transformer 2

DEVELOPMENTAL BIOLOGY

Embryonic

The gene transformer-2 of Drosophila is necessary not only for female sexual differentiation but also for normal spermatogenesis in males. The transcript is five to eight times more abundant in females than in males or pseudomales. In both sexes, a low level of transcript is present in the soma and a high level in the germ line. Transcripts are also present in the male soma and in the ovaries where they are not required (but see below). This indicates that tra-2 is regulated in a way different from other sex-determining genes (Amrein, 1988).

Two transcripts, A and B, code for two different isoforms that function redundantly to direct female differention and female specific doublesex pre-mRNA splicing (Mattox, 1996).

Adult

Two alternatively spliced transformer-2 transcripts, each encoding a different putative RNA-binding protein, are found only in the male germ line. These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1. M1-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fails to accumulate in males homozygous for transformer-2 null mutations (Mattox, 1991).

In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform (Type B) is found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving retention of intron 3 (Mattox, 1996).

Effects of Mutation or Deletion

In Drosophila melanogaster adults, a pair of muscles span the fifth abdominal segment of males but not females. To establish whether genes involved in the development of other sexually dimorphic tissues controlled 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 produced the male-specific muscles, whereas female doublesex flies lacked 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, 1992).

The regulation of the yolk protein (YP) genes in the somatic cells of the gonads has been studied, using temperature sensitive mutations (tra-2ts) of transformer-2, a gene required for female sexual differentiation. XX;tra-2ts mutant animals were raised at the permissive temperature so that they developed as females and were then shifted to the restrictive male-determining temperature either 1-2 days before or 0-2 h after eclosion. These animals form vitellogenic ovaries. Likewise, mutant gonads transplanted into either normal female hosts or normal male hosts, kept at the restrictive temperature, undergo vitellogenesis. Thus, the ovarian follicle cells can mature and express their YP genes in the absence of a functional product of the tra-2 gene. Although the gonadal somatic cells of ovary and testis may derive from the same progenitor cells, the testicular cells of XX;tra-2ts pseudomales do not express their YP genes nor take up YP from the hemolymph at the permissive female-determining temperature. It is concluded that in the somatic cells of the gonad, the YP genes are no longer under direct control of the sex-determining genes, but instead are regulated by tissue specific factors present in the follicle cells. It is the formation of follicle cells which requires the activity of tra-2 (Bownes, 1990).

The developmental regulation of three male-specific somatic transcripts is investigated. These RNAs are synthesized exclusively in the adult male accessory gland, an internal tissue derived from the genital disk of Drosophila melanogaster. The expression of these male-specific transcripts (msts) is under the control of the sex determination regulatory hierarchy, as demonstrated by the expression of all three msts in chromosomal females carrying mutant alleles at the doublesex (dsx), intersex (ix), or transformer-2 (tra2) loci. Although transcription of all three male RNAs is initiated late in pupation, temperature shifts of sturtevant/transformer; tra2ts2 homozygotes during development indicate that this expression is irreversibly determined earlier, during the third larval instar. A shift of sturtevant/transformer; tra2ts2 homozygotes to the male-determining temperature, for the duration of the late larval period only, is sufficient to elicit the expression of the msts during the adult stage. This critical period for the determination of these transcripts appears to correlate with the time of morphological determination of the accessory gland in these animals. Thus, the expression of these genes could be specified by the morphological determination of the male-specific tissue in which they are active (Chapman, 1988).

In the male germline of Drosophila the Transformer-2 protein is required for differential splicing of pre-mRNAs from the exuperantia and alternative-testes-transcript (att) genes: Transformer-2 also autoregulates alternative splicing of its own pre-mRNA. The role of alternative splicing in these targets is largely unknown, however in the case of EXU mRNA it has been shown that mutations affecting the alternatively spliced 3' UTR lead to a significant reduction in the level of the EXU mRNA that accumulates in male germ cells. Null mutations in exu result in male sterility and, like tra-2 mutations, lead to formation of spermatids with defects in nuclear elongation. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron produces an mRNA encoding Tra-2²²⁶, which is necessary and sufficient for both male fertility and regulation of downstream target RNAs. When the intron is retained, an mRNA is produced encoding Tra-2¹⁷⁹, a protein with no known function. Repression of M1 splicing is dependent on Tra-2²²⁶, suggesting that this protein quantitatively limits its own expression through a negative feedback mechanism at the level of splicing. This idea is examined by testing the effect that variations in the level of tra-2 expression have on the splicing of M1 and on male fertility. Consistent with the hypothesis, as tra-2 gene dosage is increased, smaller proportions of TRA-2²²⁶ mRNA are produced, limiting expression of this isoform. Feedback regulation is critical for male fertility, since it is significantly decreased by a transgene in which repression of M1 splicing cannot occur and TRA-2²²⁶ mRNA is constitutively produced. The effect of this transgene becomes more severe as its dosage is increased, indicating that fertility is sensitive to an excess of Tra-2²²⁶. These results suggest that autoregulation of Tra-2²²⁶ expression in male germ cells is necessary for normal spermatogenesis (McGuffin, 1998).

Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. Mutations in the Drosophila LAMMER kinase, Darkener of apricot (Doa), alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated in a similar manner as dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing (Du, 1998).

In both sexes, the Drosophila genital disc contains the female and male genital primordia. The sex determination gene doublesex controls which of these primordia will develop and which will be repressed. In females, the presence of Doublesex^F product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of Doublesex^M product results in the development and repression of the male and female genital primordia, respectively. This report shows that Doublesex^F prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas Doublesex^M blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that Doublesex^F is continuously required during female larval development to prevent activation of decapentaplegic in the repressed male primordium, and during pupation for female genital cytodifferentiation. In males, however, it seems that Doublesex^M is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, Doublesex^M does not appear to be needed during pupation for male genital cytodifferentiation. Using dachshund as a gene target for Decapentaplegic and Wingless signals, it was also found that Doublesex^M and Doublesex^F both positively and negatively control the response to these signals in male and female genitalia, respectively. A model is presented for the dimorphic sexual development of the genital primordium in which both Doublesex^M and Doublesex^F products play positive and negative roles (Sanchez, 2001).

dpp is expressed in the growing male genital primordium of male genital discs but not in the repressed male primordium (RMP) of female genital discs. This suggests that the developing or repressed status of the male genital primordium is determined by the regulation of dpp expression. As dsx controls the developmental status of the male genital primordium, and the expression of dpp depends on the Hh signal, the relationship between the Hh signal cascade and dsx in the control of RMP development was examined. To this end, a twin clonal analysis for the loss-of-function tra2 mutation was performed in tra2/+ female genital discs. In this way, the proliferation and the induction of dpp expression was examined in the clones homozygous for tra2 (male genetic constitution) and that of the twin wild-type clones, both in the repressed male and the growing female primordia. Recall that the effects of tra2 in the genital disc are entirely mediated by its role in the splicing of DSX RNA: the presence or absence of functional Tra2 product gives rise to the production of female Dsx^F or male Dsx^M product, respectively. Clones for tra2 (expressing Dsx^M) induced in the RMP of female genital discs show overgrowth and are always associated with dpp expression, indicating that the lower proliferation shown by the RMP is probably caused by the absence of dpp expression. This activation of dpp is restricted to only certain parts of the clone and never overlaps with Wg expression. Since wg is normally expressed in the RMP, the possibility exists that the cells that do not express dpp in the clone are expressing wg, owing to their antagonistic interaction. Double staining of Wg and Dpp in tra2 clones reveals an expansion of the normal domain of wg expression that abuts the dpp-expressing cells (Sanchez, 2001).

In the RMP, the two sister clones are different in size: the tra2 clone (male genetic constitution) is bigger than the wild-type twin clone (female genetic constitution). In contrast, when the clones are induced in the growing female genital primordium, both of them are of a similar size. Moreover, the pattern of dpp expression does not change in the tra2 cells induced in this primordium (Sanchez, 2001).

optomotor-blind, a target of the Dpp pathway, also responds to Dpp in the genital disc. Since dpp is de-repressed in tra2 clones induced in the RMP, the activation of omb was monitored in these clones. The activation of dpp in tra2 clones induces the expression of this target gene, whose function is required for the development of specific male genital structures. It is concluded that Dsx^F product prevents the induction of Dpp by Hh in the repressed male genital primordium of female genital discs (Sanchez, 2001).

In the male genital disc, which has Dsx^M product, the low proliferation rate of the repressed female primordium (RFP) cannot be attributed to a lack of dpp or wg, since both genes are expressed in this primordium. Failure to respond to the Dpp signal may also be ruled out because the RFP expresses the Dpp downstream gene, omb, indicating that the Dpp pathway is active in this primordium. However, Dll, a target gene for both Wg and Dpp, is not expressed in the RFP but is expressed in the developing female primordium of female genital discs. This suggests that the Wg pathway cannot activate some of its targets in the RFP. Thus, the analysis of dsx¹ mutant genital discs, where both male and female genital primordia develop, becomes relevant. These mutant discs show neither Dsx^M nor Dsx^F products. The female genital primordium of these discs now expresses Dll. It is concluded that Dsx^M controls the response to the Wg pathway in the RFP of male genital discs (Sanchez, 2001).

The gene dachsund (dac) is also a target of the Hh pathway in the leg and antenna. In the present study, it was found that dac is differentially expressed in female and male genital discs. In the female genital discs, which have Dsx^F product, dac expression mostly coincides with that of wg in both the growing female primordium and the RMP. In contrast, in male genital discs, which have Dsx^M product, dac is not similarly expressed to wg but its expression partially overlaps that of dpp and no expression is observed in the RFP. In pkA minus clones, which autonomously activate Wg and Dpp signals in a complementary pattern, dac was ectopically expressed only in mutant pkA minus cells at or close to the normal dac expression domains in male and female genital discs. In pkA minus;dpp minus double clones, which express wg, dac is not ectopically induced in the male primordium of the male genital disc, but is still ectopically induced in both the growing female genital primordium and the RMP of female genital disc. Conversely, in pkA minus wg minus double clones, which express dpp, dac is not ectopically induced in the growing female or in the RMP of female genital discs, but is ectopically induced in the growing male primordium of the male genital disc. These results indicate that dac responds differently to Wg and Dpp signals in both sexes (Sanchez, 2001).

