive lower motor centers, which are not necessarily sexually dimorphic by themselves, to generate male-type courtship behavior. It is envisaged that male-sexual behavior is initiated by the activation of P1 under normal conditions in response to adequate key stimuli, yet can be commenced by the lower motor centers without any involvement of P1 neurons, when they are somehow sensitized or disinhibited under the influence of genetic or environmental factors. This may also explain why the fruM females are less active than males in performing male-type courtship behavior. fruM females lack P1 neurons so that they need additional drive to initiate male-type behavior (Kimura, 2008).

P1 neurons require functioning dsx expression to acquire sexual dimorphism: for example, DsxF contributes to the active elimination of P1 from the female brain, thereby preventing the females from behaving similarly to males in a sexual context. There are, however, precedent cases in which both dsx and fru are required for sexually dimorphic neural development, i.e., serotonergic neurons that innervate the seminal glands of males and a group of thoracic neurons that are suspected to function in specifying the courtship song (Kimura, 2008).

It is plausible that the neural circuit underlying sexual behavior is composed of three different neuronal species, namely, class 1 sex-specific or sexually dimorphic neurons whose sex specificity is determined by the coordinated actions of dsx and fru, class 2 sex-specific or sexually dimorphic neurons whose sex specificity is determined by either dsx or fru, and class 3 neurons that show no sex-related differences and do not express either dsx or fru. The class 3 neurons likely form a circuit common to both sexes, although this circuit is involved in the generation of male-specific motor outputs for male-type sexual behavior. Because of the presence of such a non-sex-specific circuit in the female brain, female flies are able to exhibit male sexual behavior after being genetically manipulated to possess male-specific P1 neurons. A recent study with mice deficient for pheromone sensation has led to a similar hypothesis, i.e., functional neural circuits underlying male-specific behavior exist in the normal female mouse brain (Kimura, 2008).

The three classes of neurons defined above may interconnect to form circuits that control different aspects of sexual behavior. In fact, some of the olfactory pheromone receptor neurons on the antenna express Fru. Male-specific expression of a gustatory pheromone receptor in the foreleg sensory hairs is dsx dependent but fru independent. The sexually dimorphic mAL interneurons suspected to be involved in the integration of pheromone inputs express Fru but not Dsx. The P1 cluster can initiate male-type courtship behavior and can thus be placed on the highest rung of the neural hierarchy. P1 is composed of class 1 neurons whose sex-specific differentiation is governed by both dsx and fru and thus are under the stringent control of the developmental program (Kimura, 2008).

Alternatively, P1 may contribute to one of several neural pathways, each with the potential to initiate male-type courtship behavior. It is worth examining whether or not this behavior can be elicited by the selective activation of individual neural clusters with the aid of photosensitive tools such as channelrhodopsins or the ionotropic purinoceptor P2X2, both of which are expressed only in a small MARCM clone in the brain (Kimura, 2008).

Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors

Although nervous system sexual dimorphisms are known in many species, relatively little is understood about the molecular mechanisms generating these dimorphisms. Recent findings in Drosophila provide the tools for dissecting how neurogenesis and neuronal differentiation are modulated by the Drosophila sex-determination regulatory genes to produce nervous system sexual dimorphisms. This paper reports studies aimed at illuminating the basis of the sexual dimorphic axonal projection patterns of foreleg gustatory receptor neurons (GRNs): only in males do GRN axons project across the midline of the ventral nerve cord. The sex determination genes fruitless (fru) and doublesex (dsx) both contribute to establishing this sexual dimorphism. Male-specific Fru (FruM) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (DsxF). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (DsxM). The roundabout (robo) paralogs also regulate midline crossing by these neurons, and evidence is provided that FruM exerts its effect on midline crossing by directly or indirectly regulating Robo signaling (Mellert, 2010).

This study shows that the male-specific presence of contralateral GRN projections is primarily due to FruM function. Specifically, FruMC acts in foreleg GRNs to promote the crossing of the VNC midline by their axons. A role for dsx was identified in this dimorphism since (1) males that lack DsxM have somewhat fewer contralateral GRN projections, and (2) DsxF prevents the appearance of contralateral GRN axons in females (Mellert, 2010).

The finding that FruM regulates GRN axon midline crossing is consistent with previous findings that, in some neurons, FruM regulates axonal morphology. Regulation of axonal morphology is likely to alter synaptic connectivity, suggesting that one of the roles of FruM is to support the formation of male-specific connections, and possibly prevent the formation of female-specific connections, between neurons that are present in both sexes. Determining how such changes alter information processing will contribute to understanding how the potential for male courtship behavior is established (Mellert, 2010).

It is also notable that dsx plays a role in regulating sexually dimorphic midline crossing, given that it also specifies the sexual dimorphism in gustatory sensilla number in the foreleg. It might be that dsx regulates gustatory sensilla development independently of its regulation of GRN axon morphology. That dsx can independently specify multiple sexual dimorphisms within particular cell lineages has been previously shown for the foreleg bristles that comprise the sex comb teeth of the male foreleg and their homologous bristles in the female. There, dsx was shown to function at one time to determine the sex-specific number of bristles that are formed and at another time to determine their sex-specific morphology. In support of a similar sequential role in the developing GRNs, dsx is expressed in the gustatory sense organ precursor cells and continues to be expressed in the terminally differentiated GRNs (Mellert, 2010).

It is also possible that the effect of dsx on the presence of contralateral GRN projections is indirect. The two pools of gustatory sensilla, those that are male-specific and those that are homologous between males and females, might differ in their competence for midline crossing (i.e. only the male-specific GRNs will cross the midline when FruM is expressed). This is thought not to be the case for two reasons. First, dsx is expressed in the GRNs throughout their development, consistent with a role in regulating axon guidance. Second, the expression of FruMC in female GRNs using poxn-Gal4 is sufficient to induce midline crossing, suggesting that the sex-nonspecific GRNs are not intrinsically nonresponsive to FruM (Mellert, 2010).

