InteractiveFly: GeneBrief

Wnt oncogene analog 2 : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

During pupal development, two separate tissues, the gonad and the genital disc, must grow toward each other, recognize, and fuse with one another. In the male, the gonads differentiate to form the testes and the genital disc differentiates to form the external genitalia and the internal structures that connect to the testes. The sheath of the male reproductive tract develops from two populations of cells: the pigment cells (cells of somatic origin associated with the gonad) and the somatically derived precursors of smooth muscle cells. These cells migrate to form a bilayered sheath that covers both the mature gonad (the testis) and a portion of the genital disc (the seminal vesicle). Organogenesis of the male reproductive tract sheath depends on the proper specification and migration of cells; such processes may require either intrinsic cues or extracellular signals (Kozopas, 1998).

The existence of signaling molecules necessary for the development of the male reproductive tract was first postulated more than 60 years ago when researchers were studying two aspects of development: the morphogenesis of the testis and the sex-specific presence of testis pigment cells. It was noted that the male gonads of gynandromorphs, or sexual mosaics, which do not possess a male genital disc, fail to undergo morphogenesis from an ovoid to a spiral shape (Dobzhansky, 1931). In transplantation experiments, it was found that male gonads only undergo morphogenesis when attached to a male genital disc and that the extent of spiraling, a species-specific trait, depends on the species of the genital disc (Stern, 1941a and b). These results led to the hypothesis that the male genital disc releases a signal, an inducer of morphogenesis, to which the gonad responds (Kozopas, 1998).

A second signal was proposed to explain the fact that a certain male-specific cell type can be induced in females. In gynandromorphs that contain ovaries and a male genital disc, some male-specific pigment cells are often present. Through transplantation experiments, it was determined that these cells normally derive from the male gonad, which raised the question of whether the female gonad is the source of the pigment cells seen in gynandromorphs. This appears to be the case, as transplanting a male genital disc into a female is sufficient for some cells of the ovary to acquire a pigment cell fate. Clearly, some signal emanating from male tissue can support the differentiation of female cells into male testis pigment cells. For many years, these signals, crucial effectors of male development in the reproductive tract, were left unpursued (Kozopas, 1998 and references).

In a screen for mutations in Wnt2, a member of the Wnt family of genes (Russell, 1992), Wnt2 was found to be required for the development of the sheath of the male reproductive tract and testis morphogenesis. Wnt2 is expressed in somatic cell precursors. When Wnt2 is expressed ectopically in females, male-specific pigment cells appear. Pigment cells, the outer cells of the testis sheath, are absent in null mutants, indicating that Wnt2 is required for pigment cell specification in males. The inner muscle layer of the testis sheath fails to develop in the male mutants and the testis does not undergo its normal morphogenesis (Kozopas, 1998).

This raises two questions: (1) How is it possible to demonstrate Wnt2 involvement in muscle cell determination and pigment cell origin? (2) How does the somatically derived muscle layer come to lie inside the gonad derived pigment layer?

(1) The involvement of Wnt2 in pigment cell origin was proven by overexpression experiments. The pigment cells are a sexually dimorphic cell type found only in the male. The finding that Wnt2 is expressed only in the male third instar larval genital disc suggested that Wnt2 is the signal provided by a transplanted male genital disc to induce pigment cell fate in females. To test this hypothesis, Wnt2 was expressed ectopically in clones during female development. When clones were induced in third instar female larvae, all of the resulting adults had ectopic pigment cells, as assessed by their production of yellow pigment (Kozopas, 1998).

(2) To understand the role of Wnt2in the formation of a complete muscle layer, the development of this tissue was examined in wild-type animals during pupation. The muscle precursor cells have a somatic origin in the male genital disc. Muscle precursors express twist. Expression of Twist is initiated in the mesoderm during embryogenesis, persists in adult muscle precursors, and is required at high levels for myogenesis. Because the muscle precursors must migrate from the genital imaginal disc to form the testis sheath, the muscle phenotype in mutant adults indicates a thwarted attempt at the normal developmental process. The myoblasts in Wnt2 mutants begin migration over the testis, but fail to complete it. The lack of pigment cells in adult mutants, however, could indicate that either these cells are not specified or they degenerate for lack of contact with a normal muscle layer. To address this, the expression of a pigment cell marker was examined in Wnt2 mutant gonads before any contact of the gonad with the muscle precursor cells from the genital disc had been established. At 24 hr after puparium formation, 6 hr before the genital disc could be expected to contact the gonad, mutants already lack the pigment cell layer that covers the wild-type gonad. Even at the late third instar larval stage, the earliest that expression can be detected from the pigment cell marker line, the mutants lack pigment cells. Thus it appears that the pigment cells are never specified in the mutants or die before the morphogenesis of the reproductive tract occurs. It is concluded that there is a dual defect in Wnt2 mutants: failure of muscle precursor migration and failure in pigment cell specification (Kozopas, 1998).

The role of Wnt2 in testis muscle cell development, as revealed by the mutant phenotype, is to ensure the proper morphogenesis of the muscle precursor cells. Instead of initiating cell differentiation, Wnt2 contributes either to the migration of the muscle precursors away from the genital disc or to their adherence and migration over the testis. This contribution may be a direct one, in which Wnt2 regulates motility or adherence, or an indirect one, in which the role of Wnt2 is to specify the pigment cell substrate. Wnt2 activity is required in males for the presence of pigment cells. Wnt2 is necessary and sufficient for the differentiation of the male-specific pigment cell fate, at least for a certain population of cells. These experiments were not able to clarify what female cell type gives rise to the pigment-producing cells. These cells may derive from the colorless sheath cells of the ovary or an external cell type of the ovary, referred to as a lamellocyte and seen only in prepupae. This may be the cell type with the potential to become a pigment cell (Kozapas, 1998).

In summary, Wnt2 expression in males is required for testis pigment cells. Ectopic expression of Wnt2 is sufficient for the formation of ectopic pigment cells in females. Wnt-2 is also required for the normal development of the testis muscle, although it is not known if this requirement is direct. The pigment cells appear to be the substrate under which the myoblasts must migrate, and their absence in the Wnt2 mutant may explain the observed abnormal muscle formation. However, Wnt2 expression at the precise time and location of myoblast migration suggests that Wnt2 could have an independent function in the migration process. Currently, no experiments have been carried out to distinguish between a direct and indirect role for Wnt-2 in testis muscle development (Kozopas, 1998).

Direct flight muscles depend on DWnt-2 for their correct patterning

The direct flight muscles (DFMs) of Drosophila allow for the fine control of wing position necessary for flight. In Wnt-2 mutant flies, certain DFMs are either missing or fail to attach to the correct epithelial sites. Using a temperature-sensitive allele, it has been shown that Wnt-2 activity is required only during pupation for correct DFM patterning. Wnt-2 is expressed in the epithelium of the wing hinge primordium during pupation. This expression is in the vicinity of the developing DFMs, as revealed by expression of the muscle founder cell-specific gene dumbfounded in DFM precursors. The observation that a gene necessary for embryonic founder cell function is expressed in the DFM precursors suggests that these cells may have a similar founder cell role. Although the expression pattern of Wnt-2 suggests that it could influence epithelial cells to differentiate into attachment sites for muscle, the expression of stripe, a transcription factor necessary for epithelial cells to adopt an attachment cell fate, is unaltered in the mutant. Ectopic expression of Wnt-2 in the wing hinge during pupation can also create defects in muscle patterning without alterations in stripe expression. It is concluded that Wnt-2 promotes the correct patterning of DFMs through a mechanism that is independent of the attachment site differentiation initiated by stripe (Kozopas, 2002).

