hermaphrodite

Targets of Activity

To examine whether maternal her function is needed as apositive regulator of the early female-specific Sxl promoter, a lacZ reporter gene construct of the Sxl promoter was used. Fathers homozygous for the reporter gene construct werecrossed to her- mothers and to her + control mothers, and ananti-b-galactosidase monoclonal antibody was used tovisualize protein expression. Among the progeny of controlmothers, there was a striking difference between a class ofdarkly staining embryos and a class of lightly staining embryos. All the embryos fell into two equally represented classes: light and dark, although a few embryosshowed variable intermediate staining levels. It is assumed thatthe darkest class of control embryos are females and thelightest are males. In contrast, none of the progeny of her-mothers stained as intensely as the darkest class of controlembryos; the staining levels were consistently more uniformamong all progeny, as would be expected if daughtersof her - mothers were unable to fully activate the early female-specificSxl promoter. Siblings of these embryos were assayedfor viability; daughters of her- mothers survive to adulthoodabout 4% as often as their brothers, whereas the daughters ofcontrol mothers survive as well as their brothers. This experimentsuggests a role for maternal her products in the transcriptional activation of Sxl. her- flies also fail to repress Maleless (Male-lethal) X-chromosome binding in some cells of dying daughters, indicating a failure of Sxl directed dosage compensation in mutant daughters (Pultz, 1995).

DEVELOPMENTAL BIOLOGY

Embryonic

Northern analysis shows that her istranscribed throughout development. The high level of transcripts in 0-2 hour early embryos is mostlymaternal, since zygotic transcription is negligible in 0-1.5 hourembryos. The level of transcripts drops substantially during 2-4 hours of embryodevelopment, indicating a lag between loss of maternaltranscripts and the synthesis of zygotic transcripts. There is ahigher level of her expression during 4-8 hours of embryodevelopment. After about 8 hours of embryo development, heris transcribed at a low level. The patterns of her transcripts aresimilar in adults of both sexes. The level of her transcripts inadult females is slightly higher (less than two-fold) than inmales, most likely due to maternal transcription in ovaries. The Northern analysis data strongly suggest that there is no sex-specific regulation of herat the level of transcription (Li, 1998a).

Effects of Mutation or Deletion

The hermaphrodite (her) locus has both maternal and zygotic functions required for normal female development in Drosophila. Maternal her function is needed for the viability of female offspring, whilezygotic her function is needed for female sexual differentiation. This study focuses on understanding howher fits into the sex determination regulatory hierarchy. Maternal her function is needed early in thehierarchy: genetic interactions of her with the sisterless genes [sis-a and sis-b (also known as scute)], with function-specificSex-lethal (Sxl) alleles and with the constitutive allele SxlM#1 suggest that maternal her function isneeded for Sxl initiation. When mothers are defective for her function, their daughters fail to activate areporter gene for the Sxl early promoter and are deficient in Sxl protein expression. Dosagecompensation is misregulated in the moribund daughters: some salivary gland cells show binding of themaleless (mle) dosage compensation regulatory protein to the X chromosome, a binding patternnormally seen only in males. Thus maternal her function is needed early in the hierarchy as a positiveregulator of Sxl, and the maternal effects of her on female viability probably reflect Sxl's role inregulating dosage compensation (Pultz, 1995).

In contrast to her's maternal function, her's zygotic function in sexdetermination acts at the end of the hierarchy. Since the maternal effect of her on female viability is due to its role as a positive regulator of Sxl, it seems plausible thatthe zygotic effects of her on female sexual differentiationmight also be through regulation of Sxl. Therefore, a test was carried out of whether the constitutive allele Sxl M#1 can rescue the intersexualphenotype of her females. her-females bearing the Sxl M#1 chromosome have an intersexualphenotype indistinguishable from that of their Sxl +;her - sisters.Since constitutive Sxl expression cannot ameliorate the intersexualityof diplo-X her- individuals, the zygotic aspect of herfunction must not be acting through Sxlm at least not by controllingany aspect of Sxl expression that occurs constitutivelyin the Sxl M#1 allele. This suggests that zygotic her functiondiffers from maternal her function in the level at which it is required within the sex determination regulatory hierarchy. The zygotic effect of her is not rescued by constitutive transformer (tra) expression. The expression of doublesex (dsx) transcripts appears normal in her mutant females. It is concluded that the maternal and zygotic functions of her are needed at two distinctly different levels of the sex determination regulatory hierarchy (Pultz, 1995).