With respect to the latter result, it is worth considering the contrast between females that are masculinized with fruδtra, where no contralateral GRN projections are observed, and females in which poxn-Gal4 is used to drive the expression of UAS-fruMC::AU1 (AU epitope tagged Fruitless) in females, where GRN midline crossing is observed. In the case of females masculinized by fruδtra it was shown that the absence of contralateral GRN projections was due to DsxF functioning to prevent midline crossing in a manner that was epistatic to fruM function. One attractive explanation for the difference between these two situations is based on the fact that masculinization by fruδtra occurs via FruM produced from the endogenous fruitless locus, whereas masculinization by UAS-fruMC::AU1, occurs via fruMC expressed from a UAS construct that contains none of the untranslated sequences present in endogenous fruM transcripts. Thus, it might be that the difference in midline crossing seen in these two situations is due to DsxF directly regulating fruM expression through noncoding fru sequences that are present in the endogenous fru gene, but absent in the fru cDNA expressed from UAS-fruMC::AU1. It is not likely that DsxM represses fruM transcription, fruP1.LexA was seen to be expressed in GRNs in both males and females. Thus, if fruM is downstream of dsx in these cells, DsxF probably affects the processing or translation of fruM transcripts through sequences not present in the UAS-fruMC::AU1 construct. Alternatively, differences between these two situations in expression levels or patterns of expression might result in differences in the ability of FruM versus FruMC to overcome a parallel repressive effect of DsxF (Mellert, 2010).

robo, robo2 and robo3 are involved in GRN axon guidance. Of these three genes, robo appears to be most important in regulating GRN midline crossing because only reductions in levels of robo transcript result in midline crossing in females or fruM-null males. Reducing levels of robo2 and robo3 transcripts in addition to robo enhances the robo phenotype but individual reductions of robo2 or robo3 function have the opposite effect, a reduction in midline crossing, suggesting that these receptors function to promote crossing in the presence of wild-type levels of robo expression (Mellert, 2010).

It is not surprising that robo differs in function from robo2 and robo3 with respect to foreleg GRN development. robo2 and robo3 are more similar in sequence to each other than to robo, and robo contains two cytoplasmic motifs not found in its paralogs. Furthermore, functional differences have been recognized since the original reports of robo2 and robo3. Finally, robo2 might promote midline crossing if pan-neuronally overexpressed at low levels and yet repress midline crossing when overexpressed at high levels. This 'switch' in function might explain why reduced midline crossing is seen under conditions of both robo2 overexpression and reduction (Mellert, 2010).

Given that the Robo receptors play such an important role in GRN development, how might fruM regulate midline crossing? The data indicate that robo lies genetically downstream of fruM. The most straightforward mechanistic explanation is that FruM suppresses the activity of the Robo signaling pathway. Several ways that this might occur can be envisioned. First, fruM might regulate commissureless, which itself participates in the midline crossing decision by regulating the subcellular localization of Robo. No sexual dimorphism could be detected in the subcellular localization of a Robo::GFP fusion protein in GRNs in either the axons or cell body (UAS-robo::GFP), so if fruM regulates comm, it does so subtly. It is more probable that fruM regulates the expression of either other regulators of robo signaling, robo itself, or robo effectors. Strategies are being pursued to identify candidate FruM targets that might be involved in regulating midline crossing (Mellert, 2010).

How does midline crossing by GRN axons affect gustatory perception? Given that male-typical GRN morphology requires fruM, and that fruM has a major regulatory role for social behavior, one hypothesis is that the contralateral GRN projections in males play a role in mediating the processing of contact cues during male courtship and/or aggression. Previous reports have shown that fruM-masculinized females, which do not have contralateral GRN projections, readily perform tapping and proceed to subsequent steps in the male courtship ritual, and behave like males with respect to aggressive behaviors. Thus, contralateral GRN projections are not necessary for the initiation and execution of these male-specific behaviors. Nevertheless, midline crossing might still be important for mediating socially relevant gustatory information. For instance, amputation experiments suggest that the detection of contact stimuli is important for courtship initiation under conditions when the male cannot otherwise see or smell the female (Mellert, 2010).

It is possible that midline crossing by GRN axons facilitates the comparison of chemical contact cues between the two forelegs. Such a comparison might help the male to determine the orientation of another fly, which would be a useful adaptation for performing social behaviors in conditions of sensory deprivation, such as in the dark. Alternatively, midline crossing might simply be a mechanism to form additional neuronal connections that integrate gustatory information into circuits underlying male-specific behaviors. Armed with the results of the present study, fruM, dsx, and the robo genes can be used as handles for developing tools and strategies to specifically manipulate midline crossing in the foreleg GRNs, with the goal of understanding its importance with regard to male behavior (Mellert, 2010).

Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster

Doublesex proteins, which are part of the structurally and functionally conserved Dmrt gene family, are important for sex determination throughout the animal kingdom. Gal4 was inserted into the doublesex locus of Drosophila, allowing visualization and manipulation of cells expressing dsx in various tissues. In the nervous system, differences between the sexes were detected in dsx-positive neuronal numbers, axonal projections and synaptic density. dsx was found to be required for the development of male-specific neurons that coexpressed fruitless (fru), a regulator of male sexual behavior. It is proposed that dsx and fru act together to form the neuronal framework necessary for male sexual behavior. Disrupting dsx neuronal function had profound effects on male sexual behavior. Furthermore, these results suggest that dsx-positive neurons are involved in pre- to post-copulatory female reproductive behaviors (Rideout, 2010).

Neural circuitry underlying Drosophila female postmating behavioral responses

After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating. This study demonstrates an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. Shared circuitry was uncovered between dsx and a subset of the previously described SP-responsive fru+/ppk+-expressing neurons in the reproductive system. In addition, sexually dimorphic dsx circuitry was identified within the abdominal ganglion (Abg) that was critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. It is proposed that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs (Rezával, 2012).

These results show that in the female, dsx neurons associated with the internal genitalia not only form a component part of the previously described fru+/ppk+ network, but in fact define a more minimal SP-responsive neural circuit capable of inducing postmating changes, such as reduced receptivity, increased levels of rejection, and egg deposition (Rezával, 2012).

In addition to these 'classic' postmating behavioral responses, it was also noted that SP signaling to dsx neurons induces postmating changes in locomotor activity between unmated and mated females. Studies have shown that Drosophila males court immobilized females less than moving females; essentially, males react to changes in female locomotion, suggesting a causal link between female locomotion and increased courtship levels. It has been proposed that males are 'acoustically tuned' to signals generated by active females, stimulating increased courtship by changing the attention state of the male. Therefore, female mobility appears to contribute to her 'sex appeal' and decreased locomotion in mated females is likely to affect the male's willingness to copulate (Rezával, 2012).