The flight muscles develop from the myoblasts that adhere closely to the imaginal discs during larval development, then migrate and fuse to form syncytial tissue during pupation. The DFMs are tubular muscles that insert on apodemes, projections of the cuticle at the base of the wing hinge. Apodemes initially form during pupation as invaginations of the epithelium in the wing hinge primordium, before cuticle is secreted. Several of these apodemes correspond with internal portions of the axillary sclerites (small exoskeletal plates in the hinge). The most prominent group of DFMs are the muscles with obvious defects in the Wnt-2 mutant (Kozopas, 2002).

Null mutations in the Wnt-2 gene give rise to adult flies that cannot fly; they hold their wings out at an abnormal 45-90° angle from their body. In sections of thoraxes from null mutant flies, the IFMs appeared normal. However, in mutant flies dissected to directly examine the DFMs, it was found that particular DFMs are either attached inappropriately to the epidermis or they are absent. Although the extent of the muscle defects is variable, all flies exhibit some DFM defects. Most frequently affected are muscles 52 and 54. Muscle 52 is very often misattached such that its ventral edge inserts on the apodeme normally shared by muscles 49 and 50. In only 13% of hemithoraxes examined is muscle 52 present in its normal location, and in these flies, it is much reduced in size. A normal muscle 54 is only seen in 6% of the specimens. The finding that the Wnt-2 mutant has a frequent absence of muscle 54 and manifests a held-out wing position supports the conclusion from electrophysiological studies that this particular muscle functions in wing retraction at rest. The apparent absence of this muscle in the Wnt-2 mutants may be due to its misattachment in such a way that makes it unidentifiable, since ectopic muscle tissue is seen in more than half of the mutants. This ectopic muscle tissue either attaches to a known muscle site or appears to be unattached, but in no case does it misattach to a novel site on the epidermis (Kozopas, 2002).

Using a temperature-sensitive allele of Wnt-2, it has been determined that Wnt-2 function is required only during pupation for proper DFM development. The allele Wnt-2^RJ, which was originally reported as a hypomorph for the phenotype of testicular pigment cell loss, is temperature-sensitive. When flies of the genotype Wnt-2^RJ/Df are raised at 29°C, the mutants are flightless due to the absence of muscle 54. This DFM phenotype is hypomorphic because these flies rarely show the defects in the attachment of muscle 52 that are seen in the null mutants, although this muscle is usually smaller than in wild type. When Wnt-2^RJ/Df flies are raised at 18°C, they are able to fly and display no DFM defects. When flies of this genotype are raised at 29°C until the white prepupal stage and are then shifted to 18°C, they also have normal DFMs. When the converse experiment is done and Wnt-2^RJ/Df flies are raised at 18°C before shifting to the nonpermissive temperature at the white prepupal stage, flies display held-out wings and are flightless. Their phenotype is the same as flies raised at a constant 29°C, including the absence of muscle 54 (Kozopas, 2002).

To understand how Wnt-2 gene activity affects DFM development, a characterization of normal DFM development was undertaken. A marker was sought that would identify the DFMs during their development. ß-galactosidase expression was examined from the rP298 enhancer trap, which is an insertion in the embryonic muscle founder-specific gene dumbfounded (duf, also known as kirre). duf is an immunoglobulin superfamily member required for the aggregation and fusion of founder cells with naive myoblasts. In a newly formed puparium, duf-lacZ is expressed in only a subset of the adepithelial cells that are associated with the wing disc. It is expressed in cells at the ventral edge of the field of adepithelial cells that are in closest contact with the epithelium (Kozopas, 2002 and references therein).

Morphogenic movements of the wing disc during the first 6 h of pupation result in a three-dimensional structure in which the duf-lacZ-expressing cells reside on the inner face of the dorsal notum, directly adjacent to the wing hinge primordium. As pupation proceeds, the distance between the dorsal notum and the ventral pleura progressively decreases due to wing hinge morphogenesis. duf-lacZ- expressing cells will then migrate to additional sites on the inner face of the ventral pleura, reaching their destinations by 35 h after pupal formation (APF). At this time, it is apparent that duf-lacZ-expressing cells are present as clusters of cells. Myoblasts that do not express duf-lacZ can be seen aggregating with the myotubes formed from duf-lacZ-expressing cells. These observations, and the selective requirement of duf in embryonic muscle founders for fusion with myoblasts, support the hypothesis that duf-lacZ expression marks the subset of adepithelial cells that act as muscle founders for the DFMs (Kozopas, 2002).

Additional support for the hypothesis that there are cells that act as founders for the DFMs comes from the finding that apterous (ap) is expressed in the DFMs when they are first forming, around 24-36 h APF. ap is a LIM homeodomain protein expressed in embryonic muscle founder cells and is required for specific embryonic muscle identities. Using an apterous enhancer trap line, ß-galactosidase expression has been observed in many, if not all, of the developing DFMs, including the primordia of muscles 49-54, based on their morphology and attachment sites. Like duf-lacZ, ap-lacZ is expressed in small groups of cells that will form the DFM myotubes. There is a second class of myoblasts that does not express ap-lacZ, which congregates with the myoblasts and developing muscles that do express ap-lacZ. The expression of both duf-lacZ and ap-lacZ in a subset of the myoblasts that give rise to the DFMs strongly suggests that these cells function analogously to the embryonic founder cells, first promoting the fusion of naive myoblasts and then specifying their transcriptional activity. ap-lacZ is expressed in DFMs during their development and is required in a cell-autonomous manner in myoblasts for DFM development (Kozopas, 2002 and references therein).

Having determined that Wnt-2 activity is only needed during pupation for DFM development, it was asked where it is expressed at that time. In the pupal wing disc at 7 h APF, Wnt-2 mRNA is strongly expressed in the wing blade, the wing hinge primordia, and the ventral pleura. Most of these regions of expression appear to be areas where sites of third instar larval disc expression is maintained. Wnt-2 expression in the wing blade and notum appears to fade by 24 h APF, and expression in the wing hinge region becomes the most prominent feature. As the wing hinge epithelium takes on its final form, it contracts and undergoes extensive morphogenesis, including the invagination of the apodemes to which the DFMs attach. At 24 h APF, Wnt-2 is expressed in both the dorsal and ventral compartments of the wing hinge, in regions with very close proximity to one another. Wnt-2 expression in the hinge is maintained at least until the newly formed muscles have established contact with their epithelial sites of attachment at 40 h APF. Wnt-2 expression is never seen in myoblasts themselves or in the epithelial cells that serve as attachments, only in adjacent epithelial regions (Kozopas, 2002).

To determine where the DFM myoblast precursors are located relative to the expression of Wnt-2 in the wing hinge epithelium, double-labelings were performed for duf-lacZ and Wnt-2 expression. Expression of Wnt-2 is found in the wing hinge primordium adjacent to sites contacted by the duf-lacZ-expressing cells. The myoblasts at this stage (6 h APF) are not in direct contact with the region of the epithelium that expresses Wnt-2, but occupy lateral positions several cell diameters away. The fact that myoblasts reside adjacent to epithelial cells expressing Wnt-2 was confirmed by using a different marker for myoblasts, the MEF-II transcription factor that is required for muscle differentiation. These double-labelings also show that the myoblasts come in close proximity to the epithelial cells that express Wnt-2 (Kozopas, 2002).

It was asked whether Wnt-2 expression in the wing hinge adjacent to developing DFMs plays a permissive or an instructive role in their patterning. By expressing Wnt-2 in the developing wing hinge in regions that differ from the endogenous expression, defects were caused in DFMs that do not normally rely on Wnt-2 for their correct patterning. The E132:Gal4 driver, whose broad expression in the wing hinge epithelium encompasses the primordia of attachment sites for muscles 49, 50, 53, and 56, was used to misexpress UAS:Wnt-2. The resulting flies have severe DFM mispatterning defects, including the absence of muscle 49, which is never affected in the Wnt-2 mutant. Additionally, there are misattachments of muscle of a type that do not occur in the mutant, such as confusion between the attachments of muscles 50 and 53 (Kozopas, 2002).

