wingless


REGULATION

Transcriptional Regulation Table of contents

Wingless involvement in hindgut specification

During early embryogenesis in Drosophila, Caudal mRNA is distributed as a gradient with its highest level at the posterior of the embryo. This suggests that the Caudal homeodomain transcription factor might play a role in establishing the posterior domains of the embryo, which undergo gastrulation and give rise to the posterior gut. By generating embryos lacking both the maternal and zygotic mRNA contribution, caudal has been shown to be essential for invagination of the hindgut primordium and for further specification and development of the hindgut. Mature embryos lacking cad activity (maternal and/or zygotic contributions) were examined to assess the requirement for cad in establishing the structures that arise from the posterior ~15% of the blastoderm embryo, namely the posterior midgut, Malpighian tubules and hindgut (Wu, 1998).

The stages of gastrulation can be observationally followed by using expression of brachyenteron byn as a marker for the hindgut primordium. In the wild-type embryo, byn is expressed in a ring at the circumference of the amnioproctodeal plate. The edges of this ring come together as the posterior midgut primordium invaginates during stages 6 and 7; the ring of the hindgut primordium then sinks inward during stage 8 and is completely internalized by the end of stage 9. The zygotically expressed cad stripe and the posterior wg stripe are also expressed in the bordering ring (i.e., the hindgut primordium) of the invaginating amnioproctodeal plate. Strikingly, in cad-deficient embryos, the byn-expressing ring of hindgut primordium draws together, but fails to invaginate, remaining on the outside of the embryo. Thus, although internalization of the Malpighian tubule and posterior midgut primordia is normal in cad-deficient embryos, the gastrulation movements necessary for internalization of the hindgut primordium do not occur in embryos lacking cad activity (Wu, 1998).

The absence of the hindgut primordium from cad-deficient embryos suggests that Caudal regulates genes required for establishing and/or maintaining the hindgut primordium. tailless, forkhead, byn, bowl and wingless are likely targets for cad regulation, since all are required for some aspect of hindgut development: the hindgut is missing from both tll and fkh embryos, and severely reduced in wg, byn and bowl embryos. bowl, also called bowel, codes for a zinc finger transcription factor related to odd-skipped. Since maternally provided Caudal, which persists only through the blastoderm stage, is sufficient for essentially normal hindgut formation, the fact that all of these genes are expressed at the posterior of the embryo during the blastoderm stage means that they are potential targets for regulation by Caudal. The effect of absence of maternal and/or zygotic cad activity on the expression of these genes was assessed by in situ hybridization with appropriate probes. For tll, byn and bowl, absence of cad activity does not result in a detectable effect on expression. As described below, however, cad activity is essential for expression of fkh and wg. Both maternal and zygotic cad contributions are necessary for posterior wg expression. During early stage 5, just prior to its expression in 14 stripes that are required to establish the segmental pattern, wg is expressed in two domains at the anterior, and in a broad posterior stripe. This terminal wg stripe is located at approximately 8-12% EL, overlapping with the posterior of the zygotic cad stripe and with the position of the hindgut and Malpighian tubule primordia in the blastoderm fate map. Expression of the wg terminal stripe has been shown to be independent of other segmentation genes, but has not been otherwise characterized. All embryos from mutant cad germline mothers (even those expressing zygotic cad) fail to express the terminal stripe of wg. These results demonstrate that maternal cad activity is essential for the transcription of wg in the terminal stripe. Among embryos from wg heterozygous parents, approximately one-quarter (presumably those lacking only the zygotic component of cad expression) lack the terminal wg stripe. Thus both maternal and zygotic cad activities are required for expression of the terminal wg stripe (Wu, 1998).