The female's nervous system must have the capacity to receive, and interpret, postcopulatory signals derived from the male seminal package to direct physiological and behavioral responses required for successful deposition of fertilized eggs. It was demonstrated that two dsx clusters, composed of three bilateral neurons of the uterus, comprise a more defined component of the SP-responsive sensory circuit. In addition, the majority of other dsx neurons originating on the internal genitalia were shown to coexpress ppk. As ppk neurons are mechanosensory, these may be acting as uterine stretch receptors, facilitating sperm and egg transport, fertilization, and oviposition. Silencing neural function of ppk neurons appears to inhibit egg deposition, presumably by impeding egg transport along the oviducts. Similarly, in dsxGal4 females expressing TNT no egg deposition is ever observed, with unfertilized eggs atrophying in the lateral oviducts. In contrast, when fru+ neurons are silenced, deposition of successfully fertilized eggs is still observed, suggesting that different subsets of the dsx+/fru+/ppk+ SP-responsive sensory circuit may direct distinct postmating behavioral responses. As SP has been detected in the hemolymph of mated females, it has been suggested that this peptide could pass from the reproductive tract into the hemolymph to reach CNS targets. The fact that neither receptivity nor oviposition was restored to control levels when ppk-Gal80 (or Cha-Gal80) was expressed in dsxGal4/UAS-mSP flies opens the possibility that SP expression might affect additional dsx neurons in the CNS (Rezával, 2012).

Triggering of postmating responses via SP reception appears to occur via a small number of neurons expressing SPR on the female reproductive tract; however, SPR is also found on surface regions of the CNS as well as in endocrine glands and other reproductive tissues. Surprisingly, SPR may even be detected in the Drosophila male CNS, where no exposure to SP would be expected, and in insects that apparently lack SP-like. SPRs are therefore potentially responsive to other ligands, performing functions other than those associated with postmating responses in the diverse tissues in which SPRs are expressed (Rezával, 2012).

Extensive coexpression was found of dsx-expressing cells and SPR in the epithelium of the lower oviduct and spermathecae in females. However, mSP expression (or SPR downregulation) specifically in spermathecal secretory cells (SSC) or oviduct epithelium cells had no effect on receptivity or egg laying. In agreement with rescue experiments using neuronal Gal80 drivers to intersect Gal4-responsive UAS expression in dsx cells, this suggests that these cells are neither neuronal nor directly involved in SP-mediated postmating behaviors. SPR staining in the CNS was more difficult to determine given the limitations of the antibody; while no colocalization in the brain was observed, apparent coexpression was observed between SPR and a small subset of ventral (Rezával, 2012).

The results indicate that dsx-Abg neurons are required for the induction and regulation of specific components of the postmating response. It has been shown that inhibition of neurotransmission in apterous-expressing Abg neurons impairs SP-mediated postmating changes in receptivity and oviposition, emphasizing the importance of these neurons in the modulation of postmating responses (Rezával, 2012).

The level of dsx neuronal expression within the Abg and their associated fascicles projecting to the brain, where they form extensive presynaptic arborizations within the SOG, coupled with the effects that impairment of function in these neurons has on postmating responses, speaks to the involvement of these neurons in relaying information from the reproductive tract to the brain. That dsx-Abg neurons also project, and form presynaptic arborizations on the uterus, and that the effects on postmating responses when their function is impaired again argue that these neurons play a direct role in mediating processes such as egg fertilization and oviposition. Interestingly, most dsx intersecting neurons are specific to females. Sex-specific behaviors can arise from either shared circuits between males and females that operate differently and/or sex-specific circuits that result from the presence/absence of unique circuit components in one sex versus the other. The results support the latter (Rezával, 2012).

The VNC has been implicated in the modulation of postmating responses, with an identified focus specifically involved in ovulation and transfer of eggs into the uterus for fertilization. Octopaminergic modulatory neurons located at the distal tip of the VNC projecting to the reproductive tract are required for triggering ovulation, possibly by regulating muscle contractions in the ovaries and oviducts. Since earlier studies have shown that ablation of the pars intercerebralis revealed an additional focus for egg laying in the head, and the brain appears to be required for sexual behaviors, such that decapitated virgin females neither mate nor lay eggs, it seems likely that neurons in the Abg also require signals from the brain to regulate postmating responses such as egg transport, fertilization, and deposition (Rezával, 2012 and references therein).

Higher-order circuits in the female brain must be capable of integrating sensory inputs from the olfactory, auditory, and reproductive systems to decide between the alternative actions of acceptance or rejection of the male. Early gynandromorph studies mapped a region of the dorsal brain that must be female for an animal to be receptive; it has been recently shown that the majority of dsx neuronal clusters are located in this region. While neurons coexpressing dsx and fru in male brains define a more restricted circuitry for determining male mating decisions, in females no overlap between dsx+ and fru+ neurons is observable in the brain. It is also important to note that the sex-specific Fru isoform is absent in females; thus any circuits that are actively specified in the female are likely to depend on the female isoform DsxF. Most dsx neurons in the brain are found in the lateral protocerebrum, a region where multiple sensory inputs are thought to be integrated and discrete motor actions selected and coordinated. Further high-resolution functional and connectivity mapping will help to define which neurons participate in specific pre- and postmating behaviors in the female, allowing circuit architecture to be integrated with underlying cellular and synaptic properties. Future experiments will define what activity patterns trigger these behaviors and what activity patterns correlate with these behaviors (Rezával, 2012).

doublesex functions early and late in gustatory sense organ development

Somatic sexual dimorphisms outside of the nervous system in Drosophila melanogaster are largely controlled by the male- and female-specific Doublesex transcription factors (DSXM and DSXF, respectively). The DSX proteins must act at the right times and places in development to regulate the diverse array of genes that sculpt male and female characteristics across a variety of tissues. To explore how cellular and developmental contexts integrate with doublesex (dsx) gene function, this study focused on the sexually dimorphic number of gustatory sense organs (GSOs) in the foreleg. Tha DSXM and DSXF were shown to promote and repress GSO formation, respectively, and their relative contribution to this dimorphism varies along the proximodistal axis of the foreleg. The results suggest that the DSX proteins impact specification of the gustatory sensory organ precursors (SOPs). DSXF then acts later in the foreleg to regulate gustatory receptor neuron axon guidance. These results suggest that the foreleg provides a unique opportunity for examining the context-dependent functions of DSX (Mellert, 2012).

dsx regulates the sexually dimorphic number of GSOs across all tarsal segments of the foreleg: DSXM promotes and DSXF represses the development of certain GSOs. The effects of this regulation are apparent by 8 h APF, when the GSOs are first identified, and the spatiotemporal pattern of DSX implies that dsx determines the number of gustatory SOPs. dsx exhibits a surprising degree of context sensitivity: the relative importance of DSXM and DSXF varies along the proximodistal axis of the foreleg and, during the course of GSO development, DSXF progresses from regulating cell fate to regulating axon guidance (Mellert, 2012).