The gene stripe (sr) encodes a transcription factor that is required in epithelial cells for their differentiation as attachment sites. Studies of IFM formation have shown that sr is expressed in early pupal development in the epidermal cells that will serve as adult IFM attachment sites. The expression of sr was examined in the adult by following ß-galactosidase reporter gene expression in the P1618 enhancer trap line. sr-lacZ is expressed in the cells that form the epidermal attachment sites for the DFMs). These attachment sites are prefigured by sr-lacZ expression in regions of the wing hinge primordia in the pupa. Some of the regions that express sr-lacZ will invaginate to form apodemes, which are points of DFM attachment. While Wnt-2 expression in the wing hinge primordia is adjacent to some of the sites of sr expression, in no region are they coincident. However, the potential for secreted Wnt proteins to diffuse away from their source renders it possible that Wnt-2 might regulate stripe expression in adjacent cells. For this reason, the expression of sr was examined in the Wnt-2 mutant. However, no changes were found in sr-lacZ expression in the primordia of the DFM attachment sites. Neither is sr-lacZ expression altered when Wnt-2 is ectopically expressed in the pupal wing hinge such that muscle patterning defects occur. These results indicate that, although Wnt-2 is required for the proper attachment of muscle to the epidermis, it does not affect sr expression, the most upstream known component of EMA cell differentiation (Kozopas, 2002).

Thus Wnt-2 is expressed in the epithelium of the pupal wing hinge, adjacent to developing DFMs and the EMA cells that serve as their attachment sites. This expression occurs during the time period that Wnt-2 is needed to mediate formation of the proper connections between these tissues. When ectopically expressed, Wnt-2 can alter the development of DFMs that do not normally depend on it and can result in muscles choosing the wrong attachment sites. The inability of Wnt-2 to regulate stripe expression in EMA cells demonstrates that Wnt-2 exerts control over muscle attachment through some mechanism other than the initiation of EMA cell differentiation (Kozopas, 2002).

The unique feature of DFM development may merely be that each adult muscle uses a group of cells to act as 'cofounders'. These groups represent the same cells referred to as 'feeder' myoblasts. Presumably, these cells would share an identity conferred by transcription factors such as ap. It is not known whether cells in these clusters fuse with one another, which would be contradictory to the behavior of duf-expressing founder cells in the embryo. It is plausible that cells in the clusters do not fuse with one another, but instead that each helps to seed syncytial growth of the muscle that will take on their shared identity. In this respect, each cluster of founder cells would represent an equivalence group. The group’s muscle identity might be conferred by the same mechanism of ectodermal signaling that specifies individual founders in the embryo. It is possible that Wnt-2 might be one of these ectodermal signals, acting similarly to wg to control the transcription of genes in adult founders that specify DFM identities. Thus, muscle attachment defects in the mutant would be the result of muscles lacking the proper identity. However, it is not known when the cells that appear to function as DFM founders are specified with regard to the muscle identities that they will assume in the adult. For the adult abdominal muscles, there is evidence to suggest that muscle identities are generated during embryogenesis. Specifically, the myoblast progenitors of the ventral adult precursors and the lateral adult precursors (VaP and LaP) are formed through asymmetric cell divisions that yield one adult muscle precursor and one embryonic muscle founder. Although there is no evidence to indicate that the diverse adult DFM identities are also generated during embryogenesis, it is a compelling theory that all adult muscle precursor cells are the siblings of embryonic muscle founders, arising through asymmetric division of muscle progenitor cells. The presence of the duf-lacZ-expressing DFM precursors as a stereotyped ventral subset of adepithelial cells in the prepupal wing disc seems to indicate that these cells have already made significant cell-fate decisions by this stage. Adepithelial cells associated with larval leg discs do have restricted cell fates, as evidenced by experiments in which ablation of myoblasts during larval stages results in missing subsets of adult leg muscles. If the myoblasts associated with the wing disc are also already programmed by early larval stages for particular DFM identities, then Wnt-2 cannot exert its effect by specifying particular muscle founder cell fates, since it is only required during pupation. For this reason, and the probable conservation of the mechanism that generates adult muscle precursors from asymmetric divisions in the embryo, Wnt-2 is an unlikely candidate to affect intrinsic muscle identity. It is a more tenable hypothesis that the Wnt-2 signaling molecule in the pupal wing hinge primordium enables muscles to attach to their correct sites, in some way facilitating these recognition events (Kozopas, 2002).

One potential function for Wnt-2 might be in directing the differentiation of EMA cells as distinct apodemes. For instance, there may be specific adhesion molecules or guidance cues expressed in attachment sites that allow resident muscles to distinguish their sites from neighboring attachments, and which Wnt-2 might regulate (Kozopas, 2002).

DEVELOPMENTAL BIOLOGY

The expression patterns of Wnt2 and Wnt3 Wnt/wingless homologs are dynamic during Drosophila embryogenesis. The distribution of Wnt2 transcripts is predominantly segmented, with the additional presence of transcripts in the presumptive gonads. Transcripts of both Wnt2 and Wnt3 appear to be associated with limb primordia in the embryo and may therefore specify limb development. Wnt3 is also expressed in mesodermal and neurogenic regions. The distribution of Wnt3 transcripts in cells of the central nervous system (CNS) during Drosophila embryogenesis suggests that Wnt3 could be involved in CNS development (Russell, 1992).

Wnt2 transcripts are already present at the blastoderm stage of development. During cellularization, the highest levels of transcripts occur in the central region, between approximately 15% and 70% egg length (0% is the posterior pole). During the early stages of germ band extension (stage7), Wnt2 transcripts are visible within at least 12 columnar cells in the anterior wall of the proctodeal primordium. Wnt2 transcripts are detectable in the three thoracic and eight abdominal segments of stage 8 embryos; by stage 9/10, the RNA is restricted to discrete ectodermal patches in the dorsolateral regions of each of these segments. The dorsolateral patches are half a segment in width, but during stage 10 the patches broaden toward the ventral midline and along the anterior-posterior axis. Wnt2 transcripts occur across the anterior half of each segment and in the posterior portion of each segment. Just anterior to the parasegment boundary, there is overlap with wingless expressing cells. However, there is no overlap with the engrailed domain in the posterior portion of each segment. Thus Wnt2 exhibits a segment polarity expression pattern (Russell, 1992).

The appearance of Wnt2 transcripts in two of the gnathal segments is more or less concomitant with the broadening of the dorsolateral patches. In the maxillary and labial segments, many of the cells in which Wnt2 is transcribed lie anterior to the engrailed expression domain. Wnt2 transcripts are also present at low levels as spots in the primordial labrum, the dorsal area of the procephalic lobe, and in the proctodeum. The positions of the Wnt2 transcripts in labral, maxillary and labial segments may correspond to the positions of primordia of the larval mouth part appendages. At early stage 11, Wnt2 transcripts arise in the ventral ectoderm, forming a pattern of spots, two each for each of the thoracic and abdominal segments, arrayed in two longitudinal rows along the entire length of the germ band. The ventral thoracic spots may be within the anterior compartments of leg primordia as inferred from the pattern of Distal-less expression in the thoracic segments. It is also possible that the ventral spots in the abdominal segments are associated with precursors of the abdominal histoblasts, cell clusters that contribute to the larval epidermis, and from which the adult epidermis of the abdominal segments is derived (Russell, 1992).