Maternally, her+ function is needed to ensure the viability of femaleprogeny: a partial loss of her+ function preferentially kills daughters. In addition, her has both zygoticand maternal functions required for viability in both sexes. All mutant her alleles exhibit some level of temperature sensitivity. After her mothers have been held at 29 degrees for two days (a temperature that is severly restrictive), progeny of both sexes die as embryos, and the lethal mutant phenotype of embryos becomes progressively more severe as mothers continue to be brooded at 29 degrees. The first obvious defect, detected in the majority of dead progeny, is a differentiated but twisted cuticle, with improperly formed mouthparts due to failure of head involution, and with the posterior abdominal segments wrapped around toward the dorsal side, as though having failed in germ band retraction. As mothers are brooded continuously at 29 degrees for more than three days, the cuticle phenotype of the embryos degenerates such that denticles and sclerotized mouthparts are not differentiated and the cuticle appears to contain holes. The eggshells (chorions) of the embryos also become transparent. There are severe gastrulation defects, including irregular buckling of the germband, unusually deep indentation of the cephalic furrow, and failure to complete the normal process of germband extension and retraction. Ventral furrow formation and differentation of the amnioserosa often appears normal in a collection of embryos with uniformly impaired gastrulation, suggesting that the gastrulation defect is not primarily caused by failure to differentiate mesoderm or by misspecification of cell fate in the dorsal region (Pultz, 1994).

Temperature sensitivity prevails for all known her alleles and for all of the her phenotypes described above, suggesting that her may participate in an intrinsically temperature-sensitive process. This analysis of four her alleles alsoindicates that the zygotic and maternal components of her function are differentially mutable (Pultz, 1994).

Two proteins function together to regulate sex specific genes: (1) the sex specific transcription factor Doublesex (Dsx), and (2) the non-sex specific transcription factor Hermaphrodite. A study by Li and Baker (1998) analyzes the often complex combinatorial interactions between parallel pathways that intersect in the regulation of even a single gene. The targeted yolk protein (yp) genes are transcriptionally activated by two separate pathways. One is a female-specific pathway, which is positively regulated by the female-specific Doublesex protein (Dsx F). The other is a non-sex-specific pathway, that is positively regulated by Her. The Her pathway is prevented from functioning in males by the action of the male-specific Doublesex protein (Dsx M). The Her and Dsx pathways also function independently to control downstream target genes in the precursor cells that give rise to the vaginal teeth and the dorsal anal plate in females, and the lateral anal plates in males. However, a female-specific pathway that is dependent on both Dsx F and Her controls the female-specific differentiation of the foreleg bristles and tergites 5 and 6, and the male-specific differentiation of these tissues does not require the suppression of Her's function by Dsx M (Li, 1998b).

Since the only characterized target genes of dsx are the yp genes, an investigation was undertaken to see if her also regulates the expression of the yp genes and if so, whether her functions in their regulation in a manner similar to dsx . Northern analysis was used to examine the effects of her on expression of the yp genes. Since the complete loss of her function is lethal, the temperature-sensitive allele her 1 was used. At 25¡C, her 1 flies are intersexual and have severely reduced viability; in contrast to this, at 18¡C, they are morphologically normal, and have wild-type viability and fertility. There is a 10-fold activation of yolk protein 2 (yp2) expression by her +, since mutant her females raised at a non-permissive temperature (25¡C) show a 10-fold reduction of yp2 transcript levels, as compared to wild-type females and their her 1/+ sisters. This is comparable to the activation effect of the dsx + gene in females. Surprisingly, yp2 expression is also reduced 10-fold in the her 1 homozygous females raised at 18¡C. However, when grown at 16¡C, her 1 females have levels of yp2 expression comparable to that seen in wild-type females. These results indicate that yp2 expression is more sensitive to the level of her function than is external sexual morphology. In her males, the yp2 transcript level remains unchanged. This is in striking contrast to dsx males where the yp2 level is increased 20-fold compared to that of wild-type males and dsx/+ brothers, consistent with previous findings that Dsx M (the male Dsx splice variant) functions to repress the transcription of the yp genes. This result reflects a fundamental difference between the her and dsx functions in males. The expression of the yp1 and yp3 genes is regulated the same way by dsx and her as is the yp2 gene. It is concluded that Her is required, like Dsx F, for the activation of the yp genes in female fat body cells. But, in contrast to Dsx M , Her is not required for the inhibition of yp gene expression in males (Li, 1998b).