In dsx^Mas/+ intersexual genital discs, which have both Dsx^M and Dsx^F products, and in dsx¹ intersexual genital discs, which have neither Dsx^M nor Dsx^F products, dac is expressed in Wg and Dpp domains although at lower levels than in normal male and female genital discs. These results suggest that Dsx^M plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that Dsx^F plays opposing, positive and negative roles in dac expression in female and male genital discs, respectively. To test this hypothesis, tra2 clones (which express only Dsx^M ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, Dsx^F positively regulates dac expression in the Wg domain, and Dsx^M negatively regulates dac expression in this domain, otherwise dac would be expressed in tra2 clones at the low levels found in dsx intersexual genital discs. However, when the tra2 clones are induced in the RMP, in the territory competent to activate dpp, they show ectopic expression of dac (Sanchez, 2001).

Therefore, Dsx^M positively regulates dac expression in the Dpp domain, whereas Dsx^F negatively regulates dac expression in this domain, since in normal female genital discs with Dsx^F dac is not expressed in Dpp territory. This is further supported by the induction of dac in the Wg domain and repression of dac in the Dpp domain by ectopic expression of Dsx^F in the male genital primordium of male genital discs. It is concluded that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2^ts larvae with two X-chromosomes develop into female or male adults if reared at 18°C or 29°C, respectively, because at 18°C they produce Dsx^F and at 29°C they produce Dsx^M. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2_ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2_ts flies was performed using pulses between the male- and the female-determining temperatures in both directions during development (Sanchez, 2001).

Regardless of the stage in development at which the female-determining temperature pulse was given (transitory presence of functional Tra2_ts product; i.e. transitory presence of Dsx^F product and absence of Dsx^M product), the male genital disc develops normal male adult genital structures and not female ones. This occurs even if the pulse is applied during pupation. Pulses of 24 hours at the male-determining temperature (temporal absence of functional Tra2 ts product; i.e. transitory absence of Dsx^F product and presence of Dsx^M product) before the end of first larval stage produces female and not male genital structures. However, later pulses always give rise to male genital structures, except when close to pupation. Further, the capacity of the female genital disc to differentiate adult genital structures is also reduced when the temperature pulse is applied during metamorphosis (Sanchez, 2001).

When the effect of the male-determining temperature pulses was analyzed in the genital disc, it was found that overgrowth of the RMP is always associated with the activation of dpp in this primordium. However, this activation and the associated overgrowth only occurs when the temperature pulse is given after the end of first larval instar. This suggests that there is a time requirement for induction of dpp (Sanchez, 2001).

The activation of this gene in the RMP and the cell proliferation resumed by this primordium, as well as its capacity to differentiate adult structures is irreversible, because they are maintained when the larvae are returned to the female-determining temperature, which is when functional Tra2^ts product is again available (i.e. the presence of Dsx^F product and absence of Dsx^M product). This time requirement for induction of dpp is also supported by the fact that dsx¹¹ clones (which lack Dsx^M) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx¹¹/+ male genital discs (which express only Dsx^M ) after 24 hours of development. However, when the dsx¹¹ clones are induced in the time period between 0 and 24 hours of development, they do not differentiate normally and give rise to incomplete adult male genital structures. This different developmental capacity shown by the dsx¹¹ clones depending on their induction time is explained as follows. When the clones are induced after 24 hours of development, dpp is already activated. Indeed, these clones show no change in the expression pattern of dpp or their targets. Accordingly, these clones display normal proliferation and capacity to differentiate male adult genital structures. However, when the clones are induced early in development, dpp is not yet activated, since this gene is not expressed in the male genital primordium of male genital discs early in development. Therefore, when the male genital disc reaches the state in development when dpp is induced, the cells that form the clones activate this gene as in dsx mutant intersexual flies because the clones have neither Dsx^M nor Dsx^F products. Consequently, these clones do not achieve a normal proliferation rate, and then do not differentiate normal adult male genital structures (Sanchez, 2001).

As described above, it has been shown that dsx regulates the expression of gene dac. Recall that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2^ts flies using pulses between the male- and the female-determining temperatures in both directions. It was found that the dac expression pattern switches from a 'female type' to a 'male type' when male-determining temperature pulses were applied to tra2^ts larvae after first larval instar. Note that dac expression is reduced in the Wg domain of the RMP and is progressively activated in the Dpp domain. It should be remembered that these pulses lead to the transient presence of Dsx^M instead of Dsx^F product. Thus, these results are consistent with the previously proposed suggestion that Dsx^M activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for Dsx^F). When the pulse is given during first larval instar, dac is not activated in the Dpp domain of RMP, in spite of the fact that there is also a transient presence of Dsx^M instead of Dsx^F. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2^ts larvae reach such a developmental stage, these cells now produce Dsx^F because they have returned to the female-determining temperature (Sanchez, 2001).

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 Fru^m), 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).

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date revised: 10 February 2004

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