During germband shortening, Wnt2 message disappears from the gnathal segments and the dorsalateral patches retract away from the ventral midline and shrink along the anterior-posterior axis such that Wnt2 transcripts are no longer detectable within cells of the wingless expression domain. By stage 14, the segmented pattern of Wnt2 transcripts is absent. Wnt2 RNA detectable in the posterior region of stage 13 and stage 14 embryos is apparently associated with the proctodeal opening. During stage 14, Wnt2 message appears in the posterior portion of the presumptive gonads, associated with either germ-line precursor cells, recruited from the pole cells, or the mesodermal cells with which they intermingle to form the embryonic gonads. Wnt2 transcripts are not detectable in pole cells prior to stage 14. From stages 15 onward, Wnt2 RNA is limited to clusters of cells in the anteriormost region of the embryo and to the posterior portion of the presumptive gonads (Russell, 1992).

To determine how Wnt2 might affect male reproductive tract development, sites of Wnt2 expression were identified by in situ hybridization. Wnt2 is expressed in embryos in all mesodermal cells of the gonad, before the mesoderm and germ cells have condensed to form a compact gonad. Wnt2 expression is limited to the posterior mesodermal cells of the gonad late in embryogenesis (Russell, 1992). This late pattern of expression is apparently maintained in the male, as Wnt2 is expressed at the posterior of the pupal gonad, in cells that will become the terminal epithelia. In the male pupal genital disc, Wnt2 is expressed in the epithelial cells at the apical tip of each developing seminal vesicle. This is a refinement of expression that was present earlier in the third instar larval disc of the male genital primordium. Wnt2 is not expressed in the genital disc of the female third instar larva, nor is it expressed during pupation in the developing oviducts, the female structures analogous to the developing seminal vesicles. Thus, Wnt2 expression occurs in sexually dimorphic patterns (Kozopas, 1998).

Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad

Sex determination in Drosophila is commonly thought to be a cell-autonomous process, where each cell decides its own sexual fate based on its sex chromosome constitution (XX versus XY). This is in contrast to sex determination in mammals, which largely acts nonautonomously through cell-cell signaling. This study examined how sexual dimorphism is created in the Drosophila gonad by investigating the formation of the pigment cell precursors, a male-specific cell type in the embryonic gonad surrounding the testis. Surprisingly, sex determination in the pigment cell precursors, as well as the male-specific somatic gonadal precursors, was found to be non-cell autonomous. Male-specific expression of Wnt2 within the somatic gonad triggers pigment cell precursor formation from surrounding cells. These results indicate that nonautonomous sex determination is important for creating sexual dimorphism in the Drosophila gonad, similar to the manner in which sex-specific gonad formation is controlled in mammals (DeFalco, 2008).

This study has shown that two distinct male-specific cell types in the Drosophila gonad exhibit nonautonomous sex determination. For both the male specific somatic gonadal precursors (msSGPs) and the pigment cell (PC) precursors, the sex determination pathway does not act in these cells themselves, and both are dependent on sex-specific signaling from the SGPs in order to develop properly as male or female. These findings are in contrast to the commonly held view that sex determination in Drosophila is a cell-autonomous process, and demonstrate the similarity in sex-specific gonad development between flies and mammals (DeFalco, 2008).

This study has identified a novel, sex-specific cell type in the Drosophila embryonic gonad, the PC precursors, and studied the mechanism by which the sex determination switch controls the sex-specific development of these cells. The data indicate that male-specific expression of Wnt2 in the SGPs of the gonad signals nonautonomously to the fat body to form PC precursors. dsx ensures that PC formation is male-specific by repressing Wnt2 expression in female gonads in late-stage embryos (stage 17). The sex of the fat body itself does not affect PC precursor formation, since cells with a female identity can form PC precursors when associated with a male gonad or with a female gonad that expresses Wnt2. Furthermore, Wnt2 acts directly on the fat body, since blocking Wnt signaling in male fat body cells prevents them from forming PC precursors. PC precursor identity in the fat body is regulated by ems acting upstream of Sox100B in response to the Wnt2 signal. An interesting question is whether Wnt2 is a direct downstream target of DSX in controlling sexual dimorphism. The DNA binding specificity for DSX has been determined, and there are a number of sites upstream of the Wnt2 start of transcription that either exactly match or closely match the DSX binding consensus sequence. Several of these sites are conserved between different Drosophila species. However, a fragment of the Wnt2 promoter has not yet been identified that allows testing of whether Wnt2 expression in the somatic gonad is directly regulated by DSX, since the upstream region that includes the putative DSX binding sites does not promote expression in the gonad (DeFalco, 2008).

The creation of sexual dimorphism in the PC precursors differs from that of the msSGPs. While the PC precursors are apparently only specified in males and recruited to form part of the testis, msSGPs are initially specified in both sexes, and are only present in the male gonad because they undergo programmed cell death specifically in females. Furthermore, the germline stem cell niche in the testis (the hub) is formed from a population of anterior SGPs that are present in the gonads of both sexes, but only form the hub in males and presumably form part of the ovary in females. These events are all regulated by dsx, and demonstrate the diverse cellular mechanisms that a sex determination gene can utilize to control sexual dimorphism. Interestingly, in dsx null mutant embryos each of these cell types develops as if it were male. Thus, the male mode of development can at least be initiated in these cell types in the absence of dsx function, and dsx primarily acts in females to repress male development. dsx is clearly required in males at some point for proper testis formation, therefore some cell types in the gonad may not be entirely masculinized in dsx mutants (DeFalco, 2008).

The nonautonomous nature of PC precursor specification contrasts with the commonly held view that sex determination in Drosophila is a cell-autonomous process, where 'every cell decides for itself' whether it should develop as male or female based on its own intrinsic sex chromosome constitution. This study has shown that the msSGPs undergo nonautonomous sex determination. The data indicate that a male-specific survival signal coming from the SGPs allows the msSGPs to survive and join the male gonad, while they undergo apoptosis in females. Finally, it has been shown that nonautonomous sex determination in the germ cells requires a male-specific signal from the SGPs that acts through the JAK/STAT pathway. Thus, not only does non-cell autonomous sex determination occur in the Drosophila gonad, it appears to be the predominant mechanism of sex determination. Of the cell types tested so far, only the hub cells, which form from a subset of SGPs, appear to decide their sexual fate in an autonomous manner. The current model is that the SGPs determine their sex in a cell-autonomous manner, and then signal to other cell types in the gonad (PC precursors, msSGPs, and germ cells) to control the sex-specific development of these cells via nonautonomous sex determination (DeFalco, 2008).

Nonautonomous sex determination is not limited to the gonad. Other tissues have been shown to decide their sex through cell-cell signaling. In the genital imaginal disc, the sexual identity of a signaling center, the A/P organizer, largely determines whether the disc will develop in the male or female mode. This is controlled non-cell autonomously through Wingless and Decapentaplegic signaling. In addition, sex-specific migration of mesodermal cells into the male genital disc is regulated by male-specific expression of the Fibroblast Growth Factor Branchless in the genital disc. Finally, in the nervous system, male neurons can non-cell autonomously induce the formation of the male-specific muscle of Lawrence from female muscle precursors. Given the large number of tissues and cell types that undergo nonautonomous sex determination, it seems that the conventional view can be abandoned that sex determination in Drosophila is an obligatorily cell-autonomous process; while some cell types utilize a cell-autonomous mechanism, many cell types clearly do not (DeFalco, 2008).