Given that dsx controls the formation of the other sexually dimorphic cuticular structures of the fly, as well as the number of GSOs in segment T1 of the foreleg, it was anticipated that dsx would regulate the sex-specific GSO numbers in segments T2-T4 of the foreleg. However, the manner in which this regulation is achieved across the tarsal segments was surprising. Although each of the T1-T4 foreleg tarsal segments produces more GSOs in males than in females, in two segments this difference is achieved by promoting formation of several GSOs in males (via the action of DSXM), in one segment it is achieved by repressing the formation of several GSOs in females (via the action of DSXF), and in another segment, both DSXM and DSXF act to regulate GSO number. This is more complicated than the simpler a priori expectation that the function of dsx would be the same across the T1-T4 foreleg segments (Mellert, 2012).

That DSXM and DSXF can be utilized differentially has been previously established. In the fat body, female-specific expression of Yp1 and Yp2 depends on up-regulation by DSXF in females and down-regulation by DSXM in males. Thus, in dsx null flies, both sexes express these genes at equivalent levels. Similarly, DSXF activates and DSXM represses expression at the bric-a-brac locus to generate sex-specific pigmentation in the abdominal epithelium. In these two cases, both DSX proteins contribute to regulation of a single trait, similar to the regulation of GSO number in T2. In contrast, desatF is activated by DSXF in oenocytes to produce female-specific pheromones without influence from DSXM. This single isoform-mediated regulation bears similarity to the regulation of foreleg GSOs in T1, T3 and T4. Whereas the previous studies found that DSXM or DSXF were differentially utilized to sculpt sexually dimorphic traits arising from developmentally distinct tissues, this study found that these transcription factors can be differentially utilized across a single developmental field-the epithelium of the foreleg disc. Moreover, the differential roles of DSXM and DSXF in different tarsal segments suggest that each segment may have independently evolved a molecular mechanism for integrating sexual and proximodistal axis information within the foreleg disc to produce more GSOs in the male (Mellert, 2012).

Attempts were made to determine when the function of dsx impacts neurogenesis to generate the numbers of GSOs. Although the details of foreleg GSO development have not been specifically reported, studies of the mechanosensory macrochaete lineages of the notum provide a basic framework for the multi-step process of sensory organ neurogenesis. The initiating event is patterned expression of the proneural genes ac and sc, which imparts the potential to produce SOPs to specific clusters of epithelial cells across the disc epithelium. Subsequent cell-cell interactions within the cluster typically specify a single SOP. The nascent SOP must then sustain its fate and undergo a series of stereotyped cell divisions to produce all of the cells of the sensory organ. Any of the molecular processes that underlie these stages could be influenced by the functions of dsx (Mellert, 2012).

Of interest was the broad distribution of the DSX proteins across the T1-T4 foreleg disc epithelium before and at 0 h APF, a time when the gustatory SOPs are specified. Because the number of DSX-positive cells far exceeded the number of gustatory SOPs necessary to give rise to the GSOs, it was inferred that dsx is acting prior to or during SOP formation. This is consistent with the frequent colocalization of DSX with AC in proneural clusters, which suggests that dsx might act within these cells to determine whether the SOP fate is promoted in males or repressed in females. The broad distribution of the DSX proteins could ensure that sexual information is available for integration with positional information across the foreleg disc epithelium to guide sexually dimorphic development (Mellert, 2012).

In contrast to T2-T4, DSX was not apparent in the foreleg epithelium of T5, which produces a sexually monomorphic GSO number. Thus, the presence of DSX in the epithelium correlates with the adult sexual dimorphism in GSO number, consistent with the notion that dsx is expressed at the right time and place to impact SOP selection in the foreleg. However, at 0 h APF DSX was observed in two nascent sensory organs expressing ase-lacZ. It is speculated that these sensory organs correspond to the GSOs containing GRNs that express pickpocket 25 (ppk25), which is enriched in males and required for their normal response to female pheromones. Thus, the presence of DSX in the nascent GSOs may forecast sexually dimorphic gene expression in the adult GSO (Mellert, 2012).

In addition to specifying foreleg GSO numbers, a temporally distinct function was observed for dsx in the GRNs. During pupal development, GRN axons project proximally along the leg nerves and into the VNC, and here the behavior of the axon depends on the activities of FRUM or DSXF. In males, FRUM promotes crossing of the VNC midline by the axons, but in females, DSXF represses this behavior. This dual regulation causes GRN axons to project across the VNC midline only in males. Two competing hypotheses have been proposed to explain the action of DSXF: 1) DSXF directly affects axon guidance in differentiating GRNs; or 2) only male-specific GRNs are competent to cross the VNC midline and DSXF indirectly affects midline crossing by repressing formation of the male-specific GSOs. Having shown that post-mitotic expression of DSXF in FruM-expressing GRNs subsequent to the establishment of GSO number prevents midline crossing, the second hypothesis is now rejected. Moreover, the early sexual information that impacts GSO number does not irreversibly determine sex-specific development of the GRNs as they continue to be sensitive to the action of DSXF (and presumably FRUM). Because dsx and fru are classically thought of as acting in parallel, it was intriguing to find both genes regulating the same phenotype in a common set of GRNs. Determining whether they coregulate a common set of target genes or independently regulate distinct targets will be of great interest (Mellert, 2012).

Because DSXM and DSXF differentially impact GSO numbers in different tarsal segments, and DSXF regulates the later process of axon guidance, the identity of the genes directly regulated by dsx during foreleg development likely changes with spatiotemporal context. Although it is currently unknown which genes are directly regulated by dsx in the foreleg epithelium or the GSO lineage, the available data on in vivo DSX binding sites may reveal genes that are known to be involved in peripheral neurogenesis or axon guidance. The challenge will then be to determine if such candidates exhibit sexually dimorphic expression in the different tarsal segments at different developmental time points. In this way, development of the foreleg GSOs presents a unique opportunity for investigating how dsx function is integrated with spatiotemporal context across a changing developmental landscape (Mellert, 2012).

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation

Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. This study greatly expands this network through an RNAi-screen of 558 transcription factors. Twenty-eight genes were identified, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. Many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. Pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution (Rogers, 2014).

Genetic identification and separation of innate and experience-dependent courtship behaviors in Drosophila

Wild-type D. melanogaster males innately possess the ability to perform a multistep courtship ritual to conspecific females. The potential for this behavior is specified by the male-specific products of the fruitless (fruM) gene; males without fruM do not court females when held in isolation. Such fruM null males acquire the potential for courtship when grouped with other flies; they apparently learn to court flies with which they were grouped, irrespective of sex or species and retain this behavior for at least a week. The male-specific product of the doublesex gene dsxM is necessary and sufficient for the acquisition of the potential for such experience-dependent courtship. These results reveal a process that builds, via dsxM and social experience, the potential for a more flexible sexual behavior, which could be evolutionarily conserved as dsx-related genes that function in sexual development are found throughout the animal kingdom (Pan, 2014).

