doublesex
See the embryonic expression pattern of dsx at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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).
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).
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 (Sánchez, 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 (Sánchez, 1997 and references).
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).
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).
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doublesex:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions and Regulation of Splicing
| Developmental Biology
| Effects of Mutation
date revised: 20 January 2008
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