One reason why sex determination has been traditionally thought of as a cell-autonomous process in Drosophila is due to its relationship with X chromosome dosage compensation. This is the process by which gene expression from the single X chromosome in males is regulated to match that from the two X chromosomes in females. Both sex determination and X chromosome dosage compensation are regulated by the number of X chromosomes, acting through the master control gene Sex lethal (Sxl). It is likely that most or all cells count their X chromosomes and use this information to control X chromosome dosage in a cell-autonomous manner. However, as discussed above, it is now clear that cells do not necessarily use this information to decide their sex. Consistent with this idea, the expression of dsx, a key regulator of sex determination downstream of Sxl, is surprisingly tissue-specific. Within the embryo, dsx is only expressed in the SGPs and msSGPs of the gonad. Thus, not all cells even express the machinery to translate their sex chromosome constitution into sexual identity, and it is therefore necessary that sex-specific development of many cell types be controlled nonautonomously (DeFalco, 2008).

The nonautonomous cell-cell interactions that control gonad sexual dimorphism in Drosophila show great similarity to sex-specific gonad development in other species. In mammals, somatic sex determination is based on the presence or absence of the Y chromosome. The critical Y chromosome gene Sry is mainly expressed in a subset of cells in the somatic gonad in the mouse embryo, similar to dsx expression in the Drosophila embryonic gonad. Sry is only thought to be important for formation of Sertoli cells in males, and the sexually dimorphic development of all other cell types is thought to be regulated by local cell-cell interaction or hormonal cues. An excellent example of nonautonomous sex determination in the mouse is the recruitment of cells from the neighboring mesoderm (mesonephros) to form specific cell types in the mouse testis. Recruitment of these cells is dependent on the sex of the gonad, not the sex of the mesonephros. In addition, sex-specific development of other somatic cell types in the mouse gonad is regulated nonautonomously by cell-cell interaction, as is sexual identity in the germline. Thus, the use of non-cell autonomous sex determination and sex-specific cell recruitment are common mechanisms for creating gonad sexual dimorphism in flies and mice (DeFalco, 2008).

Nonautonomous sex determination in the mouse also utilizes signaling through the Wnt pathway. Wnt4 acts as a 'pro-female' gene that opposes Fibroblast growth factor 9 to regulate sex determination in the gonad. In early stages of gonad development, Wnt4 knockout females form a male-specific coelomic blood vessel and exhibit ectopic migratory steroidogenic cells, suggesting that Wnt4 acts to inhibit endothelial cell and steroid cell migration from the mesonephros into the female gonad. Interestingly, Wnt4 also has been shown to have a role in the male gonad, as male knockout mice show defects in Sertoli cell differentiation, downstream of Sry but upstream of Sox9. Wnt7a also has been implicated in sexual dimorphism in the reproductive tract, as Wnt7a knockout mice fail to express Mullerian-inhibiting substance (MIS) type II receptor in the Mullerian duct mesenchyme, which is required for regression of the duct in male embryos. In addition, a number of Wnt genes have been found to be expressed sex-specifically in the gonad through sex-specific gene profiling, indicating that other Wnt family members play a role in creating sexual dimorphism in the mammalian gonad (DeFalco, 2008).

It is also interesting that several conserved transcription factors act during gonad development in diverse species. Sox100B is the fly homolog of SOX9/Sox9, a critical regulator of sex determination and male gonad development in humans and mice. Similarly, a mouse homolog of ems, Emx2, is expressed in the developing gonad and is required for development of the urogenital system. Lastly, dsx homologs of the DMRT family have been implicated in sex-specific gonad development in diverse species. Thus, not only are the cellular mechanisms, such as non-cell autonomous sex determination and cell-cell recruitment, common between flies and mice, but the specific genes that regulate sexually dimorphic gonad development may also be conserved. Since the formation of testes versus ovaries, and sperm versus egg, are critical features of sexual reproduction, they may represent processes that are highly conserved across the animal kingdom (DeFalco, 2008).

EFFECTS OF MUTATION

Flies with null mutations in Wnt2 are viable but male sterile. To determine the cause of sterility, testes were dissected from Wnt2 mutant males and examined. With variable penetrance, testes from null mutant males are much smaller than wild-type, with the most severe examples having an abnormal oblong shape, as compared to the wild-type spiral. The null mutant testes do not possess the yellow pigmentation of the testis sheath. Males with the hypomorphic allele Wnt2RJ have a less severe phenotype, in which regions of the sheath produce yellow pigment (Kozopas, 1998).

To determine whether the cell types necessary for spermatogenesis are present in the mutants, the expression of beta-galactosidase was assayed from P-element enhancer trap lines specific for cells of the testis. Expression from the germ-line-specific enhancer trap S346 clearly shows that a germ cell lineage is maintained in the mutants. All of the specialized somatic cells of the testis required for spermatogenesis are also present in the mutants. These include the hub cells, cyst cells, and terminal epithelial cells, as assayed by expression from the markers I72 and 34, respectively. The presence of these cell types, along with the observation that mutant testes occasionally possess motile sperm, suggests that the male sterility of the mutants is not attributable to a failure in the process of spermatogenesis (Kozopas, 1998 and references).

The somatic cells necessary for the structure of the testis are affected in the mutants. These cells form a sheath composed of an outer pigment cell layer, an inner muscle cell layer, and their respective basal laminae. Assaying for expression from the pigment cell marker 365 shows that the testes from null mutant adults completely lack the outermost pigment cell layer, which normally covers both the testis and the seminal vesicles. Expression from the enhancer trap L44a, which marks muscle cells of the testis sheath, reveals that the muscle cell layer is malformed or incomplete in these mutants. Cells in this morphologically aberrant tissue do have a muscle cell identity, as they express muscle myosin. The phenotype of the mutant testis muscle layer varies from a partially complete muscle layer to a tangle of muscle cells at the base of the testis (Kozopas, 1998 and references).

In order to address whether Axin inhibits only Wg, or whether it can function as a more general inhibitor of Wnt signals, an examination was carried out to see whether Axn could interfere with a different Wnt signal. Four Wnt genes have been identified in Drosophila (see The World Wide Web Wnt Window ), but mutations have been described only for two of them: wg and DWnt-2. Loss of DWnt-2 produces a muscle migration defect in the male gonads, resulting in male sterility, and a lack of the characteristic pigment cells that migrate over the male testis. Ovaries are normally not surrounded by pigment cells, but misexpression of DWnt-2 in females can induce ectopic male-specific pigment cells. This dominant phenotype of DWnt-2 misexpression was used to address whether Daxin can block ectopic pigment cell formation. Overexpression of Axn strongly reduces the frequency of DWnt-2-mediated pigment cell formation in ovaries, demonstrating that Axn can block the DWnt-2 signal in addition to the Wg signal. Overexpression of a dominant-negative Pangolin/dTCF lacking the amino-terminal Arm binding domain (DN-dTCF) also blocks the ectopic DWnt-2 induced pigment cells in the ovary (Willert, 1999).

Sequencing of the Drosophila genome has revealed that there are 'silent' homologs of many important gene family members that were not detected by classic genetic approaches. Why have so many homologs been conserved during evolution? Perhaps each one has a different but important function in every system. Perhaps each one works independently in a different part of the body. Or, perhaps some are redundant. This study takes one well known gene family and analyzes how the individual members contribute to the making of one system, the tracheae. There are seven DWnt genes in the Drosophila genome, including wingless. The wg gene helps to pattern the developing trachea but is not responsible for all Wnt functions there. Each one of the seven DWnts was tested in several ways and evidence was found that wg and DWnt2 can function in the developing trachea: when both genes are removed together, the phenotype is identical or very similar to that observed when the Wnt pathway is shut down. DWnt2 is expressed near the tracheal cells in the embryo in a pattern different from wg's, but is also transduced through the canonical Wnt pathway. The seven DWnt genes vary in their effectiveness in specific tissues, such as the tracheae; moreover, the epidermis and the tracheae respond to DWnt2 and Wg differently. It is suggested that the main advantage of retaining a number of similar genes is that it allows more subtle forms of control and more flexibility during evolution (Llimargas, 2001).