For nearly 100 years male courtship behavior in D. melanogaster has been recognized as a robust, complex, and largely innate behavior: a male fly is fully capable of performing all steps of courtship behavior when raised in complete isolation from egg to adulthood and then presented with a female fly as his first encounter with another creature. Thus male courtship has been used as a model system for the analysis of such topics as, how innate behaviors are elicited by specific environmental cues and how sequential motor programs are coordinated (Pan, 2014).

One of the most significant findings with respect to courtship behavior during the last decade is that a single gene (fruM) is both necessary and sufficient for building the potential of courtship behavior into a dedicated courtship circuitry. This study shows that while courtship behavior is abolished in fruM null males that are raised in isolation, a condition used by most studies in this field, many steps of courtship behavior can be alternatively established simply by group-housing fruM null males with either male or female flies for 1 or more days prior to testing. It was further demonstrated that such fruM-independent, experience-dependent courtship is genetically specified by the dsx gene, whose expression significantly overlaps that of fruM in the CNS. Finally, this study shows that the experience-dependent acquisition of the potential for courtship has properties indicative of learning and memory, but is independent of mushroom bodies. Integrating these results with previous findings deepens our understanding of both the genetic and neuronal underpinnings of courtship (Pan, 2014).

Numerous studies have contributed to identifying fruM as a dedicated regulatory gene that specifies the neural substrates of D. melanogaster male courtship and showing that fruM largely functions to regulate fine neural connectivity and/or neural physiology. Recent findings have highlighted the importance of dsx-expressing neurons, and in particular those that also express fruM, in male courtship. Of particular relevance, in the light of the discovery of a fruM-independent, dsx-dependent, and experience-dependent courtship pathway, is the finding that artificial activation of all dsx neurons elicits courtship by males independent of whether they had functional fruM. Approximately two-thirds of all dsx-expressing CNS neurons are found in the ventral nerve cord, and in particular the abdominal ganglion, where they likely function in the execution of sexual behaviors. This leaves five bilaterally present clusters of dsx-expressing neurons in the brain (300 neurons total counting both hemispheres) as likely containing the dsx neurons that mediate the acquisition of experience-dependent courtship. Of these five clusters of dsx-expressing neurons, the male-specific PC1 (also termed P1) cluster, which expresses both fruM and dsxM, is a particularly attractive candidate for having a significant role in experience-dependent courtship, based on its key role in initiating fruM-dependent courtship (Pan, 2014).

These findings add significantly to understanding the role of dsxM in specifying male courtship behavior. Previous studies showed that in males that are wild-type for fruM one specific aspect of male courtship (e.g., sine song) is dependent on dsxM function. Thus, it is likely that the potential for sine song is innately established in CNS by dsxM in a manner analogous to how the potential for fruM-dependent aspects of courtship are innately established. Additionally, in dsx null males that are wild-type for fruM there is a poorly understood deficit in the overall level of courtship (as measured by the CI), but all steps of courtship occur, except for sine song and copulation itself, which is mechanically not possible due to dsx-dependent defects in genital development. These results reveal additional roles of dsxM in the acquisition of the potential for courtship in the absence of fruM function. This reasoning suggests that dsxM functions both to facilitate acquisition of the potential for many aspects of courtship (in the absence of fruM) and to (in the presence of fruM) innately determine at least one aspect of courtship—sine song (Pan, 2014).

As noted above, the sex determination genes fruM and dsxM in males function developmentally to build some aspects of courtship behavior into the CNS. Although the majority of neurons comprising the courtship circuitry are still present in fruM null males, they do not function effectively in transducing sensory cues to motor centers that execute courtship behavior. Strikingly, group-housing experience allows efficient transduction from sensory cues to motor centers when fruM is not expressed. Thus social experience acting via dsxM-mediated processes somehow compensates for many aspects of fruM function. It is noted that other aspects of courtship behavior (e.g., attempted copulation) are not observed in fruM null males, even after they have been group-housed, suggesting that the latter aspects of courtship are solely fruM-dependent (Pan, 2014).

How does social experience change the courtship circuitry in the absence of fruM? It is noted that many recent studies on flies have found that social experience can change gene expression, synaptic connectivity, and/or pheromone profiles. As this study showed that when fruM null males that had been group housed were isolated and then singly housed for 7 days, they still courted fresh females intensively, it is unlikely that changed pheromone profiles, if any, play essential roles in establishing courtship behavior in fruM null males. Rather, it is suggested that social experience induces courtship in fruM null males by changing gene expression and/or neuronal connectivity to allow efficient transduction from sensory perception to motor output. Whether social experience functioning through dsxM during adulthood and fruM functioning during development, act through identical or synonymous mechanisms to specify the courtship circuitry is unknown and awaits further study. In this regard, it is noted that the experience- dependent acquisition of the potential for male courtship behavior during adulthood provides a robust single fly paradigm for learning that may facilitate studies of learning at a variety of levels (Pan, 2014).

Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila

How do evolved genetic changes alter the nervous system to produce different patterns of behavior? This question was addressed using Drosophila male courtship behavior, which is innate, stereotyped, and evolves rapidly between species. D. melanogaster male courtship requires the male-specific isoforms of two transcription factors, Fruitless and Doublesex. These genes underlie genetic switches between female and male behaviors, making them excellent candidate genes for courtship behavior evolution. Their role in courtship evolution was tested by transferring the entire locus for each gene from divergent species to D. melanogaster. It was found that despite differences in Fru+ and Dsx+ cell numbers in wild-type species, cross-species transgenes rescued D. melanogaster courtship behavior and no species-specific behaviors were conferred. Therefore, fru and dsx are not a significant source of evolutionary variation in courtship behavior (Cande, 2014).

