Interactive Fly, Drosophila

Regulation in genital disc

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).

Dsx^F prevents activation of dpp in the RMP, and consequently no induction of dac expression occurs. In the female genital primordium, dac expression is strongly reduced in the Wg domain and absent in the Dpp domain. Taken together, these results suggest that the development of male and female genital primordia have different time requirements for Dsx^M and Dsx^F products (Sanchez, 2001).

dsx controls which of the two genital primordia will develop and which will be repressed. Nevertheless, since it is expressed in each cell, another gene(s) is required to distinguish between the female and the male genitalia. The female genitalia develop from eighth abdominal segment and the male genitalia develop from ninth abdominal segment. It is also known that Abdominal-B (Abd-B) is responsible for the specification of these posterior segments. It has been proposed that the development of the male and female genitalia requires the concerted action of Abd-B and dsx, and that these two genes control proliferation of each genital primordium through the expression, either directly or indirectly, of dpp and wg. Abd-B produces two different proteins: Abd-Bm and Abd-Br. Abd-Bm is present only in the female genital primordium, whereas Abd-Br is present only in the male genital primordium. It is proposed that Dsx^M and Dsx^F combine with Abd-Bm and Abd-Br to make up the signals that determine the dimorphic sexual development of the genital disc. In the absence of both Dsx^M and Dsx^F products (dsx intersexes), there is a basal expression of dpp and a basal functional level of the Wg signaling pathway in both male and female genital primordia. In females, the concerted signal made up of Dsx^F and Abd-Br cause repression of the development of the male genital primordium by preventing the expression of dpp, resulting in the RMP of female genital discs. In males, the concerted signal formed by Dsx^M and Abd-Bm represses the female genital primordium by blocking the Wg signaling pathway, giving rise to the RFP of male genital discs. It is further proposed that Dsx^M plus Abd-Br increase dpp expression in the male genital primordium of male genital discs, and that Dsx^F plus Abd-Bm enhance Wg signaling pathway function in the female genital primordium of female genital discs. A similar mechanism of modulation of Dpp and Wg responses has been described for the shaping of haltere development by Ultrabithorax. Therefore, Dsx^M would play a positive and a negative role in male and female genital primordia, respectively, whereas Dsx^F would play a positive and a negative role in female and male genital primordia, respectively. This positive role of both Dsx products serves to explain the expression of dpp and the function of the Wg signaling pathway in growing male and female genital primordia, respectively, in dsx ^Mas/+ intersexual flies, where both genital primordia simultaneously have Dsx^M and Dsx^F. Otherwise, dpp would not be expressed in the male genital primordium and the Wg signaling pathway would not be functional in the female genital primordium, as occurs in normal female and male genital discs. If so, this would mean that the two genital primordia of these intersexual genital discs would be kept in the repressed state and would not develop. Contrary to observations, this would result in a lack of male and female adult genital structures in these intersexes (Sanchez, 2001).

Bmp signals directly repress bag of marbles in germline stem cells

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters (cystoblasts). Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for two essential Bmp signal transducers (Med and punt) bam transcription is also elevated. Finally, Med and Mad are shown to directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal (Song, 2004).

This study reveals a new function for gbb in the regulation of GSCs in the Drosophila ovary. Loss of gbb function leads to GSC differentiation and stem cell loss, similar to dpp mutants. gbb is expressed in somatic cells but not in germ cells, suggesting that gbb is another niche signal that controls GSC maintenance. Like dpp, gbb contributes to the production of pMad in GSCs and also functions to repress bam expression in GSCs. As in the wing imaginal disc, gbb also probably functions to augment the dpp signal in the regulation of GSCs through common receptors in the Drosophila ovary. In both dpp and gbb mutants, pMad accumulation in GSCs is severely reduced but not completely diminished. Since the dpp or gbb mutants used in this study do not carry complete loss-of-function mutations, it remains possible that complete elimination of either dpp or gbb function is sufficient for eradicating pMad accumulation in GSCs. Alternatively, both dpp and gbb signaling are required independently for full pMad accumulation in GSCs, and thus disrupting either one of them only partially diminishes pMad accumulation in GSCs. The lethality of null dpp and gbb mutants, and the difficulty in completely removing their function in the adult ovary, prevent these possibilities from being tested directly (Song, 2004).

Interestingly, dpp overexpression results in complete suppression of cystoblast differentiation and complete repression of bam transcription in the germ cells, whereas gbb overexpression does not have obvious effects on cystoblast differentiation or bam transcription. Even though the UAS-gbb transgene and the c587 driver for gbb overexpression have been demonstrated to function properly, it is possible that active Gbb proteins are not produced in inner sheath cells and somatic follicle cells because of a lack of proper factors that are required for Gbb translation and processing in those cells, which could explain why the assumed gbb overexpression does not have any effect on cystoblast differentiation. However, since active Dpp proteins can be successfully achieved using the same expression method, and Dpp and Gbb are closely related Bmps, it is unlikely that active Gbb proteins are not produced in inner sheath cells and follicle cells. Alternatively, dpp and gbb signals could have distinct signaling properties, and dpp may play a greater role in regulating GSCs and cystoblasts. Recent studies have indicated that Dpp and Gbb have context-dependent relationships in wing development. In the wing disc, duplications of dpp are able to rescue many but not all of the phenotypes associated with gbb mutants, suggesting that dpp and gbb have not only partly redundant functions but also distinct signaling properties. In the wing and ovary, gbb and dpp function through two Bmp type I receptors, sax and tkv. The puzzling difference between gbb and dpp could be explained by context-dependent modifications of Bmp proteins, which render different signaling properties in different cell types. It will be of great future interest to better understand what causes Bmps to have distinct signaling properties (Song, 2004).