DWnt proteins bind as ligands to a family of receptor proteins -- four Frizzled (Fz) homologs in Drosophila, of which Fz and Fz2 are the most important and act through a cascade of genes [e.g., disheveled, armadillo (arm), pangolin] on the nucleus. If, therefore, Wg is the only ligand acting from the outside of the cell on the receptors, the wg^- phenotype should be identical to the phenotype when fz and fz2 are removed -- in some organs, this is so. However, in the trachea, although removal of the two receptor proteins or one of the intracellular proteins in the cascade eliminates all dorsal trunk (DT), removing only Wg leaves some DT intact. Therefore, it seems that another molecule, presumably a DWnt, acts through the canonical Wnt pathway to build DT. This study asks which DWnt is responsible (Llimargas, 2001).

Overexpression of wg or other downstream elements of the Wnt pathway in the tracheal cells results in increased DT at the expense of the ventral branch. To investigate further, each one of the seven DWnts was overexpressed locally in the embryonic trachea in a normal background. Overexpression of five DWnt genes (DWnt5, -4, -6, -8, and -10) had no detectable effects; indeed, the flies were viable, fertile, and seemed normal. This experiment suggests that the tracheae are not particularly sensitive to these five proteins. To check whether these proteins are made properly and can function, they were tested in other assays. DWnt6 and DWnt8 are able to affect tracheal development in a sensitized background. DWnt5 produces a phenotype in the ventral nerve cord when expressed with the neural specific driver 1407Gal4, in agreement with the phenotype produced by a HS-DWnt5. Moreover, protein expression is detected in the tracheae when DWnt5 is expressed in tracheal cells. DWnt4 produces ectopic denticles in the ventral epidermis when overexpressed with armGal4, and the flies die as pharate adults, showing several defects in the wings when crossed to ptcGal4. No noticeable phenotype was found when overexpressing DWnt10 in several structures, and, thus, the activity of this line awaits confirmation. However, DWnt10 together with three other DWnts (DWnt4, -6, and wg) were removed in Df(2L)RF embryos and a phenotype similar to wg^- was found, because there still was some DT. This experiment argues that at least zygotic DWnt 4, -6 and -10 do not have a significant function in the trachea under normal conditions. However, overexpression of DWnt2 locally in the tracheal cells does affect its development in a way similar to that of wg, producing an excess of DT cells and DT material at the expense of the ventral branch. These tracheae are defective; they fail to fill with air and the flies die as embryos and young larvae. This result suggests that both wg and DWnt2 act or can act in the developing trachea (Llimargas, 2001).

The tracheal placodes are specified by stage 10 in a specific part of the dorsal ectoderm and express several markers such as trachealess. The expression of DWnt2 is suggestive: it is expressed close to and dorsal to the tracheal placode by stage 10 and early stage 11 but later disappears (Llimargas, 2001).

The spalt (sal) gene (coding for a transcription factor) is expressed in the dorsal ectoderm, including some tracheal cells, during stage 10 and persists later in those tracheal cells that form the DT. sal is absolutely required for DT formation and is thus a good marker for DT cell identity. The most dorsal cells that express sal also coexpress DWnt2. The pattern of wg expression differs strikingly from that of DWnt2, although both gene products are made near the tracheal cells. In arm mutants, Sal is not expressed in tracheal cells and no DT is formed, suggesting that sal expression in tracheal cells depends on activation of the Wnt pathway. Thus, sal can be induced in the tracheal cells wherever either Wg or DWnt2 proteins are received (Llimargas, 2001).

The above results suggest that wg and DWnt2, made near the tracheal cells, together sponsor DT formation. In wg^- embryos, some DT is still formed. However, the tracheal phenotype of wg^-DWnt2^- embryos is significantly different from that of wg^- embryos: in 40%-45% of hemisegments, the DT is completely missing, and in the remaining 55%-60%, only some reduced and thin DT forms. Interestingly, in practically all hemisegments of Df(2L)RF DWnt2^- embryos, the DT is completely missing, indicating that other DWnts account for these traces of DT. Nevertheless, wg^-DWnt2^- double-mutant embryos are very similar to or indistinguishable from fz^-fz2^- embryos, suggesting that wg and DWnt2 sponsor virtually all DT formation (Llimargas, 2001).

Removal of Wg and DWnt2 proteins (in wg^-DWnt2^- embryos) eliminates detectable expression of sal in the presumptive tracheal cells of the DT, whereas in wg^- embryos, very low levels of sal still can be detected in some embryos. The early expression of sal in the dorsal ectoderm still is observed in both wg^- and wg^-DWnt2^- embryos. In wg^-DWnt2^- embryos, late kni expression in tracheal cells is normal, as is the case in arm mutants. In addition, dpp expression also is normal -- Dpp has been shown to inhibit sal expression by activating kni in tracheal cells. Thus, the lack of sal must be caused by the absence of direct or indirect stimulation by the DWnt pathway and not due to repression by the Dpp pathway (Llimargas, 2001).

Does DWnt2 act through the canonical Wnt pathway? It seems so, because the ectopic effects of DWnt2 protein are blocked in embryos that lack the arm gene. Moreover, in wg^-DWnt2^- embryos, the DT can be substantially rescued by expressing a constitutively active form of Arm in the tracheal cells. Also, the tracheal phenotype of porcupine (por) mutants is very similar to that of wg^-DWnt2^- embryos, indicating that por also might be required for DWnt2 secretion (Llimargas, 2001).

If DWnt2 sponsors at least part of DT formation, one might expect that loss of DWnt2 alone would affect trachea in some noticeable way. Surprisingly, DWnt2^- embryos and larvae have normal trachea and normal expression of sal. However, the flies have reduced viability and the males are sterile (Llimargas, 2001).

In normal embryos, the wg gene is expressed in a row of cells at the rear of the A compartment, whereas DWnt2 is expressed at the front. Wg protein spreads to make a gradient that patterns the anterior compartment. DWnt2 protein is expressed where the concentration of Wg is low or absent; that is where the tracheal placodes form and where the cuticle secretes denticles. Thus, in wg^- embryos, where there is no Wg protein and the denticles are continuous, one might expect the tracheal placodes and DWnt2 expression to form one continuous stripe and, indeed, they do (Llimargas, 2001).

This adventitious expression of DWnt2 in a broad domain in wg^- embryos could compensate at least in part for the lack of wg itself. Indeed, in these embryos, it must be mainly DWnt2 that activates some sal and determines most or all of the DT found. No change could be detected in the pattern of wg RNA or protein distribution in DWnt2 mutants (Llimargas, 2001).

The potency of DWnt2 and Wg in the tracheae was assayed: DWnt2^-wg^- double mutants were taken and each of the two missing proteins were added back in the normal pattern of expression for the wg gene. DWnt2 and Wg were found to both rescue some DT in the trachea; however, only Wg can partially rescue the various embryonic defects in morphology found in wg^- embryos. When either DWnt2 or Wg is expressed locally in the tracheal cells, each gives strong rescue, and more DT is made (Llimargas, 2001).

The DWnt2 gene was also expressed in wild-type embryos either universally and strongly (arm VP16 Gal4) or in stripes (ptcGal4), and in both cases, the tracheae are altered to the same extent as when DWnt2 is expressed in the tracheal cells alone. However, DWnt2 fails to alter the cuticle pattern, whereas wg produces a naked cuticle phenotype. This lack of effect of DWnt2 on the epidermis is remarkable since both the drivers used are strong and, when wg is driven, are more than adequate to make a naked cuticle. Interestingly, when DWnt2 is missexpressed in the eye, it also does not emulate the phenotype produced by missexpression of wg. Moreover, the effects of overexpressing DWnt2 in the ovary are stronger than when overexpressing wg. All these results argue that the tracheal cells and other tissues, including the epidermis, the eye, and the ovary are differentially sensitive to the two DWnt molecules, the trachea and the ovary being particularly responsive to DWnt2 (Llimargas, 2001).