Sex differences in Drosophila somatic gene expression: Variation and regulation by Doublesex

Sex differences in gene expression have been widely studied in Drosophila melanogaster. Sex differences vary across strains, but many molecular studies focus on only a single strain or on genes that show sexually dimorphic expression in many strains. How extensive variability is and whether this variability occurs among genes regulated by sex determination hierarchy terminal transcription factors is unknown. To address these questions, this study examined differences in sexually dimorphic gene expression between two strains in Drosophila adult head tissues. Gene expression was also analyzed in doublesex (dsx) mutant strains to determine which sex-differentially expressed genes are regulated by DSX and the mode by which DSX regulates expression. Substantial variation was found in sex-differential expression. The sets of genes with sexually dimorphic expression in each strain show little overlap. The prevalence of different DSX regulatory modes also varies between the two strains. Neither the patterns of DSX DNA occupancy, nor mode of DSX regulation explain why some genes show consistent sex-differential expression across strains. This study found that the genes identified as regulated by DSX in this study are enriched with known sites of DSX DNA occupancy. Finally, it was found that sex-differentially expressed genes and genes regulated by DSX are highly enriched on the fourth chromosome. These results provide insights into a more complete pool of potential DSX targets, as well as reveal the molecular flexibility of DSX regulation (Arbeitman, 2016).


REFERENCES

Ahmad, S. M. and Baker, B. S. (2002). Sex-specific deployment of FGF signaling in Drosophila recruits mesodermal cells into the male genital imaginal disc. Cell 109: 651-661. 12062107

An, W. and Wensink, P. C. (1995a). Integrating sex- and tissue-specific regulation within a single Drosophila enhancer. Genes Dev 9: 256-266. PubMed Citation: 7851798

An, W. and Wensink, P. C. (1995b). Three protein binding sites form an enhancer that regulates sex- and fat body-specific transcription of Drosophila yolk protein genes. EMBO J. 14(6): 1221-1230. PubMed Citation: 7720712

An, W., et al. (1996). Sex-specific and non-sex-specific oligmerization domains in both of the doublesex transcription factors in Drosophila melanogaster. Mol. Cell Biol. 16: 3106-11. PubMed Citation: 8649422

Arbeitman, M. N., et al. (2004). A genomic analysis of Drosophila somatic sexual differentiation and its regulation. Development 131: 2007-2021. 15056610

Arbeitman, M. N., New, F., Fear, J. M., Howard, T. S., Dalton, J. E. and Graze, R. M. (2016). Sex differences in Drosophila somatic gene expression: Variation and regulation by Doublesex. G3 (Bethesda) ;6(7):1799-808. PubMed ID: 27172187

Arthur, B. I., et al. (1998). Sexual behaviour in Drosophila is irreversibly programmed during a critical period. Curr. Biol. 8(21): 1187-90. PubMed Citation: 9799737

Billeter, J. C., et al. (2006). Isoform-specific control of male neuronal differentiation and behavior in Drosophila by the fruitless gene. Curr. Biol. 16(11): 1063-76. 16753560

Bray, S. and Amrein, H. (2003). A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship. Neuron 39: 1019-1029. 12971900

Burtis, K. C., and Baker, B. S. (1989). Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell 56: 997-1010. PubMed Citation: 2493994

Cande, J., Stern, D. L., Morita, T., Prud'homme, B. and Gompel, N. (2014). Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila. Cell Rep 8(2): 363-70. PubMed ID: 25017068

Chase, B. A. and Baker, B. S. (1995). A genetic analysis of intersex, a gene regulating sexual differentiation in Drosophila melanogaster females. Genetics 139: 1649-1661. PubMed Citation: 7789766

Chatterjee, S. S., et al. (2011). The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila. Development 138(6): 1099-109. PubMed Citation: 21343364

Cho, S., and Wensink, P. C. (1996). Purification and physical properties of the male and female double sex proteins of Drosophila. Proc. Natl. Acad. Sci. 93: 2043-47. PubMed Citation: 8700882

Clyne, J. D. and Miesenböck, G. (2008). Sex-specific control and tuning of the pattern generator for courtship song in Drosophila. Cell 133: 354-363. PubMed Citation: 18423205

Dauwalder, B., et al. (2002). The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior. Genes Dev. 16: 2879-2892. 12435630

DeFalco, T. J., et al. (2003). Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad. Dev. Cell 5: 205-216. 12919673

DeFalco, T., Camara, N., Le Bras, S. and Van Doren, M. (2008). Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad. Dev. Cell 14(2): 275-86. PubMed Citation: 18267095

Demir, E. and Dickson, B. J. (2005). fruitless splicing specifies male courtship behavior in Drosophila. Cell 121: 785-794. 15935764

Ditch, L. M., et al. (2005). Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors. Development 132: 155-164. PubMed Citation: 15576402

Du, C., et al. (1998). Protein phosphorylation plays an essential role in the regulation of alternative splicing and sex determination in Drosophila. Mol. Cell 2(6): 741-50. PubMed Citation: 9885562

Eldridge, A. G., et al. (1999). The SRm160/300 splicing coactivator is required for exon-enhancer function. Proc. Natl. Acad. Sci. 96(11): 6125-30. PubMed Citation: 10339552

Erdman, S. E. and Burtis, K. C. (1993). The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain. EMBO J. 12:527-35. PubMed Citation: 8440242

Erdman, S. E., Chen, H. J. and Burtis, K. C. (1996). Functional and genetic characterization of the oligomerization and DNA binding properties of the Drosophila Doublesex proteins. Genetics 144(4): 1639-1652. PubMed Citation: 8978051

Finley, K. D., et al. (1997). dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster. Proc. Natl. Acad. Sci. 94: 913-918. PubMed Citation: 9023356

Foronda, D., Martín, P., Sánnchez-Herrero, E. (2012). Drosophila Hox and sex-determination genes control segment elimination through EGFR and extramacrochetae activity. PLoS Genet. 8(8): e1002874. PubMed Citation: 22912593

Fujii, S. and Amrein, H. (2002). Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology. EMBO J. 21: 5353-5363. 12374736

Garrett-Engele, C. M., et al. (2002). intersex, a gene required for female sexual development in Drosophila, is expressed in both sexes and functions together with doublesex to regulate terminal differentiation. Development 129: 4661-4675. 12361959

Grmai, L., Hudry, B., Miguel-Aliaga, I. and Bach, E. A. (2018). Chinmo prevents transformer alternative splicing to maintain male sex identity. PLoS Genet 14(2): e1007203. PubMed ID: 29389999

Flaherty, M. S., Salis, P., Evans, C. J., Ekas, L. A., Marouf, A., Zavadil, J., Banerjee, U. and Bach, E. A. (2010). chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila. Dev Cell 18(4): 556-568. PubMed ID: 20412771

Haag, E. S. and Doty, A. V. (2005). Sex determination across evolution: connecting the dots. PLoS Biol. 3(1): e21. 15660158

Hall, J. C. (1979). Control of male reproductive behavior by the central nervous system of Drosophila: dissection of a courtship pathway by genetic mosaics. Genetics 92: 437-457. PubMed Citation: 114447