The expression of the early cap of fkh also requires cad activity; approximately half of the embryos from mutant cad germline females mated to cad heterozygous males (i. e., cad m-z - embryos) show a dramatic reduction in both the size and intensity of the posterior cap of fkh expression. However, if cad is supplied either maternally or zygotically, fkh expression is normal. Thus expression of the posterior cap of fkh requires cad activity, which can be provided either maternally or zygotically. Later, by stage 10, fkh expression is as strong in cad-deficient as in wild-type embryos, indicating that this later expression is independent of cad activity. Since tll and hkb are also required to activate early fkh expression but are not themselves regulated by cad, cad must act combinatorially with these two genes to promote early fkh expression (Wu, 1998).

cad also regulates wg in combination with other genes. In addition to the demonstrated requirement for cad, expression of the posterior wg stripe requires positive input from fkh and tll, since the stripe is absent from the respective mutant embryos. Since embryos lacking either maternal or zygotic cad fail to express the posterior wg stripe, but still express fkh and tll, cad must act combinatorially with fkh and tll to promote formation of the posterior wg stripe. Expression of the terminal stripe thus requires the combinatorial action of cad, tll and fkh; the posterior limit of the stripe is known to be defined by repression by hkb (Wu, 1998).

The failure of the hindgut to become internalized in caudal-deficient embryos raises the question of whether cad might regulate a zygotically expressed gene required for the invagination of the amnioproctodeal plate. One gene known to be required for gastrulation is fog; fog mutant embryos lack not only the posterior midgut, but, as revealed by anti-Crb staining, the Malpighian tubules and hindgut as well. In the blastoderm stage embryo, fog expression is first activated in the region that will become the ventral furrow; shortly thereafter, expression is initiated in a posterior cap, in the region that will become the amnioproctodeal invagination. In cad-deficient embryos, fog expression in the prospective ventral furrow is normal, but is significantly reduced in the posterior cap. Thus, cad is required for the normal level of expression of fog in the prospective amnioproctodeal plate; decreased fog expression in cad-deficient embryos is likely responsible for the failure of the hindgut primordium to be internalized during gastrulation. Since fkh or wg mutant embryos do not display detectable defects in gastrulation, fog is the only gene presently known to mediate the effects of cad on gastrulation. In fog mutant embryos, none of the posterior gut primordia invaginate, while in cad-deficient embryos the posterior midgut and Malpighian tubule primordium do invaginate; thus, consistent with the in situ hybridization results, a low level of fog activity is present at the posterior of embryos lacking cad (Wu, 1998).

In addition to cad, three other genes (fkh, byn and wg,), which are required at the posterior of the Drosophila embryo for formation of the hindgut, are related to genes found throughout the metazoa, known as HNF-3 (alpha, beta, and gamma), Brachyury (also known as T) and Wnt, respectively. In many cases, these homologs are expressed in portions of the ‘blastopore equivalent’ at the posterior of the embryo, that overlap with domains of expression of cad (Cdx). In C. elegans, a Wnt homolog is expressed, and required for proper posterior development, in the same posterior blastomere where the cad homolog pal-1 functions. In sea urchin, HNF-3 and Brachyury homologs are expressed in the vegetal plate just prior to gastrulation. In fish and frog, Caudal, Brachyury and Wnt (Wnt8 and Wnt11) are initially expressed around most or all of the blastopore lip while HNF-3 expression is dorsally localized. As gastrulation proceeds, the expression of these genes becomes more restricted and non-overlapping, with HNF-3 and Brachyury expression becoming localized to the notochord and Wnt8 expression retreating from the dorsal position and becoming exclusively ventral. Patterns of expression of HNF-3 and Brachyury consistent with this general description have been found in ascidians, amphioxus, chick and mouse. Required roles for some of these genes have been demonstrated by analysis of mutants: mouse HNF-3ß knockouts reveal requirements in the formation of the node, notochord and head process; fish no tail and mouse T mutants reveal a requirement for Brachyury in migration of mesoderm through the primitive streak and in formation of the notochord. There is thus a constellation of conserved genes -- cad (Cdx), fkh (HNF-3), wg (Wnt8 and Wnt11) and byn (Brachyury) -- whose overlapping expression patterns in the blastopore equivalent suggests function in a related process. The phenotypes of the available mutations in these genes suggest that the common function is to specify cell fate at the blastopore; in most cases, essential parts of this fate are internalization and forward migration, two of the cellular movements that occur during gastrulation (Wu, 1998 and references).