There are several ways this difference could be achieved. Perhaps DWnt2 does not act through the canonical Wnt pathway in some tissues, such as the ectoderm or the eye. Perhaps DWnt2, on its way to the tracheal cells, could be secreted or processed differently. Perhaps the tracheal cells have something that allows efficient presentation of the ligand to the Fz receptors, or they lack a component that, in other tissues, impedes DWnt2 binding or transduction. One possibility is that glucosaminoglycans help breathless (btl, an FGF receptor expressed in tracheal cells) and are needed for Wnt signaling. Maybe Btl helps to gather or modify the heparan sulfate glucosaminoglycans, thereby altering the presentation of DWnt2 to the two receptors, Fz and Dfz2. Whatever the explanation, the tracheal cells are more responsive than other tissues to the DWnt2 signal (Llimargas, 2001).

The other DWnts were examined. DWnts5, -6, -8, and -10 were drived in the epidermis of wild-type embryos with one copy of ptcGal4; none of these affected the cuticle pattern in a noticeable way. Are these DWnts able to affect tracheal development in the wg^-DWnt2^- double mutants? Each of these five DWnts were added back to either the tracheal cells themselves or in the pattern of normal wg expression. DWnts6 and -8 are each able to rescue DT partially, whereas -4, -5 and -10 did not. Note that DWnt6 and DWnt8 are not able to produce a tracheal phenotype when expressed in tracheal cells of normal embryos, but they can do so in a sensitized background (Llimargas, 2001).

The results indicate that wg and DWnt2 make the main contribution to DT formation, as the absence of both genes completely eliminates DT in many cases. However, traces of DT still are formed in about half the hemisegments of wg^-DWnt2^- embryos, indicating that contributions of other genes might help. Also, rescue experiments show that some other DWnts are able to activate the pathway. In agreement with this result, in most Df(2L)RF DWnt2^- embryos, all DT is missing, indicating that DWnt6 and/or -4 and/or -10 can compensate weakly for the absence of wg and DWnt2. However, expression of DWnt6 and DWnt10 does not suggest that they act in tracheal development in the wild type. DWnt4 is expressed in a similar pattern to that of wg but does not seem to assist wg during embryogenesis. In addition, none of DWnt4, -6, or -10 affected tracheal development when expressed in tracheal cells of wild-type embryos. Most likely, they produce traces of DT in the wg^-DWnt2^- embryos, because those embryos offer a very sensitive test of stimulation of the Wnt pathway. It remains unclear whether these DWnts make any residual contribution to DT in the wild type (Llimargas, 2001).

However, several observations suggest that DWnt2 contributes to tracheal development in the wild-type fly. Notably, DWnt2 is expressed near the tracheal cells at the appropriate stage, and when overexpressed in tracheal cells, it mimics the effects of overexpressing wg or a constitutively activated Arm. But, most importantly, the phenotype of wg^-DWnt2^- embryos indicates that wg and DWnt2 together are responsible for virtually all DT formation. Thus, DWnt2 probably cooperates with Wg or reinforces its main action (Llimargas, 2001).

Nevertheless, DWnt2^- embryos do not show a visible tracheal phenotype, indicating, at first sight, that the gene does not normally contribute to DT formation. This lack of abnormality suggests that wg alone (or with some help from different DWnts) is sufficient to sponsor normal development in these mutant flies. Nevertheless, it remains possible that DWnt2 could act in the wild-type. There are at least two alternative hypotheses that could explain the lack of tracheal phenotype in DWnt2^- embryos (Llimargas, 2001).

(1) The loss of DWnt2 could induce compensatory changes in the amount, distribution, or activity of the other DWnts. As in the case of DWnt6, -4, and -10, the expression of DWnt5 and DWnt8 (expression is detected only in the CNS at early stages) does not suggest that they act in tracheal development, although contributions under the level of detection cannot be discarded. Moreover, although some small changes have been detected in the expression of some DWnts in wg^- and wg^-DWnt2^- embryos (e.g., the loss of DWnt5 expression in the labial segment at stage 10 as well as loss of expression in lateral clusters of the thoracic segments at stage 11), no changes have been detected in the pattern of expression that might account for any strong tracheal rescue of DWnt2^- embryos. Therefore, it is not clear how other DWnts could contribute to the complete DT formation in DWnt2^- embryos (Llimargas, 2001).

(2) It is supposed that all DWnts bind the receptor with different affinities, with Wg binding most strongly. In the wild type, the DWnts could compete, but Wg would be most effective: the contribution of DWnt2 to DT formation would be minor. However, in embryos lacking Wg, mainly DWnt2 (which is expressed in a broader domain in wg^- embryos and is not now competing with Wg) could bind and partially substitute for Wg. In the absence of DWnt2, Wg (and maybe other DWnts) would have no competition from DWnt2 and would become even more efficient, compensating for the contribution to DT formation that DWnt2 has in the wild type. Finally, in the absence of both Wg and DWnt2, other DWnts, even if they did not act in the wild type, could now bind to the unoccupied receptors and have some tiny effect on DT formation (Llimargas, 2001).

Complications of this kind may bedevil attempts to analyze the precise wild type contributions of individual members of other gene families (Llimargas, 2001).

Thus DWnt2 can act in tracheal development, whereas Wg acts in both developing epidermis and trachea. The other five DWnts do little for the trachea. As with the achaete/scute homologs (which are alike in structure and function but have different patterns of expression and, therefore, act in different places, it may be that the DWnts are preserved fundamentally because seven genes, even if they do similar things, can be regulated in a more sophisticated way than one. Perhaps, like DWnt2, they perform specialized tasks, acting locally to help Wg in ways that could not be provided by any additional regulatory control of wg itself. In the case of tracheae, this tissue can have differential sensitivity to specific homologs, a property that may allow even more intricate forms of control (Llimargas, 2001).

Mutations in Wnt2 alter presynaptic motor neuron morphology and presynaptic protein localization at the Drosophila neuromuscular junction

Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. This study sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins (Liebl, 2010).

Synapse development is a complex process that requires pre- and postsynaptic cells to maintain constant communication with one another via transsynaptic signaling. Molecules with well established roles in this process include cell adhesion molecules, Ephrin ligands and Eph receptors, and the classical cadherins. This study provides evidence that Wnt2 may act as a signaling molecule that is expressed by the postsynaptic muscle where it acts on the presynaptic cell to directly or indirectly regulate size of the presynaptic motor neuron and promote protein localization (Liebl, 2010).

Several pieces of evidence are presented to support the conclusion that Wnt2 regulates development of the NMJ. wnt2 mutations produce overgrown NMJs with an increased number of branches. The significant increase in NMJ branches is present early in development as both 1^st and 2^nd instar mutant larvae also exhibit an overgrowth. This could indicate that wnt2 is required shortly after synapse formation to regulate NMJ growth. Although the NMJ is enlarged in the wnt2 mutant, the number of Brp puncta remained similar to controls. The level of Brp immunfluorescence is reduced, however, suggesting that the amount of Brp protein per punctum is decreased. Since Brp is localized to active zones where it promotes Ca²⁺ channel clustering, reduced staining of Brp puncta may indicate that functioning of the active zones are compromised. A recent paper however, reported that the majority of active zones in Drosophila rab3 mutants do not contain Brp (Liebl, 2010).