All the defined niches share a commonality, structural asymmetry, which ensures stem cells and their differentiated daughters receive different levels of niche signals. In order for a niche signal to function differently in a stem cell and its immediately differentiating daughter cell that is just one cell away, it has to be short-ranged and localized. This study shows that Bmp signaling mediated by Dpp and Gbb results in preferential expression of pMad and Dad in GSCs. Bmp signaling appears to elicit different levels of responses in GSCs and cystoblasts, suggesting that the cap cells are likely to be a source for active short-ranged Bmp signals. These observations support the idea that Bmp signals are active only around cap cells. Consistently, when GSCs lose contact with the cap cells following the removal of adherens junctions they move away from the niche and then are lost. As gbb and dpp mRNAs are broadly expressed in the other somatic cells of the germarium besides cap cells, localized active Bmp proteins around cap cells could be generated by localized translation and/or activation of Bmp proteins. As they can function as long-range signals, it remains unclear how Dpp and Gbb act as short-range signals in the GSC niche (Song, 2004).

Bmp signaling and bam expression are in direct opposition in Drosophila ovarian GSCs. bam is actively repressed in GSCs through a defined transcriptional silencer. These observations lead to a model in which Bmp signals from the niche maintain adjacent germ cells as GSCs by actively suppressing bam transcription and thus preventing differentiation into cystoblasts. The levels of pMad are correlated with the amount of bam transcriptional repression in GSCs and cystoblasts. In the wild-type germarium, pMad is highly expressed in GSCs and some cystoblasts where bam is repressed. In other cystoblasts and differentiated germline cysts, pMad is reduced to very low levels, and thus bam transcriptional repression is relieved. In the GSCs mutant for dpp, gbb or punt, pMad levels are severely reduced, and bam begins to be expressed. The repression of bam transcription as a result of dpp overexpression seems to be a rapid process; bam mRNA is reduced to below detectable levels two hours after dpp is overexpressed. This suggests that repression of bam transcription by Bmp signaling could be direct. Furthermore, Med and Mad can bind to the defined bam silencer in vitro, which also supports the idea that Bmp signaling acts directly to repress bam transcription. Dpp signaling has also been shown to repress brinker (brk) expression in the wing imaginal disc and in the embryo. The repression of brk expression by Dpp signaling is mediated by the direct binding of Mad and Med to a silencer element in the brk promoter. Since the brk silencer is very similar to the bam silencer, the results suggest that bam repression in GSCs is also mediated directly by Dpp and Gbb in a similar manner (Song, 2004).

It remains unclear how the binding of Med and Mad to the bam silencer results in bam transcriptional repression in GSCs. For the brk silencer, Dpp signaling and Shn are both required to repress brk expression in the Drosophila wing disc and embryo. Mad and Med belong to the Smad protein family, which are known to function as transcriptional activators by recruiting co-activators with histone acetyltransferase activity. In the wing disc, Shn is proposed to function as a switch factor that converts the activating property of Mad and Med proteins into a transcriptional repressor property. Possibly, the Mad-Med complex could also recruit Shn to the bam repressor element. Consistent with the possible role of Shn in repressing bam expression in GSCs is the observation that GSCs that lose shn function differentiate, and thus are lost. Also, it remains possible that Mad and Med could recruit a repressor other than Shn when binding to the bam repressor element. In the future, it will be very important to determine whether Shn itself is a co-repressor for Mad/Med proteins or whether it directly recruits a co-repressor to repress bam transcription in GSCs (Song, 2004).

Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis.

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling, expression of bam is upregulated, whereas forced bam expression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs (Kawase, 2004).

To determine the sources for Gbb and Dpp in the testis, RT-PCR was used to study the presence of gbb and dpp mRNAs in the purified hub cells, somatic cyst cells and germ cells using fluorescent-activated cell sorting (FACS). The hub cells were marked by the upd-gal4 driven UAS-GFP expression. The somatic cyst cells and somatic stem cells were marked by the c587-gal4-driven UAS-GFP. vasa is a germline-specific gene. The germ cells were marked by a vasa-GFP transgene. The tips of the testes were isolated and dissociated, and the GFP-positive cells were purified from the dissociated testicular cells by FACS. As a control, vasa mRNAs were present in the whole testis and isolated germ cells but were absent in the somatic cyst cells and hub cells. Interestingly, gbb and dpp mRNAs were present in the hub cells and the somatic cysts/somatic stem cells but were absent in the germ cells. In addition, dpp mRNAs appeared to be less abundant than gbb mRNAs in the testis. These results indicate that both Dpp and Gbb are probably somatic cell-derived Bmp signals that directly regulate GSC maintenance in the testis (Kawase, 2004).

Table of contents

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