Heinrichs, V. and Baker, B. S. (1995). The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences. EMBO J. 14: 3987-4000. PubMed Citation: 7664738

Herndon, L. A. and Wolfner, M. F. (1995). A Drosophila seminal fluid protein. Acp26Aa, stimulates egg laying in females for 1 day after mating. Proc. Natl. Acad. Sci. 92: 10114-8. PubMed Citation: 7479736

Hittinger, C. T. and Carroll, S. B. (2007). Gene duplication and the adaptive evolution of a classic genetic switch. Nature 449: 677-681. PubMed Citation: 17928853

Hoshijima, K., Inoue, K., Higuchi, I., Sakamoto, H. and Shimura, Y. (1991). Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science 252: 833-836. 1902987

Hotta, Y, and Benzer, S. (1972). Mapping of behaviour in Drosophila mosaics. Nature 240: 527-535. PubMed Citation: 4568399

Huang, X., Hong, C. S., O'Donnell, M. and Saint-Jeannet, J. P. (2005). The doublesex-related gene, XDmrt4, is required for neurogenesis in the olfactory system. Proc. Natl. Acad. Sci. 102(32): 11349-54. PubMed Citation: 16061812

Inoue, K., et al. (1992). Binding of the Drosophila transformer and transformer-2 proteins to the regulatory elements of doublesex primary transcript for sex-specific RNA processing. Proc. Natl. Acad. Sci. 89: 8092-6. PubMed Citation: 1518835

Jursnich, V. A. and Burtis, K. C. (1993). A positive role in differentiation for the male doublesex protein of Drosophila. Dev. Biol. 155: 235-49. PubMed Citation: 8416836

Keisman, E. L. and Baker, B. S. (2001a). The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc. Development 128: 1643-1656. 11290302

Keisman, E. L., Christiansen, A. E. and Baker, B. S. (2001b). The sex determination gene doublesex regulates the A/P organizer to direct sex-specific patterns of growth in the Drosophila genital imaginal disc. Dev. Cell 1: 215-225. PubMed Citation: 11702781

Kimura, K.-i., et al. (2008). Fruitless and Doublesex coordinate to generate male-specific neurons that can initiate courtship. Neuron 59: 759-769. PubMed Citation: 18786359

Kopp, A., Duncan, I. and Carroll, S. B. (2000). Genetic control and evolution of sexually dimorphic characters in Drosophila. Nature 408: 553-559. 11117736

Le Bras, S and Van Doren, X. (2006). Development of the male germline stem cell niche in Drosophila. Dev. Biol. 294(1): 92-103. 16566915

Li, H. and Baker, B. S. (1998). hermaphrodite and doublesex function both dependently and independently to control various aspects of sexual differentiation in Drosophila. Development 125: 2641-2651. PubMed Citation: 9636079

Li, Y. and Blencowe, B. J. (1999). Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract. J. Biol. Chem. 274(49): 35074-9. 10574987

Loh, S. H. Y. and Russell, S. (2000). A Drosophila group E Sox gene is dynamically expressed in the embryonic alimentary canal. Mech. Dev. 93: 185-188. 10781954

Luo, S. D., Shi, G. W. and Baker, B. S. (2011). Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development. Development 138(13): 2761-71. PubMed Citation: 21652649

Lynch, K. W. and Maniatis, T. (1995). Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 9: 284-293. PubMed Citation: 7867927

Lynch, K. W. and Maniatis, T. (1996). Assembly of specific SR protein complexes on distinct regulatory element of the Drosophila doublesex splicing enhancer. Genes Dev. 10: 2089-2101. PubMed Citation: 8769651

Lossky, M. and Wensink, P. C. (1995). Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor. Mol. Cell. Biol. 15: 6943-6952. PubMed Citation: 8524261

Mason, D. A., Rabinowitz, J. S. and Portman, D. S. (2008). dmd-3, a doublesex-related gene regulated by tra-1, governs sex-specific morphogenesis in C. elegans. Development 135: 2373-2382. PubMed Citation: 18550714

Matsuda, M., et al. (2002). DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417: 559-563. 12037570

Mellert, D. J., Knapp, J. M., Manoli, D. S., Meissner, G. W. and Baker, B. S. (2010). Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors. Development 137(2): 323-32. PubMed Citation: 20040498

Mellert, D. J., Robinett, C. C. and Baker, B. S. (2012). doublesex functions early and late in gustatory sense organ development. PLoS One 7: e51489. PubMed ID: 23240029

Meng, A., et al. (1999). A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates. Development 126: 1259-1268. PubMed Citation: 10021344

Mohamad Ishak, N. S., Nong, Q. D., Matsuura, T., Kato, Y. and Watanabe, H. (2017). Co-option of the bZIP transcription factor Vrille as the activator of Doublesex1 in environmental sex determination of the crustacean Daphnia magna. PLoS Genet 13(11): e1006953. PubMed ID: 29095827

Murphy, M. W., Zarkower, D. and Bardwell, V. J. (2007). Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA. BMC Mol. Biol. 8:58. PubMed Citation: 17605809

Murphy, M. W., et al, (2010). Genome-wide analysis of DNA binding and transcriptional regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis. Proc. Natl. Acad. Sci. 107(30): 13360-5. PubMed Citation: 20616082

Nanda, I., et al. (2002). A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc. Natl. Acad. Sci. 99(18): 11778-83. 12193652

Pan, Y. and Baker, B. S. (2014). Genetic identification and separation of innate and experience-dependent courtship behaviors in Drosophila. Cell 156: 236-248. PubMed ID: 24439379

Pane, A., et al. (2002). The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate. Development 129: 3715-3725. 12117820

Pane, A., et al. (2005). Evolutionary conservation of Ceratitis capitata transformer gene function. Genetics 171(2): 615-24. 15998727

Peng, X. and Mount, S. M. (1995). Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor. Mol Cell Biol 15: 6273-6282. PubMed Citation: 7565780

Raymond, C. S., et al. (1998). Evidence for evolutionary conservation of sex-determining genes. Nature 391(6668):691-695. PubMed Citation: 9490411

Raymond, C. S. et al. (1999). A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Hum. Mol. Genet. 8: 989-996. 10332030

Raymond, C. S., et al. (2000). Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev. 14: 2587-2595. PubMed Citation: 11040213

Ren, Q., Awasaki, T., Huang, Y.F., Liu, Z. and Lee, T. (2016). Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain. Curr Biol 26(19):2583-2593. PubMed ID: 27618265

Rezával, C., et al. (2012). Neural circuitry underlying Drosophila female postmating behavioral responses. Curr. Biol. 22(13): 1155-65. PubMed Citation: 22658598