The reduction of yp2 transcripts in her mutant females could be due to the involvement of her in the regulation of yp2 transcription or yp2 RNA stability. To distinguish between the two possibilities, the yp reporter gene pCR1 was used. In the pCR1 construct, the intergenic regulatory region of the divergently transcribed yp1 and yp2 genes remains intact while the coding sequences of yp1 and yp2 are replaced by the Drosophila Adh and the Escherichia coli lacZ genes, respectively. The effects of her and dsx on the expression of the lacZ gene of pCR1 are in all cases comparable to their effects on yp gene expression as monitored by Northern blots, demonstrating that her, like dsx, controls yp gene expression at the level of transcription, rather than RNA stability. Similarly, her + activity is also required in females for the transcriptional activation of the yp genes, rather than the stability of their transcripts. These results demonstrate that her, like dsx, activates the transcription of the yp genes in females through the intergenic region of yp1 and yp2 (Li, 1998b).

The perceptions that derive from the above experiments concerning the roles of dsx and her in regulating the transcription of the yp genes suggest that both genes function in the activation of the yp genes in females, but that only dsx functions in males, where it acts to repress the yps expression. However, consideration of the quantitative aspects of the data from these experiments indicates that this interpretation is incorrect. In particular, the data with respect to the roles of Dsx M in males and Dsx F in females indicate that dsx function can account for all of the difference between the sexes in the levels of yp gene expression. These findings with regard to dsx clearly contradict the idea that there is a female-specific role for her in the activation of the yp genes. Two alternative views of the role of her in regulating yp gene expression are presented that are consistent with these results. The argument that dsx is the major, if not the only, sex-specific regulator of the yp genes derives from the analysis of the transcriptional regulation of pCR1. The pCR1 lacZ activity in dsx/+ females is about 2000-fold higher than in dsx/+ males (no expression of the yp genes). However, the difference is only about 2.6-fold between the dsx homozygous female and male sibs. Since flies homozygous for the X-linked pCR1 transgene were female, the 2.6-fold difference in the pCR1 activity between the dsx females and males is largely, if not entirely, due to the 2-fold difference in the gene dosage of pCR1 between females (two copies of the pCR1 transgene) and males (one copy of the pCR1 transgene). Therefore, these results demonstrate that in the absence of dsx, the yp genes are expressed at the same levels in both sexes (Li, 1998b).

In considering these results, it is important to note that two factors contribute to making the levels of yp gene expression equivalent in dsx mutant males and females: (1) the expression level of the yp genes is elevated in dsx males (compared to wild-type males), due to the absence of repression by Dsx M, and (2) the expression level of the yp genes in dsx females is reduced, due to the absence of activation by Dsx F. Thus in both dsx mutant males and females, there are significant levels of expression of the yp genes, and these levels are equivalent in the two sexes. There are two ways to reconcile these observations with regard to dsx with the observation that her appears to control the expression of the yp genes female-specifically. One model is that her does function female-specifically, but that its female-specific function is dependent on Dsx F. The second model is that her functions sex-independently to activate the expression of the yp genes, but that its action in males is precluded by Dsx M's repression of any yp gene's expression. These two models make different predictions as to the effects expected of her mutants in dsx mutant backgrounds. If the first model is correct, the presence or absence of her should have no effect on the yp genes when Dsx F is absent. If the second model is correct, her should be able to activate the yp genes in dsx mutant males where Dsx M is absent. To examine the effects of her on yp gene expression in the absence of dsx function, the pCR1 reporter gene was used. To test whether her activates yp gene expression in males in the absence of the inhibition by Dsx M, the responsiveness of pCR1 to her regulation was examined in the absence of Dsx M. In males, when Dsx M is present, pCR1 is not expressed whether or not her is present. However, in males without Dsx M, pCR1 is expressed and the pCR1 activity is 5-fold higher when her is present than when her is absent. This finding suggests that wild-type her function is normally present in males and capable of activating the transcription of the yp genes, but its activity is normally overridden by the inhibitory function of Dsx M. In conclusion, there are two separate pathways for the activation of the yp genes. One is the female-specific activation of yp genes, which is Dsx F-dependent. The other is the non-sex-specific activation of ypgenes, which is Her-dependent, Dsx F-independent and inhibited by Dsx M. These results also suggest that her has the same biological function in both sexes, providing further evidence that the expression of her is independent of the sex determination hierarchy. It is further shown that Dsx and Her can activate the yp genes independently in females (Li, 1998b).

Further dissection of the yp promoter reveals that the fat body enhancer (FBE), the site of Dsx action, is not sufficient to confer her responsiveness and the major her responsive element is located outside of the FBE, in the Her responsive region (HRR). Thus, the HRR is necessary for the Her-dependent non-sex-specific activation of yp1 and yp2 (Li, 1998b).