The striking conservation in expression (and likely in function) of cad suggests that the regulation of posterior terminal development in Drosophila by Caudal may represent a more ancient regulatory mechanism than the tor receptor and the two genes that it activates: tll and hkb. Of these three genes, a vertebrate homolog is known only for tll; the function of this vertebrate gene, Tlx, is related to that of Drosophila tll not in the posterior, but rather in the anterior, in the establishment of the brain. Thus the Torso receptor pathway and its activation of tll and hkb has probably been superimposed relatively recently (in evolutionary terms) upon a more ancient, Caudal-regulated network of gene activity controlling gastrulation and gut formation. The fact that the same four genes are expressed at both the blastopore equivalent of chordates and at the amnioproctodeal invagination of Drosophila suggests that these two highly dynamic domains are homologous. Given the regulatory hierarchy that is present in Drosophila, it is proposed that in embryos of the proximate ancestor to arthropods and chordates, the posterior was defined by a posterior-to-anterior gradient of Cad activity. Cad is thought to have then activated expression of downstream network of genes in control of invagination (gastrulation) and gut specification. Cad expression in the archenteron probably continued during evolution and played an essential developmental role, since this structure differentiated into the gut. Going beyond the bilaterian ancestor to chordates and arthropods, it is worth considering that this nexus of gene expression may have evolved even more basally in the metazoa. The foregoing, by homologizing the insect amnioproctodeal invagination with the echinoderm and vertebrate blastopore, does not fit with the classical definition of protostomes and deuterostomes. This view categorizes arthropods as protostomes, in which the mouth is derived from the primary invagination of gastrulation; chordates are categorized as deuterostomes, where the mouth arises from a secondary invagination. More recently, comparisons of gastrulation patterns in many different species, as well as construction of molecularly based cladograms, have called into question the utility of these classically defined groups. While there continues to be uncertainty in understanding of ‘protostome’ and ‘deuterostome’ phyla, the significant conclusion of the information presented here is that there may be a homology between the blastopore of vertebrates and the amnioproctodeal (posterior) invagination of insects (Wu, 1998 and references).

Wingless expression in the 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 DoublesexF product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of DoublesexM product results in the development and repression of the male and female genital primordia, respectively. This report shows that DoublesexF prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas DoublesexM blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that DoublesexF 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 DoublesexM is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, DoublesexM 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 DoublesexM and DoublesexF 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 DoublesexM and DoublesexF 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 DsxF or male DsxM product, respectively. Clones for tra2 (expressing DsxM) 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 DsxF 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 DsxM 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 DsxM nor DsxF products. The female genital primordium of these discs now expresses Dll. It is concluded that DsxM 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 DsxF 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 DsxM 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 dsxMas/+ intersexual genital discs, which have both DsxM and DsxF products, and in dsx1 intersexual genital discs, which have neither DsxM nor DsxF 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 DsxM plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that DsxF 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 DsxM ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, DsxF positively regulates dac expression in the Wg domain, and DsxM 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, DsxM positively regulates dac expression in the Dpp domain, whereas DsxF negatively regulates dac expression in this domain, since in normal female genital discs with DsxF 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 DsxF in the male genital primordium of male genital discs. It is concluded that in male genital discs, DsxM positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, DsxF positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2ts 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 DsxF and at 29°C they produce DsxM. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2ts 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 Tra2ts product; i.e. transitory presence of DsxF product and absence of DsxM 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 DsxF product and presence of DsxM 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 Tra2ts product is again available (i.e. the presence of DsxF product and absence of DsxM product). This time requirement for induction of dpp is also supported by the fact that dsx11 clones (which lack DsxM) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx11/+ male genital discs (which express only DsxM ) 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 DsxM nor DsxF 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, DsxM positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, DsxF positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2ts 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 tra2ts 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 DsxM instead of DsxF product. Thus, these results are consistent with the previously proposed suggestion that DsxM activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for DsxF). 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 DsxM instead of DsxF. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2ts larvae reach such a developmental stage, these cells now produce DsxF because they have returned to the female-determining temperature (Sanchez, 2001).

DsxF 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 DsxM and DsxF 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 DsxM and DsxF 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 DsxM and DsxF 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 DsxF 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 DsxM 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 DsxM plus Abd-Br increase dpp expression in the male genital primordium of male genital discs, and that DsxF 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, DsxM would play a positive and a negative role in male and female genital primordia, respectively, whereas DsxF 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 DsxM and DsxF. 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).

Transcriptional Regulation Table of contents


wingless continued: Biological Overview | Evolutionary Homologs | Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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