Electrophysiological recordings from muscle 6 of wnt2 mutants showed that the amplitudes of evoked events were significantly reduced without a reduction in the frequency or amplitude of spontaneous events. This intriguing finding led to a careful examination the concentrations of presynaptic proteins including Syt, Syb, CSP, Brp. The levels of Syb and Brp were significantly reduced in the wnt2 mutant as indicated by immunocytochemistry. Syb is a synaptic vesicle associated protein that assembles with syntaxin and SNAP-25 to form the SNARE complex, which renders vesicles competent for fusion. The electrophysiological phenotype observed in wnt2 mutants is consistent with both the syb and brp mutant phenotypes. Syb is required for evoked but not spontaneous transmission in Drosophila and knockdown of brp in neurons reduces evoked responses while preserving spontaneous transmission. Thus, the finding that the total number of Brp puncta in wnt2 mutants is unchanged coupled with the significant reduction in evoked responses, suggests that there may be a reduction in the number of functional active zones in the wnt2 mutant. Indeed, it was observed that the immunolabeling of Brp puncta is reduced, suggesting that the amount of Brp protein per puncta is decreased (Liebl, 2010).

The reduced labeling of Brp and Syb in the presynaptic motor neuron of wnt2 mutants is not likely due to changes in transcriptional mechanisms. Messenger RNA levels of both brp and syb are similar in mutant and control animals. It is possible that the observed changes in Brp and Syb are due to mislocalization of mRNA. Another possibility is that the loss of wnt2 leads to mislocalization of presynaptic proteins. Rat Wnt7a, which is 77.1% similar in amino acid sequence to Wnt7b, when applied to hippocampal cultures, induces clustering of Syt, SV2, and increases the number of clusters containing synaptophysin. Both Wnt7a and Wnt7b induce clustering of synapsin I in mouse cerebellar granule cell cultures. Treatment of culture medium with Wnt7b increased Bassoon clustering but did not increase total protein levels as indicated by Western Blots (Liebl, 2010).

Wnts are secreted glycoproteins. An important aspect of understanding the function of Wnt2 is to determine where at the synapse it functions to regulate presynaptic motor neuron morphology and localization of proteins. Cell-type specific cDNA expression in wnt2 mutants showed that Wnt2 may function in either presynaptic motor neurons or postsynaptic muscle. Expression in either motor neurons or muscle restored presynaptic Brp density. Expression in postsynaptic muscle also restored NMJ morphology while expression in presynaptic motor neurons caused a further increase in the number of NMJ branches. Knockdown of wnt2 in muscle produced a phenotype similar to that of null mutant. The results collectively suggest that Wnt2 may be expressed by the postsynaptic muscle where it acts as a retrograde signal that negatively regulates NMJ growth and promotes the localization of presynaptic proteins. Based on the current data, it cannot be concluded whether wnt2 directly or indirectly regulates these synaptic characteristics (Liebl, 2010).

A number of other molecules have been implicated in retrograde synaptic signaling including Ankyrin, nitric oxide, SAP97, Synaptotagmin 4, and secreted proteins such as Glass Bottom Boat, fibroblast growth factors, and Wnts. Mouse Wnt3 is secreted from motor neurons where it increases the size of growth cones and branching of incoming sensory neurons. Similarly, mouse Wnt7a is expressed by cerebellar granule cells and acts on presynaptic mossy fibers to remodel axons and growth cones. The receptor(s) that mediate the above effects are, as yet, unidentified but Wnt ligand binding to its receptor induces cytoskeletal changes (Liebl, 2010).

This study sought to determine the receptor through which Wnt2 signaled by examining mutants for drl, fz, fz2, and fz3. None of the mutants exhibited a reduction in the density of Brp. Mutations in fz3, however, led to a significant increase in NMJ branches similar to that of the wnt2 mutant. This raised the possibility that Wnt2 was signaling via Fz3 to negatively regulate NMJ growth. wnt2^O; fz3^G10 double mutants, however, exhibited a significant increase in NMJ branches greater than that of the single mutants suggesting wnt2 and fz3 act independently of one another to regulate synaptic growth. Wnt2 may signal via the Wnt receptors Fz4 or Smo but binding assays indicate there is no detectible binding between Wnt2 and these receptors. It is also possible that no phenotype was detected in Frizzled mutants due to functional redundancy of these receptors. Future work will be required to uncover the receptor that mediates Wnt2 signaling (Liebl, 2010).

EVOLUTIONARY HOMOLOGS

Formation of the vertebrate limb requires specification of cell position along three axes. Proximal-distal identity is regulated by the apical ectodermal ridge (AER) at the distal tip of the growing limb. Anterior-posterior identity is controlled by signals from the zone of polarizing activity (ZPA) within the posterior limb mesenchyme. Dorsal-ventral identity is regulated by ectodermally derived signals. Recent studies have begun to identify signaling molecules that may mediate these patterning activities. Members of the fibroblast growth factor (FGF) family are expressed in the AER and can mimic its proximal-distal signaling activity. Similarly, the gene Sonic hedgehog (Shh) is expressed in the ZPA, and Shh-expressing cells, like ZPA cells, can cause digit duplications when transplanted to the anterior limb margin. In contrast, no signal has yet been identified for the dorsal-ventral axis, although Wnt-7a is expressed in the dorsal ectoderm, suggesting that it may play such a role. To test this possibility, mice were generated lacking Wnt-7a activity. The limb mesoderm of these mice shows dorsal-to-ventral transformations of cell fate, indicating that Wnt-7a is a dorsalizing signal. Many mutant mice also lack posterior digits, demonstrating that Wnt-7a is also required for anterior-posterior patterning. It is proposed that normal limb development requires interactions between the signaling systems for these two axes (Parr, 1995).

Growth and patterning of the vertebrate limb are controlled by the ridge, posterior mesenchyme, and non-ridge ectoderm. Fibroblast growth factor 4 (FGF4) and Sonic hedgehog (SHH) can mediate signaling from the ridge and posterior mesenchyme, respectively. Dorsal ectoderm is required together with FGF4 to maintain Shh expression. Removal of dorsal ectoderm results in loss of posterior skeletal elements, which can be rescued by exogenous SHH. Wnt7a, which is expressed in dorsal ectoderm, provides the signal required for Shh expression and formation of posterior structures. These results provide evidence that all three axes (dorsoventral, proximodistal, and anteroposterior) are intimately linked by the respective signals WNT7a, FGF4, and SHH during limb out-growth and patterning (Yang, 1995).

REFERENCES

Search PubMed for articles about Drosophila Wnt oncogene analog 2

DeFalco, T., Camara, N., Le Bras, S. and Van Doren, M. (2008). Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad. Dev. Cell 14(2): 275-86. PubMed Citation: 18267095

Dobzhansky, T. (1931). Interactions between female and male parts in gynandromorphs of Drosophila simulans. Wilhelm Roux's Arch. Dev. Biol. 123: 719-746.

Kozopas, K. M., Samos, C. H. and Nusse, R. (1998). DWnt-2, a Drosophila Wnt gene required for the development of the male reproductive tract, specifies a sexually dimorphic cell fate. Genes Dev. 12(8): 1155-1165. PubMed Citation: 9553045

Kozopas, K. M. and Nusse, R. (2002). Direct flight muscles in Drosophila develop from cells with characteristics of founders and depend on DWnt-2 for their correct patterning. Dev. Biol. 243: 312-325. 11884040

Liebl, F. L., McKeown, C., Yao, Y. and Hing, H. K. (2010). Mutations in Wnt2 alter presynaptic motor neuron morphology and presynaptic protein localization at the Drosophila neuromuscular junction. PLoS One 5(9): e12778. PubMed Citation: 20856675

Llimargas, M. and Lawrence, P. A. (2001). Seven Wnt homologues in Drosophila: A case study of the developing tracheae. Proc. Natl. Acad. Sci. 98: 14487-14492. 11717401

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