The fact that her and dsx mutant females have similar external phenotypes raises the possibility that dsx and her may regulate other downstream target genes in a manner similar to how they regulate the yp genes. This predicts that the loss of her should masculinize dsx mutant XX flies and vice versa, since Her and Dsx F regulate the yp genes independently. In addition, the loss of her should also masculinize dsx mutant XY flies, since Dsx M inhibits her's activation of the yp genes. To examine whether these predictions are true, a comparison was made of the phenotypes of five different external cuticular structures (which are sexually dimorphic in wild-type adult flies) among XX and XY sibs of the following four genotypes: (1) her/+; dsx/+, (2) her/her; dsx/+, (3) her/+; dsx/dsx and (4) her/her; dsx/dsx. The first cuticular structure examined was the number of the vaginal teeth. In the precursor cells that give rise to vaginal teeth, her and dsx are shown to act independently as in the case of the regulation of the yp genes in fat body. These results show that the loss of her masculinizes dsx mutant XX flies and vice versa, indicating that her + and dsx + can act in each other's absence in these cells (Li, 1998b).

The second set of cuticular structures examined were the anal plates. The dorsal anal plate of females and the two lateral anal plates of males derive from the same precursor cells. In XX and XY intersex flies, there are a pair of anal plates located dorsolaterally to the anal opening and they are often fused at the dorsoanterior side. This pair of anal plates (referred to as DLAP hereafter) represents the intersexual differentiation of the precursor cells, and they are completely fused to form the dorsal anal plate in wild-type females and are completely separated to form the two lateral anal plates in wild-type males. Loss of her masculinizes dsx mutant XX flies and vice versa. These results indicate that, in the precursor cells of vaginal teeth and DLAP, Her controls downstream female-specific differentiation genes non-sex-specifically, and Her's functioning is independent of Dsx F in females and is inhibited by Dsx M in males, analogous to Her's regulation of the yp genes in fat body cells. However, the results also indicate that this is not the only mechanism by which her and dsx act. In the precursor cells of the last (most distal) transverse row of bristles (LTRB) of the basitarsus of the forelegs (LTRB form sex combs in males), Her functions together with Dsx F. In addition to the cuticular structures already described, the number of the 6th sternite (S6) bristles was also examined on dsx mutant, her mutant, and her; dsx mutant XX and XY flies. The results indicate that (1) in XX flies, S6 differentiation follows a default pathway that is independent of dsx (Dsx F ) and her, and (2) in XY flies, S6 differentiation is dependent on both her and dsx (DSX M) (Li, 1998b).

In summary, analysis of sexual phenotypes of various tissues in the her and dsx single mutants and the her; dsx double mutants demonstrates that there are three ways by which sexual dimorphism is generated. The first utilizes DsxM in males and does not require DsxF in females. The second utilizes DsxF in females and does not require DsxM in males. The third utilizes both DsxM in males and DsxF in females. Her is involved in the last two modes of regulation, and likely also in at least some cases of the first mode of regulation. On theoretical grounds, the most parsimonious way to generate differences between homologous tissues in the two sexes during evolution is to have a regulatory gene product present in the tissues of one sex and absent in the other sex, thus affecting the pre-existing non-sex-specific differentiation in one sex, but not in the other. For example, the default pathway for T6 is full pigmentation. The sexual dimorphism of T6 is solely due to the suppression of the T6 pigmentation by Dsx F in females, in collaboration with Her, and is irrespective of the presence or absence of Dsx M in males. Another example is the formation of sixth sternite (S6) bristles. The default pathway is to form 18 bristles on S6. The sexual dimorphism of S6 is caused by the suppression of bristle formation by Dsx M in males, likely in collaboration with Her, and is irrespective of Dsx F in females. However, in the presence of selective pressures on both sexes in evolution, one way to increase sexual dimorphism is to have female- and male-specific products of regulatory genes that each have active roles in modifying the effects of pre-existing non-sex-specific regulatory systems in opposite ways, thus generating dramatic sex-specific features. For instance, in the absence of Dsx F in females and Dsx M in males, the expression levels of the yp genes are equivalent between the two sexes due to non-sex-specific control by Her. When females have Dsx F and males do not have Dsx M, there is a 30-fold difference between females and males in the expression levels of the yp genes, and when females do not have Dsx F and males have Dsx M, there is a 180-fold difference between females and males. However, a maximum difference (2000-fold) is observed only when Dsx F is present in females and Dsx M is present in males. The sexually dimorphic differentiation of the precursor cells of the vaginal teeth and DLAP is similarly controlled by Her and both Dsx proteins. Thus, her may be viewed as part of a non-sex-specific regulatory system in these tissues, which is subject to sex-specific modification by Dsx F and Dsx M (Li, 1998b).

date revised: 8 April 98  

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