Rice, W. R. (1996). Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature 381: 232-4. PubMed Citation: 8622764

Rideout, E. J., Billeter, J. C. and Goodwin, S. F. (2007). The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song. Curr. Biol. 17(17): 1473-8. Medline abstract: 17716899

Rideout, E. J., et al. (2010). Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster. Nat. Neurosci. 13(4): 458-66. PubMed Citation: 20305646

Robida, M. D. and Singh, R., et al. (2003). Drosophila polypyrimidine-tract binding protein (PTB) functions specifically in the male germline. EMBO J. 22: 2924-2933. 12805208

Rogers, W. A., Salomone, J. R., Tacy, D. J., Camino, E. M., Davis, K. A., Rebeiz, M. and Williams, T. M. (2013). Recurrent modification of a conserved cis-regulatory element underlies fruit fly pigmentation diversity. PLoS Genet 9: e1003740. PubMed ID: 24009528

Rogers, W. A., Grover, S., Stringer, S. J., Parks, J., Rebeiz, M. and Williams, T. M. (2014). A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation. Dev Biol 385: 417-432. PubMed ID: 24269556

Ross, J. M., Kalis, A. K., Murphy, M. W. and Zarkower, D. (2005). The DM domain protein MAB-3 promotes sex-specific neurogenesis in C. elegans by regulating bHLH proteins. Dev. Cell 8(6): 881-92. 15935777

Saccone, G., Peluso, I., Artiaco, D., Giordano, E., Bopp, D., et al. (1998). The Ceratitis capitata homologue of the Drosophila sex-determining gene sex-lethal is structurally conserved, but not sex-specifically regulated. Development 125: 1495-1500. 9502730

Salvemini, M., D'Amato, R., Petrella, V., Aceto, S., Nimmo, D., Neira, M., Alphey, L., Polito, L. C. and Saccone, G. (2013). The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing. PLoS One 8: e48554. PubMed ID: 23418412

Sanchez, L., et al. (1997). The genital disc of Drosophila melanogaster II. Role of the genes hedgehog, decapentaplegic and wingless. Dev. Genes Evol. 207: 229-241

Sanchez, L., Gorfinkiel, N. and Guerrero, I. (2001). Sex determination genes control the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals. Development 128: 1033-1043. 11245569

Scali, P., et al. (2005). Identification of sex-specific transcripts of the Anopheles gambiae doublesex gene. J. Exp. Bio. 208: 3701-3709. 16169947

Sharma, A., Heinze, S. D., Wu, Y., Kohlbrenner, T., Morilla, I., Brunner, C., Wimmer, E. A., van de Zande, L., Robinson, M. D., Beukeboom, L. W. and Bopp, D. (2017). Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22. Science 356(6338): 642-645. PubMed ID: 28495751

Shirangi, T. R. and McKeown, M. (2007). Sex in flies: What 'body-mind' dichotomy? Dev. Biol. 306: 10-19. PubMed Citation: 17475234

Shirangi, T. R., Taylor, B. J. and McKeown, M. (2006). A double-switch system regulates male courtship behavior in male and female Drosophila melanogaster. Nat. Genet. 38: 1435-1439. PubMed Citation: 17086183

Steinmann-Zwicky, M., Anrein, H. and Nöthiger, R. (1990). Genetic control of sex determination in Drosophila. Adv. Genet. 27: 189-237

Taylor, B. J., (1992a). Differentiation of a male-specific muscle in Drosophila melanogaster does not require the sex-determining genes doublesex or intersex. Genetics 132: 179-91

Taylor, B. J. and Truman, J. W. (1992b). Commitment of abdominal neuroblasts in Drosophila to a male or female fate is dependent on genes of the sex-determining hierarchy. Development 114: 625-42

Taylor, B. J., et al. (1994). Behavioral and neurobiological implications of sex-determining factors in Drosophila. Dev Genet 15: 275-296

Tian, M. and Maniatis, T. (1992). Positive control of pre-mRNA splicing in vitro. Science 256: 237-40

Tian, M. and Maniatis, T. (1993). A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell 74: 105-14

Tian, M. and Maniatis, T. (1994). A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 8: 1703-12

Tresser, J., et al. (2010). doublesex/mab3 related-1 (dmrt1) is essential for development of anterior neural plate derivatives in Ciona. Development 137(13): 2197-203. PubMed Citation: 20530547

Tortiglione, C. and Bownes, M. (1997). Conservation and divergence in the control of yolk protein genes in dipteran insects. Dev. Genes Evol. 207: 264-281

Villella, A. and Hall, J. C. (1996). Courtship anomalies caused by doublesex mutations in Drosophila melanogaster. Genetics 143: 331-344

Wang, W. and Yoder, J. H. (2012). Hox-mediated regulation of doublesex sculpts sex-specific abdomen morphology in Drosophila. Dev. Dyn. 241(6): 1076-90. PubMed Citation: 22488883

Waterbury, J. A., Jackson, L. L. and Schedl, P. (1999). Analysis of the Doublesex female protein in Drosophila melanogaster: Role in sexual differentiation and behavior and dependence on Intersex. Genetics 152: 1653-1667

Waterbury, J. A., et al. (2000). Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma. Genetics 155(4): 1741-56.

Williams, T. M., et al. (2008). The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila. Cell 134: 610-623. PubMed Citation: 17928853

Yi, W. and Zarkower, D. (1999). Similarity of DNA binding and transcriptional regulation by Caenorhabditis elegans MAB-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanisms. Development 126: 873-881

Yi, W., Ross, J. M. and Zarkower, D. (2000). mab-3 is a direct tra-1 target gene regulating diverse aspects of C. elegans male sexual development and behavior. Development 127: 4469-4480

Zhang, T., Murphy, M. W., Gearhart, M. D., Bardwell, V. J. and Zarkower, D. (2014). The mammalian Doublesex homolog DMRT6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis. Development 141: 3662-3671. PubMed ID: 25249458

Zhang, T., Oatley, J., Bardwell, V.J. and Zarkower, D. (2016). DMRT1 is required for mouse spermatogonial stem cell maintenance and replenishment. PLoS Genet 12: e1006293. PubMed ID: 27583450

Zhang, W., et al. (2006). Regulation of sexual dimorphism: mutational and chemogenetic analysis of the doublesex DM domain. Mol. Cell. Biol. 26(2): 535-47. 16382145

Zhu, L., et al. (2000). Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers. Genes Dev. 14: 1750-1764. PubMed Citation: 10898790

Zuo, P. and Maniatis, T. (1996). The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev. 10: 1356-1368


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

date revised: 15 December 2016

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.